ISOLATION AND CHARACTERISATION OF THE ANTIOXIDANT AND ANTIBACTERIAL COMPOUNDS FROM THE AERIAL PART OF ASPARAGUS SUAVEOLENS
M.Sc. (Chemistry)
MT Olivier 2016
ISOLATION AND CHARACTERISATION OF THE ANTIOXIDANT AND ANTIBACTERIAL COMPOUNDS FROM THE AERIAL PART OF ASPARAGUS SUAVEOLENS
by
MUTENDELA TABIZE OLIVIER
(201116132)
RESEARCH DISSERTATION
Submitted in fulfilment of the requirements for the degree of
MASTER OF SCIENCE
in
Chemistry
in the
FACULTY OF HEALTH SCIENCES
School of pathology & pre-clinical sciences
at the
SEFAKO MAKGATHO HEALTH SCIENCES UNIVERSITY
Supervisor: Prof LJ. Shai
Co-supervisors: Dr FM. Muganza
Dr SS. Gololo
May 2016
DECLARATION
I declare that “ISOLATION AND CHARACTERISATION OF THE ANTIOXIDANT AND
ANTIBACTERIAL COMPOUNDS FROM THE AERIAL PART OF ASPARAGUS
SUAVEOLENS” is my own work and that all the resources that I have used or quoted have been indicated and acknowledged by means of complete references and that this work has not been submitted before for any other degree at any other institution.
Name------
Student Number------
Signature------
Date------
ii
DEDICATION
This research project is dedicated to:
My late beloved parents Mr. Mutendela Mukosa Masahani and Mrs. Wanga Appoline
Kakembwa. May their souls continue to rest in peace.
The Mutendela family, especially my wife Marie Jeanne Umutesi and son Katumba-
Yango Joseph Mutendela. May God continue to pour wisdom, love and kindness upon
them.
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ACKNOWLEDGMENTS
I thank the almighty God for the knowledge and strength that he has given me so that I can be able to complete this research project. I also want to express my gratitude to the following persons for their contribution toward the completion of this dissertation:
Professor BB Marvey, the Director of the school of Pathology and Pre-clinical sciences
at Sefako Makgatho Health Sciences University for giving me the opportunity to be
admitted into the department of chemistry when he was the HoD.
Mr. SM Ndlovu, with whom I started this project as the main supervisor. Unfortunately,
he had to leave before the completion of the project.
Professor LJ Shai, who accepted the task of being the main supervisor after the
departure of Mr. SM Ndlovu, for your charismatic advices, team work spirit and
especially for your leadership skills.
Dr. FM Muganza, who stepped in as co-supervisor after Mr. SM Ndlovu’s departure,
for his valuable contributions. May his courage, expertise and advices continue to
nurture his spirit of Ubuntu.
Dr. SS Gololo, for his valuable contribution as co-supervisor in the Laboratory and in
the field.
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Medical microbiology department, especially Professor M Nchabeleng, Ms. LP
Kekana, Mr. LD Nemutavhanani and entire NHLS staff, for their kindness for
providing us with their facilities when conducting the antimicrobial tests.
The entire staff and postgraduate students of chemistry department for their valuable
contributions: Prof. NM Agyei, Dr. MA Dibeila, Dr. BR Maseko, Mr. J Mofokeng, Mr.
ZI Masilela, Mr. LS Sethoga, Mr. NFH Makhubela, Mrs. DM Moeletsi, Mr. MS
Adebayo, Ms. FH Mudau among others.
