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
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………………………………..
iii
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
iv
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
v
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
ix
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.
x
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
xi
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
xii
XRD X-ray diffraction
xiii
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
xiv
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
xv
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
xvi
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
xviii
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
xix
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
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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
xxi
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
xxii
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
xxiii
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
xxiv
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
-1 VOLATILE OIL IC50 (MG ML )...... 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
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-1 TABLE 4.14: TIME OF KILL FOR THE STANDARD DRUGS CIPROFLOXACIN (0.1 MG ML )
AGAINST BACTERIAL ORGANISMS...... 153
-1 TABLE 4.15: TIME OF KILL FOR ETHYL ACETATE EXTRACT (0.1 MG ML ) 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
xxvi
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
xxvii
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
1 FIGURE 4.13: DOSE-RESPONSE CURVE FOR TRYPANOSOME ASSAY ...... 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
xxviii
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
xxix
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
1 FIGURE 4.40: DOSE RESPONSE CURVE FOR TRYPANOSOME ASSAY...... 211
xxx
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.
xxxi
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
xxxii 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
xxxiii 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.
xxxiv
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 1 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).
2
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).
3
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
4 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
5 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).
6
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
7
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-
8 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,
9 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.
10
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.
11
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 12 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
13 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).
14
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
15 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).
16
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
17 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).
18
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).
19
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
20 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).
21
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
22 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.
23
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).
24
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
25
(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
26 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
27 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.
28
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
29 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
30 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.
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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).
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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
33 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
34 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
35 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
36 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
37 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
38 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.,
39
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)
40
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).
41
(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)
42
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-
43 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).
44
(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)
45
(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).
46
(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
47 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).
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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 49 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 50 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
51 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).
52
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).
53
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
54 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
55 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).
56
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.
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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,
58 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).
59
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.
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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.
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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
62 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
63 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).
64
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
65 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).
66
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.
67
(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
68 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
69 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
70 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
72 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
73 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)
75 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
76 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
77 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).
80
81
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.
82
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.
83
84
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, 85
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