______
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TABLE OF CONTENTS
Page
TILTLE PAGE ...... i
DECLARATION ...... ii
DEDICATION ...... iii
ACKNOWLEDGMENTS ...... iv
TABLE OF CONTENTS ...... vi
LIST OF TABLES ...... xii
LIST OF FIGURES ...... xiv
LIST OF ABBREVIATIONS AND ACRONYMS ...... xix
RESEARCH OUTPUT ...... xxiii
ABSTRACT ...... xxiv
CHAPTER 1 ...... 1
INTRODUCTION, BACKGROUND, RESEARCH PROBLEM AND AIM OF STUDY .... 1
1.1. Introduction and Background ...... 1
1.2. Study problem ...... 2
1.3. Research question ...... 3
1.4. Purpose of the study ...... 3
1.4.1. Aim of the study ...... 3
1.4.2. Objectives of the study ...... 4
1.5. Scope of the study ...... 4
vi
CHAPTER 2 ...... 6
LITTERATURE REVIEW ...... 6
2.1. Introduction ...... 6
2.2. Overview on traditional medicine ...... 6
2.3. Medicinal plants ...... 7
2.4. Plant metabolites (PMs) ...... 8
2.4.1. Secondary metabolites ...... 9
2.4.2. Classes of different secondary metabolites in plants ...... 9
2.4.2.1. Flavonoids ...... 9
2.4.2.2. Tannins ...... 14
2.4.2.3. Coumarins ...... 17
2.4.2.4. Lignans ...... 21
2.4.2.5. Quinones ...... 26
2.4.2.6. Terpenoids...... 28
2.4.2.7. Saponins ...... 31
2.4.2.8. Cardiac glycosides ...... 35
2.4.2.9. Alkaloids ...... 37
2.4.3. Biological activity of secondary metabolites ...... 40
2.4.3.1. Antioxidants ...... 40
2.4.3.2. Antibacterial ...... 41
2.4.4. Bio-guided isolation of secondary metabolites ...... 42
2.5. Asparagus species: Overview ...... 43
2.6. Plant under current study: Asparagus suaveolens ...... 46
vii
2.6.1. Discovery of Asparagus suaveolens ...... 46
2.6.2. Taxonomy of Asparagus suaveolens ...... 46
2.6.3. Distribution of Asparagus suaveolens ...... 47
2.7.4. Botany of Asparagus suaveolens ...... 48
2.6.5. Ethnomedicinal usages of Asparagus suaveolens...... 48
CHAPTER 3 ...... 50
MATERIAL AND METHODS ...... 50
3.1. Instrumentations and apparatus...... 50
3.2. Solvents and chemicals ...... 50
3.3. Collection, identification and preparation of plant material ...... 51
3.3.1. Collection of the plant material...... 51
3.3.2. Identification of the plant ...... 52
3.3.3. Preparation of plant material ...... 52
3.4. Extraction ...... 52
3.5. Fractionation of the aerial part ethanol extract of Asparagus suaveolens ...... 53
3.6. Phytochemical analysis of the fractions ...... 54
3.6.1. Alkaloids ...... 55
3.6.2. Tannins ...... 55
3.6.3. Saponins ...... 55
3.6.4. Flavonoids (Shindo’s test) ...... 55
3.6.5. Cardiac glycosides ...... 56
3.6.6. Anthraquinones ...... 56
3.6.7. Terpenoids...... 56
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3.8.8. Steroids ...... 57
3.6.9. Reducing sugars ...... 57
3.6.10. Proteins ...... 57
3.6.11. Coumarins ...... 58
3.7. Screening for biological activity ...... 58
3.7.1. Antioxidant screening ...... 58
3.7.1.1. Bio-autographic antioxidant assay...... 58
3.7.1.2. DPPH free radical scavenging assay...... 59
3.7.2. Antibacterial assay ...... 59
3.7.2.1. Bio-autography antibacterial assay ...... 59
3.7.2.2. Minimum inhibitory concentration (MIC) ...... 60
3.7.2.3. Minimal bactericidal concentration (MBC) ...... 61
3.8. Bio-guided isolation of antioxidant and antibacterial compounds ...... 61
3.8.1. n-Hexane fraction...... 62
3.8.2. Chloroform fraction ...... 62
3.8.3. Butan-1-ol fraction ...... 63
3.9. Structure elucidation of isolated compounds ...... 63
3.9.1. Nuclear magnetic resonance ...... 64
3.9.2. Gas Chromatography-Mass Spectrometry ...... 64
3.9.3. Liquid chromatography-Mass spectrometry (LC-MS) ...... 65
CHAPTER 4 ...... 67
RESULTS AND DISCUSSIONS ...... 67
4.1. Extraction and fractionation for qualitative and quantitative screening ...... 67
ix
4.1.1. Phytochemical analysis of solvent fractions ...... 68
4.1.2. Antioxidant screening of n-hexane, chloroform and butan-1-ol fractions ...... 70
4.1.2.1. Bio-autography antioxidant assay of n-hexane, chloroform and butan-1-ol fractions .... 70
4.1.2.2. Quantitative DPPH free radical scavenging assay ...... 73
4.1.3. Antibacterial activity ...... 75
4.1.3.1. Antibacterial bio-autography assay...... 75
4.1.3.2. Minimum inhibitory concentration (MIC) ...... 77
4.1.3.2.1. Minimum inhibitory concentration (MIC) of general bacteria ...... 77
4.1.3.2.2. Minimum inhibitory concentration (MIC) of fractions against specific bacteria
(Neisseria gonorrhoea) ...... 79
4.1.3.3. Minimum bactericidal concentration (MBC) of solvent fraction against specific
bacteria (Neisseri gonorrhoea) ...... 81
4.1.3.4. Ratio MBC/MIC of solvent fraction ...... 83
4.2. Isolation of active compounds from ethanolic fractions ...... 85
4.2.1. n-Hexane fraction...... 85
4.2.1.1. Compound 1 ...... 85
4.2.1.1.1. Structure elucidation of compound 1 ...... 86
4.2.1.2. Compound 2 ...... 92
4.2.1.2.1. Structure elucidation of compound 2 ...... 92
4.2.2. Chloroform fraction...... 98
4.2.3. Butan-1-ol fraction ...... 99
4.2.3.1. Compound 4 ...... 100
4.2.3.1.1. Structure elucidation of compound 4 ...... 100
x
4.2.3.2. Compound 5 ...... 108
4.2.4. Summary of the isolation process ...... 110
4.3. Antioxidant and antibacterial activity of isolated compounds ...... 111
4.3.1. Screening of antioxidant activities of isolated compounds...... 111
4.3.2. Minimum inhibitory concentration (MIC) of isolated compounds...... 113
4.3.3. Minimum bactericidal concentration (MBC) of isolated compounds ...... 115
4.3.4. MBC/MIC radio of isolated compounds...... 117
CHAPTER 5 ...... 119
CONCLUSION AND RECOMMENDATIONS ...... 119
CHAPTER 6 ...... 1223
REFERENCES ...... 123
ANNEXURE 1 ...... 146
ANNEXURE 2 ...... 150
ANNEXURE 3 ...... 154
ANNEXURE 4 ...... 157
xi
LIST OF TABLES
Page
Table 2.1: Different classes of terpenoids (Wagner and Elmadfa, 2003; Liu, 2011) ...... 28
Table 2.2: Some of Asparagus plants and their usages (Fouche et al., 2008; Van Wyk et al.,
2009; Kubota et al., 2012) ...... 45
Table 4.1: Qualitative phytochemical analysis of n-hexane, chloroform and butan-1-ol
fractions from aerial part of Asparagus suaveolens ...... 69
Table 4.2: MIC results of different fractions (n-hexane, chloroform and butan-1-ol) against
selected general bacteria ...... 78
Table 4.3: 1H NMR (400 MHz) chemical shifts data of compound 1 dissolved in
Chloroform- d3 (CDCl3)...... 87
Table 4.4: 13C NMR (100 MHz) chemical shifts data of compound 1 dissolved in
Chloroform-d3 (CDCl3)...... 87
Table 4.5:. Chemical shifts of Palmitone according to Gonzalez-Trujano et al (2001) and
1 13 this current work both using CDCl3 as solvent for H (300MHz) and C (75
MHz) NMR, and its MS fragmentation patterns ...... 91
Table 4.6: Chemical shift of 1H NMR (300 MHz) data of compound 2 dissolved in
Chloroform- d3 (CDCl3) ...... 94
Table 4.7: Chemical shift of 13C NMR (75 MHz) of compound 2 dissolved in Chloroform-
d3 (CDCl3) ...... 95
Table 4.8: Chemical shifts of octacosanol according to Hamdan and El-shazly (2014) and
that of n-heptacosanol reported in the current work both using CDCl3 as solvent
for 1H (300MHz) and 13C (75 MHz) NMR, and its MS fragmentation patterns…...…97
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Table 4.9: Chemical shift of 1H NMR (400 MHz) data of compound 4 dissolved in
methanol-d4 (CD3OD) ...... 103
Table 4.10: Chemical shifts of 13C NMR (100 MHz) data of compound 4 dissolved in