86 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.
87
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
88 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
89 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.
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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.
91
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
1 13 chloroform at δH 7.24 for H NMR spectra and δC 77.20 for C 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
92 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
93 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.
퐴 − 퐴 % 퐼푛ℎ𝑖푏𝑖푡𝑖표푛 = 푐표푛푡푟표푙 푣표 × 100 퐴푐표푛푡푟표푙
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.
94
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
95 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
96 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.
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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)
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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.
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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.
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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
101 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
o 37 C 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.
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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
103 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.
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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-
105 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).
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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 107 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.
Oil composition Retention time Constituents Leaves Flowers Seeds 3.43 Isopentyl acetate 0.17 - - 3.89 β-Thujene - 0.65 - 3.97 α-Pinene 20.02 - 31.03 3.98 1R-α-Pinene - 4.57 - 4.12 Camphene 0.63 3.81 - 4.25 Vinyl amyl carbinol - 0.14 - 4.29 β-Phellandrene - 0.37 - 4.34 β-Pinene 1.10 2.26 1.18 4.52 3,4-dimethylheptane 0.81 - - 4.69 o-Cymene - 1.22 - 4.73 Limonene - - 3.97 4.77 Eucalyptol 48.98 8.11 56.00 4.97 γ-Terpinene 0.21 1.76 - 5.21 3-Carene 0.17 0.05 - 5.23 β-linalool 0.57 - 1.08 5.44 α-Fenchol 0.93 - - 5.67 Pinocarveol 5.75 - 0.51 5.73 (+)-2-Bornanone - 4.35 - 5.76 Camphenilanol 0.27 - - 5.85 Pinocarvone 2.81 - - 5.88 Borneol - 1.75 - 5.94 Terpinen-4-ol 0.79 0.77 0.77
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6.03 α-Terpineol 8.01 0.22 3.69 6.09 Myrtenol 0.29 - - 6.22 cis-Carveol 0.70 - - 6.23 (+)-Camphene - 0.26 - 6.30 cis-p-metha-1(7), 8- 0.53 - - diene-2-ol 6.42 Geraniol 0.40 - - 6.75 (-)-Bornyl acetate - 10.02 - 7.11 trans -2-Acetoxyl-1, 8- 0.18 - - cineole 7.23 4-Carene 0.76 - - 7.42 Copaene - 0.34 - 7.77 Caryophyllene - 11.89 - 7.89 Aromadendrene - 3.80 - 7.93 α-Gurjinene - 0.70 - 7.98 Humulene - 1.05 - 8.03 Alloaromadendrene - 0.89 - 8.08 α-Elemene - 0.51 - 8.16 α-Farnesene - 0.42 - 8.25 Bicyclogermacrene - 5.71 - 8.36 (+)-δ-Cadinene - 2.10 - 8.52 Elemol - 0.58 - 8.65 (+)-Valencene - 0.37 - 8.76 Spathulenol 0.19 3.16 1.10 8.80 (-)-Globulol 0.39 - 0.65 8.82 (+)-Ledene - 2.33 - 8.86 γ-Gurjinene 0.20 - - 8.87 (+)-Viridiflorol - 0.49 - 8.91 Rosifoliol - 0.50 - 8.96 3,4-Dimethoxy-benzoic 1.72 - - acid methyl ester 9.02 β-Eudesmol 0.26 - - 9.07 γ-Eudesmol - 5.21 - 9.11 Hinesol - 2.03 - 9.22 α-Eudesmol - 12.93 - 9.28 cis-1(7),8-p-mentha- - 1.05 - diene-2-ol 9.38 5-Amino-1- - 1.15 - phenylpyrazole 9.52 p-Cymene-3-propionic - 0.35 - acid, α-methyl ester 9.82 3-Carene, 4- - 0.34 - isopropenyl 9.93 cis-Calemenene - 0.42 - 10.69 Geranyl-α-Terpinene - 0.28 - 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 -
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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
110 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).
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Table 4.2: Major Components of Volatile Oil of Callistemon citrinus from
Various Parts of the World
Origin Major Components Reference
α-Eudesmol (12.93%), caryophyllene (11.89%), bornyl-acetate (10.02%), South Africa f Larayetan et al., 2017 eucalyptol (8.11%), bicyclogermacrene (5.71%) and α-eudesmol (5.21%)
Eucalyptol (48.98%), α-pinene (20.02%), South Africa l α-terpineol (8.10%), isopinocarveol Larayetan et al., 2017
(5.75%) and pinocarvone (2.81%) 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- Kumar et al.,2015 terpineol (14.6%) Eucalyptol (34.20%), α-pinene (29.0%), α- Iran l Zandi et al., 2012 terpineol (16.70%), α-phellandrene (9.0%) Eucalyptol (67.60%), α-pinene (9.40%), β- Iran l Minar et al., 2014 pinene (4.70%) α-Pinene (25.70%), eucalyptol (18.10%), β- Iran f Minar et al., 2014 pinene (7.30%), linalool (5.30%). Eucalyptol (41.30%), α-pinene (19.10%), Iran s Minar et al., 2014 α-terpineol (4.10%) Eucalyptol (68.0%), α-pinene (12.80%), α- Reunion Island l Chane-Ming et al., 1998 terpineol (10.6%) 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- Oyedeji et al., 2009 pinene (4.70%) 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.
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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).
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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
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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
Positive control Zone of inhibition of volatile oils and standard drug Microorganism 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 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 hydrophila 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 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 alginolyticus Salmonella 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 typhi Gram-positive bacteria Staphylococcal 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 enteritis Staphylococcus 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 aureus Listeria 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 monocytogenes Zone of inhibition (millimeter), ACC: Aemreg Culture Collection, ATCC:
American Type Collection Center, values are mean ± SD, n = 3
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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)
116
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
117 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)
Callistemon citrinus Standard antioxidant (positive control) S/N Activity Leaves’ oil Flowers’ oil Vitamin C β-Carotene BHT (IC50) (IC50) (IC50) (IC50) (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 118 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.
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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,
120 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
121 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.