Methanol-d4 (CD3OD) ...... 105
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LIST OF FIGURES
Page
Figure 2.1: Structures of different classes of flavonoids...... 10
Figure 2.2: The structure of quercetin, a flavonoid showing the antioxidant potency spots...... 11
Figure 2.3: Reaction scheme showing how flavonoids reacting with free radical...... 12
Figure 2.4: Structures illustrating hydrolysable, condensed and complex tannins subclasses. ... 16
Figure 2.5: Structures showing different classes of coumarins. (Lacy and O’Kennedy, 2004) .. 18
Figure 2.6: Chemical structures showing different subclasses of coumarins...... 19
Figure 2.7: Structure-activity relationship of coumarins in respect to antioxidant activity,
taken from Bubols et al (2013)...... 20
Figure 2.8: Structure of the phenylpropane and its C8–C8’ dimer (phenylpropanoid)...... 21
Figure 2.9: Chemical structures illustrating different subgroups of lignans...... 22
Figure 2.10: Chemical structures of different neolignans as lignan subclass...... 23
Figure 2.11: Chemical structures showing subclass of neolignans (oxyneolignan, sesquineo-
lignan and dineolignan)...... 23
Figure 2.12: Structures of Norlignans as subclass of lignans...... 24
Figure 2.13: Reaction scheme of lignans reacting with hydroxyl free radicals taken from
Eklund et al (2005)...... 25
Figure 2.14: Chemical structures representing different classes of quinones...... 26
Figure 2.15: Reaction scheme representing the antioxidant properties of hydroquinone and
quinones...... 27
Figure 2.16: Chemical structures illustrating different classes of terpenoids...... 29
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Figure 2.17: Reaction scheme showing how DPPH radical abstract the allylic hydrogen
from terpenes...... 30
Figure 2.18: Skeletal structures of different groups of saponins using genin part as
reference...... 32
Figure 2.19: Structure of Monodesmosidic and Bidesmosidic saponins...... 32
Figure 2.20: Chemical structure of Cephalotoside A, a tridemosidic saponin...... 33
Figure 2.21: Reaction scheme of saponin with superoxide free radical...... 34
Figure 2.22: Structures showing the two classes of cardiac glycosides...... 35
Figure 2.23: Some chemical structures of alkaloids isolated from medicinal plants...... 38
Figure 2.24: Asparagus suaveolens. Photograph taken near Bolahlakgomo village, Limpopo
Province, South Africa (November, 2013)...... 47
Figure 3.1: Maps showing sample collection side, near Bolahlakgomo village in Lepelle-
Nkumpi Municipality of the Limpopo province, South Africa (Google Maps n.d). ... 51
Figure 3.2: Schematic representation of the liquid-liquid fractionation of ethanolic aerial
part extract of Asparagus suaveolens using different organic solvents...... 54
Figure 3.3: Sketch summarizing the entire methodology involved in the current research
project (SE: Structure Elucidation)...... 66
Figure 4.1: Graph showing percentage yield and mass recovered when the ethanolic extract
from aerial part of Asparagus suaveolens was fractionated using n-hexane,
chloroform and butan-1-ol...... 67
Figure 4.2: TLC plates on which 100 µl of fraction solution (125 mg/ml) were spotted,
developed into BEA, CEF and EMW solvent systems, dried before being sprayed
with DPPH free radical solution (0.2 %) showing yellow spots which confirming
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the presence of antioxidant activity into n-hexane, chloroform and butan-1-ol
fraction extracted from aerial part of Asparagus suaveolens...... 71
Figure 4.3: DPPH free radical scavenging of n-hexane, chloroform and butan-1-ol fractions.