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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
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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 Jan Feb Mar April May June Harvest Treatment RT 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, 3.551 - - 0.04 0.06 0.05 0.03 methyl valerate 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- 5.446 0.30 0.53 - 0.29 1.20 0.76 diene-2-ol 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- 5.502 - - 0.44 0.03 0.11 0.09 1-ol Trans-p-2,8- 5.523 - - - - 0.18 - Menthadien-1-ol
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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- 8.169 ------lavandulol 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
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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- 8.996 - - - - 4.70 2.42 methylazulene 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 16.19 22.89 33.48 24.19 18.52 14.09 Monoterpene Oxygenated 78.44 70.03 51.29 63.91 59.95 46.24 Monoterpenes Sesquiterpene 0.21 - 4.38 - 0.82 0.03 Hydrocarbons Oxygenated - 0.78 - 0.64 4.89 1.00 Sesquiterpenes 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
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Month of July Aug Sept Oct Nov Dec Harvest Treatment RT 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, - - - 0.06 0.03 0.12 3.551 methyl valerate 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- 0.56 0.06 - 0.05 0.10 1.36 5.446 diene-2-ol 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- 0.17 - - - 0.10 0.18 5.502 ol Trans-p-2,8- - - - 0.20 - - 5.523 Menthadien-1-ol 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
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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- - - - 0.24 - - 8.169 lavandulol 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 -
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Ledol 8.936 0.11 2.15 - - - - 1-acetyl-2-amino-3- - - - 7.48 - - cyano-7-isopropyl-4- 8.996 methylazulene 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 17.01 63.74 8.45 20.55 17.39 14.12
Monoterpene Oxygenated 61.23 13.37 84.86 32.95 66.53 73.94
Monoterpenes Sesquiterpene 0.33 1.36 - 7.95 2.65 0.15
Hydrocarbons Oxygenated 1.80 8.77 0.43 7.25 2.33 1.83
Sesquiterpenes 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.
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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
130 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
131 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
132 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
133 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
-1 -1 (summer) IC50 0.88 mg mL , April (autumn) IC50 1.35 mg mL . 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,
134 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
135 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.
136
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
137 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
138
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 Peak Molecular Molar Mass Compound RI Compound Area (%) Formula (g mol-1) Nature
4-Carene 919 0.22 C10H16 136 Monoterpene Oxygenated Eucalyptol 1059 2.22 C H O 154 10 18 monoterpenoid Unsaturated 2-Nonenal 1112 0.27 C H O 140 9 16 aldehyde Limonene Oxygenated 1128 0.31 C H O 168 diepoxide 10 16 2 monoterpenoid Oxygenated Pinocarveol 1131 0.46 C H O 152 10 16 monoterpenoid Oxygenated α-Terpineol 1143 1.22 C H O 154 10 18 monoterpenoid
4-Methyl-isopulegone 1252 0.27 C10H18O 166 Oxygenated
139
monoterpenoid Unsaturated trans-2-Decenol 1266 0.92 C H O 156 10 20 alcohol
Aromadendrene 1386 0.46 C15H24 204 Sesquiterpene (8E,10Z)-1,8- 1518 27.00 C H 248 Hydrocarbon Pentadecatriene 15 26 Oxygenated β-Eudesmol 1593 0.55 C H O 222 15 26 sesquiterpenoid Methyl 14-methyl- 1814 2.04 C H O 270 Ester pentadecanoate 17 34 2
Palmitic acid 1968 32.87 C16H32O2 256 Fatty acid Unsaturated 9-Octadecenal 2007 0.91 C H O 266 18 34 aldehyde Stearic acid, methyl 2077 1.07 C H O 298 Ester ester 19 38 2 trans-Vaccenic acid, 2085 3.46 C H O 296 Ester methyl ester 19 36 2
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.
Peak Molecular Molar mass Compound Name of Compound RI Area (%) formula (g mol-1) Nature
4-Carene 919 0.35 C10H16 136 Monoterpene 2,6-Dimethyl-3,7-octadien- Oxygenated 1041 0.31 C H O 154 2-ol, 10 18 monoterpenoid Oxygenated Eucalyptol 1059 4.55 C H O 154 10 18 monoterpenoid Unsaturated 2-Nonenal 1112 1.53 C H O 140 9 16 aldehyde Oxygenated trans-Pinocarveol 1131 0.69 C H O 152 10 16 monoterpenoid Oxygenated α-Terpineol 1143 1.56 C H O 154 10 18 monoterpenoid Oxygenated Myrtanal 1126 0.51 C H O 152 10 16 monoterpenoid Oxygenated 4-methyl-isopulegone 1252 0.38 C H O 166 10 18 monoterpenoid
Aromadendrene 1386 0.46 C15H24 204 Sesquiterpene
Patchulane 1393 0.29 C15H26 206 Sesquiterpene
Viridiflorol 1530 1.17 C15H26O 222 Sesquiterpene Methyl-2- 1842 7.18 C H O 258 Hydroxyl ester hydroxytetradecanoate 15 30 3
Methyl hexadecanoate 1878 3.76 C17H34O2 270 Ester
Palmitic acid 1968 13.57 C16H32O2 256 Fatty acid
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Unsaturated 9-Octadecenal 2007 1.70 C H O 266 18 34 aldehyde
Stearic acid, methyl ester 2077 2.03 C19H38O2 298 Ester trans-Vaccenic acid, methyl 2085 5.67 C H O 296 Ester ester 19 36 2
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
141 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.
Overall Overall Tannin Overall Phenolic Overall Flavonoid Overall Flavonol Antioxidant Extracts Content Content Content Content Capacity (mg TAE/100g) (mg GAE/100g) (mg RE/100g) (mg RE/100g) (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
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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
144 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
145 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.
Callistemon citrinus Reference compound Activity 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
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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).
147
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.
Positive control Zone of inhibition of extracts and standard drug Microorganism Ciprofloxacin (mg mL-1) Ethyl acetate extract (mg mL-1) Methanol extract (mg m-L1) Concentration 0.4 0.1 0.025 0.4 0.1 0.025 0.4 0.1 0.025 Gram-negative Bacteria Aeromonas 34.0 26.0 10.0 ± 20.0 ± 0.5 22.7 ±2.3 15.0 ± 1.0 11.3 ± 1.2 19.7 ±1.5 15.0 ± 1.0 hydrophila ACC ±0.6 ±2.0 0.3 Escherichia coli 38.0 32.0 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 ATCC 35150 ±4.0 ±4.0 Vibro 33.0 29.0 alginolyticus 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 ±2.0 ±2.0 DSM 2171 Salmonella typhi 40.0 34.0 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 ACC ±1.0 ±0.6 Pseudomonas 38.0 36.0 14.0 ± 33.0 ± 3.0 28.7 ± 1.2 17.3 ± 2.1 14.7 ± 1.2 22.7 ± 1.2 17.2 ± 1.9 aeruginosa ACC ±2.0 ±1.0 1.0 Gram positive Bacteria Staphylococcal 40.0 32.0 12.3 ± 28.0 ± 0.2 20.3 ± 0.6 14.7 ± 1.2 11.0 ± 1.7 21.0 ± 1.0 15.7 ± 1.5 enteritis ACC ±5.0 ±1.0 0.6 Staphylococcus 29.0 24.0 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 aureus ACC ±2.0 ±0.0 Listeria 44.0± 38.0± monocytogenes 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 6.0 0.2 ACC Zone of inhibition (millimeter), ACC Aemreg culture collection, ATCC
American type collection center, values are mean ± SD, n = 3
148
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).
149
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.
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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.
Ethylacetate leaf Ciprofloxacin Bacteria Methanol leaf extract extract Positive control Aeromonas hydrophila 0.025 ± 0.00 0.100 ± 0.01 0.100 ± 0.02 ACC Escherichia coli ATCC 0.025 ± 0.00 0.100 ± 0.00 0.100 ± 0.00 35150
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Vibro alginolyticus 0.025 ± 0.00 0.100 ± 0.00 0.100 ± 0.01 DSM 2171 Salmonella typhi ACC 0.025 ± 0.01 0.025 ± 0.00 0.100 ± 0.01 Pseudomonas 0.100 ± 0.00 0.100 ± 0.00 0.100 ± 0.02 aeruginosa ACC Staphylococcal 0.025 ± 0.01 0.100 ± 0.00 0.100 ± 0.00 enteritis ACC Staphylococcus aureus 0.100 ± 0.00 0.025 ± 0.01 0.100 ± 0.01 ACC Listeria 0.025 ± 0.00 0.100 ± 0.01 0.100 ± 0.01 monocytogenes ACC
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
152
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.
Period of incubation (h) Bacteria strains 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.
Period of incubation (h) Bacteria strains 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
153
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.
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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
155
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
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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 157 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).
158
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
159 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).
160
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
161 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
162
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).
Positive control Microorganism Seed volatile oil (mg mL-1) Ciprofloxacin (mg mL-1) Concentration 0.4 0.1 0.025 0.4 0.1 0.025 Escherichia coli 0157:H7:ATCC 38.0 ± 4.0 32.0 ± 4.0 24.0 ± 0.9 55.0 ± 1.0 40.0 ± 1.0 20.0 ± 0.8 35150 Vibro alginolyticus 33.0 ± 2.0 29.0 ± 2.0 23.0 ± 0.4 62.0 ± 4.0 45.0 ± 0.8 22.0 ± 0.5 DSM 2171 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 40.0 ± 3.0 32.0 ± 1.0 28.0 ± 0.2 52.0 ± 2.0 40.0 ±0.9 25.0 ± 0.8 enteritis ACC Staphylococcus 29.0 ± 1.0 24.0 ± 0.0 10.0 ± 3.0 56.0 ± 3.0 30.0 ± 5.0 19.0 ± 2.0 aureus ACC Listeria Ivanovii 44.0 ± 4.0 38.0 ± 0.5 30.0 ± 1.0 48.0 ± 2.0 36.0 ± 3.0 20.0 ± 3.0 ATCC 19119 Mycobacterium smegmatis ATCC 36.0 ± 0.6 25.0 ± 2.0 18.0 ±1.0 38.0 ± 1.0 21.0 ± 2.0 14.0 ± 0.8 19420
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).
165
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
166 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
167 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 Hexane Crude DCM Crude EA Crude Me Crude Material used (% Yield) (% Yield) (% Yield) (% 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).
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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
169 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.
IC50 (μg/mL for samples, μM for Compound 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.
170
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.
IC50 (μg/mL for samples, μM for Compound 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.
IC50 Compound 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.
IC50 Compound 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
174 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, IC50 μg/mL for Samples) 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
176
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, IC50 ug/ml for Samples) 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.
Anti- Anti- Cyto- Anti- Anti- Cyto-toxicity plasmodi trypanoso toxicity S.I on 3D7. (S.I Samples malarial cell trypanosomal cell viability al activity mal activity on T. b brucei) viability cell viability (%) IC50 activity IC50 value value IC50 value Crude A 1.85 ± 1.22 -4.34 ±0.91 27.81 ± 0.93 2.30 1.19 92.1 40.04 (30.49) 15.90 Crude B 8.43 ± 7.98 -1.11 ± 0.64 26.74 ± 6.20 3.61 3.02 57.4 (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) 6.52 Crude E 7.69 ± 3.14 10.22 ± 1.44 19.35 ±0.08 3.22 0.39 32.5 (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,
178
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.
Dichloromethane Ethyl acetate Methanol Phytochemicals Hexane leaf DCM seed leaf leaf leaf 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.,
179
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).
180
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
181 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
182 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
183 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,
184 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;
185
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
186 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
187
o o the reduction of AgNO3. Peaks observed at 2 values of 28 and 34 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.
188
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.
189
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
190 the shape and size of the synthesized materials. The same observation was reported (Jyoti et al., 2016).
A B
C
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
191 material after drying) with silver showing to possess about 89% of the overall composition of elements in the different materials.
A B
C D
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).
192
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
193 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. 194
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
195
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.
197
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.
198
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.
Positive control Microorganism Nano Leaf (mg/mL) Ciprofloxacin (mg/mL) concentration 62.5 31.25 15.625 62.5 31.25 15.625 Gram-Negative Bacteria Strains Escherichia coli 0157:H7:ATCC 35.0 ± 4.0 30.0 ± 4.0 18.0 ± 0.9 18.0 ±3.0 12.0 ± 3.0 7.0 ± 3.0 35150 Vibro alginolyticus 33.0 ± 2.0 25.0 ± 2.0 13.0 ± 0.4 13.0 ± 2.0 11.0 ± 5.0 9.0 ± 4.0 DSM 2171 Salmonella typhi 35.0 ± 1.0 32.0 ± 0.6 18.0 ± 3.0 13.0 ± 0.5 10.0 ± l.0 8.0 ± 2.0 ACC Gram-Positive Bacteria Strains Staphylococcal 40.0 ± 5.0 32.0 ± 1.0 17.0 ± 0.2 20.0 ± 0.5 16.0 ± 0.1 8.0 ± 0.4 enteritis ACC Staphylococcus 20.0 ± 2.0 15.0 ± 0.0 11.0 ± 2.0 10.0 ± 4.0 8.0 ± 1.0 7.0 ± 2.0 aureus ACC Listeria Ivanovii 35.0 ± 6.0 30.0 ± 0.2 22.0 ± 1.0 18.0 ± 2.0 12.0 ± 2.0 10.0 ± 1.0 ATCC 19119 Mycobacterium smegmatis ATCC 35.0 ± 6.0 32.0 ± 0.0 24.0 ± 1.0 13.0 ± 0.4 `10.0 ± 0.5 8.0 ± 1.0 19420
200
Microorganism Nano Seed (mg/ mL) Nano Flower(mg/mL) concentration 62.5 31.25 15.625 62.5 31.25 15.625 Gram-Negative Bacteria Strains Escherichia coli 0157:H7:ATCC 18.0 ± 3.0 15.0 ± 3.0 8.0 ± 3.0 25.0 ± 3.0 18.0 ± 3.0 10.0 ± 3.0 35150 Vibro alginolyticus 15.0 ± 2.0 12.0 ± 5.0 6.0 ± 4.0 15.0 ± 2.0 10.0 ± 5.0 8.0 ± 4.0 DSM 2171 Salmonella typhi 20.0 ± 0.5 18.0 ± 1.0 10.0 ± 2.0 15.0 ± 0.5 10.0±1.0 8.0 ± 2.0 ACC Gram-Positive Bacteria Strains Staphylococcal 25.0 ± 0.5 23.0 ± 0.1 18.0 ± 0.4 22.0 ± 0.5 18.0 ± 0.1 15.0 ± 0.4 enteritis ACC Staphylococcus 15.0 ± 4.0 15.0 ± 1.0 13.0 ± 2.0 10.0 ± 4.0 8.0 ± 1.0 8.0 ± 2.0 aureus ACC Listeria Ivanovii 18.0 ± 2.0 15.0 ± 2.0 13.0 ± 1.0 25.0 ± 2.0 20.0 ±2.0 18.0 ± 1.0 ATCC 19119 Mycobacterium smegmatis ATCC 15.0 ± 3.0 10.0 ± 0.1 8.0 ± 1.0 15..0 ± 0.5 15.0 ± 0.4 12.0 ± 0.5 19420
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)
201
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 7.8125 7.8125 7.8125 15.625 0.5 mL VG * 35150 Vibro alginolyticus 7.8125 7.8125 7.8125 15.625 0.5 mL VG DSM 2171 Salmonella typhi 7.8125 7.8125 7.8125 7.8125 0.5 mL VG ACC Staphylococcal 7.8125 7.8125 7.8125 15.625 0.5 mL VG enteritis ACC Staphylococcus 7.8125 15.625 7.8125 7.8125 0.5 mL VG aureus ACC Listeria Ivanovii 7.8125 7.8125 7.8125 15.625 0.5 mL VG ATCC 19119 Mycobacterium 15.625 1.5 mL smegmatis ATCC 7.8125 7.8125 7.8125 VG 19420 * VG – visible growth
Table 4.32: Minimum Bactericidal Concentration (MBC) Values (mg/mL)
for Nanoparticles and Standard Drug.
DMSO Nanoparticle Nanoparticle Nanoparticle Ciprofloxacin Bacteria Negative (Leaf) (Seed) (Flower) Positive control control Escherichia coli Bactericidal at Bactericidal at Bactericidal at Bactericidal at 0157:H7:ATCC 0.5 mL VG * 31.25 NG 31.25 NG * 62.5 NG 35150 ≤ 15.625 NG Vibro Bactericidal at Bactericidal at Bactericidal at Bactericidal at alginolyticus 0.5 mL VG 31.25 NG 31.25 NG 31.25 NG DSM 2171 ≤15.625 NG
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Salmonella Bactericidal at Bactericidal at Bactericidal at Bactericidal at 0.5 mL VG typhi ACC 31.25 NG 31.25 NG 15.625 NG ≤15.625 NG
Staphylococcal Bactericidal at Bactericidal at Bactericidal at Bactericidal at 0.5 mL VG enteritis ACC 62.5 NG 31.25 NG 15.625 NG ≤15.625 NG
Staphylococcus Bactericidal at Bactericidal at Bacteriostatic Bactericidal at 0.5 mL VG aureus ACC 31.25 NG 31.25 NG at 31.25 VG ≤15.625 NG
Listeria Ivanovii Bactericidal at Bacteriostatic Bactericidal at Bactericidal at 0.5 mL VG ATCC 19119 62.5 NG at 15.75 VG 61.5 NG ≤15.625 NG Mycobacteriu Bactericidal at Bactericidal at Bacteriostatic Bactericidal at m smegmatis 0.5 mL VG 62.5 NG 62.5 NG at 31.25 VG ATCC 19420 ≤15.625 NG * 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
203 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,
204 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
205 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
+ 0 bioreduction of Au ions of HAuCl4⋅4H2O into metallic Au 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).
206
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
207
(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).
208
A B
C
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
209 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.
210
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
211
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.
Positive control Microorganism Au Seed nano (mg mL-1) Ciprofloxacin (mg mL-1) Concentration 62.5 31.25 15.625 62.5 31.25 15.625 Gram-negative bacteria strains Escherichia coli 0157:H7:ATCC 35.0 ± 4.0 30.0 ± 4.0 18.0 ± 0.9 15.0 ± 3.0 12.0 ± 3.0 7.0 ± 3.0 35150 Vibro alginolyticus 33.0 ± 2.0 25.0 ± 2.0 13.0 ± 0.4 13.0 ± 2.0 12.0 ± 5.0 9.0 ± 4.0 DSM 2171 Salmonella typhi 35.0 ± 1.0 32.0 ± 0.6 18.0 ± 3.0 15.0 ± 0.5 9.0 ± 1.0 7.0 ± 2.0 ACC Gram-positive bacteria strains Staphylococcal 40.0 ± 5.0 32.0 ± 1.0 17.0 ± 0.2 20.0 ± 0.5 13.0 ± 0.1 9.0 ± 0.4 enteritis ACC Staphylococcus 20.0 ± 2.0 15.0 ± 0.0 11.0 ± 2.0 20.0 ± 4.0 10.0 ± 1.0 8.0 ± 2.0 aureus ACC Listeria Ivanovii 35.0 ± 6.0 30.0 ± 0.2 22.0 ± 1.0 15.0 ± 2.0 12.0 ± 2.0 10.0 ± 1.0 ATCC 19119 Mycobacterium smegmatis ATCC 35.0 ± 6.0 32.0 ± 0.0 24.0 ± 1.0 13.0 ± 0.0 11.0 ± 1.0 9.0 ± 0.3 19420
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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.
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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 214 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.
215
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
-1 radical by 50% with IC50 of 1.49 and 1.13 mg mL compared to β- carotene and ascorbic acid which have an IC50 of 1.28 and 3.57 mg mL-1.
216
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
217 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,
218 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
219 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
220 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.
221
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.
222
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