Fraction solution was mixed with a DPPH solution then incubated in the dark for
about 30 minutes to 1 hour before the absorbance can be measured using
spectrophotometer at 517 nm ...... 74
Figure 4.4: TLC plates developed into EMW solvent system then sprayed with activelty
growing bacterial strains suspension, than sprayed with p-iodonitrotetrazolium
chloride (INT) showing antibacterial results. The presence of purple-radish colour
on the TLC explained that the microorganisms are able to reduce INT...... 76
Figure 4.5: MIC of eight clinical isolate, WHO (2008) N. gonorrhoea tested against three
solvent fractions and two standards. Microtiter plate containing 100 µl of fraction,
100 µl of Moller Hinton Broth and 40 µl of 24 hours old bacterial suspensions
were mixed then incubated for 18 to 24 hours in presence of 5 to 10 % of CO2. A
volume of 40 µl of INT was added to each well then incubated for 30 minutes to 1
hour in the similar conditions...... 80
Figure 4.6: Minimum bactericidal concentration (MBC) results of eight clinical isolate
(WHO 2008 N. gonorrhoea) tested against solvent fractions (n-hexane,
chloroform and butan-1-ol) then, subcultured into New York City Agar.
Thereafter, incubated for 18 to 24 hours in presence of 5 to 10 % of CO2, the
highest dilution that gave the visible inhibition was considered as the MBC value. ... 82
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Figure 4.7: Results showing MBC/MIC ratio when solvent fractions (n-hexane, chloroform
and butan-1-ol) where subjected to 8 WHO (2008), N. gonorrhoea using the
micro-dilution method...... 83
Figure 4.8: GC-MS results of compound 1 isolated from n-hexane fraction using chloroform
as blank...... 86
Figure 4.9: Proposed structure of compound 1 (stereochemistry of the structure was
ignored)...... 89
Figure 4.10: GC-MS observed fragmentation of compound 1...... 90
Figure 4.11: GC-MS spectra of compound 2, isolated from n-hexane fraction using
chloroform as blank showing three different retention times in A but with one
fragmentation pattern as shown in A, B and C...... 93
Figure 4.12: Proposed structure of compound 2 (stereochemistry of the structure was
ignored)...... 96
Figure 4.13: Possible fragmentation patterns of compound 2 as showed by the MS spectrum
in Figure 4.11...... 98
Figure 4.14: Liquid chromatography spectrum of compound 4 dissolved in methanol with a
retention time of 3.472 minutes...... 101
Figure 4.15: Mass spectrometry spectrum of compound 4, dissolved in methanol with the
base peak at m/z 303.23...... 102
Figure 4.16: Proposed structure of compounds 4...... 107
Figure 4.17: Proposed possible fragmentation of compound 4 according to mass spectrum. ... 108
Figure 4.18: Photographs showing the isolation process leading to compound 5...... 109
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Figure 4.19: The sketch showing the isolation process undertaken for the aerial part material
of Asparagus suaveolens ethanolic extraction until pure compounds...... 110
Figure 4.20: DPPH free radical scavenging of compounds 1, 4 and 5 isolated from n-hexane
and butan-1-ol fractions...... 112
Figure 4.21: MIC of compounds (compound 1, 4, and 5) isolated from aerial part of
Asparagus suaveolens tested against WHO 2008 N. gonorrhoea strains (F, G, K,
L, M, N, O and P)...... 114
Figure 4.22: MBC results of compounds (compound 1, 4 and 5) isolated from aerial part of
Asparagus sualveolens against WHO 2008 N. gonorrhoea (F, G, K, L, M, N, O
and P)...... 115
Figure 4.23: MBC/MIC ratio showing the bacteriostatic and bactericidal character of
isolated compounds against the WHO 2008 N. gonorrhoea (F, G, K, L, M, N, O
and P)...... 117
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LIST OF ABBREVIATIONS AND ACRONYMS
. - O2 : Superoxide radical
Abs : Absorbance
ABTS : 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)
AMST : Antimicrobial Susceptible Testing amu : atomic mass unit
ATCC : American Type Culture Collection
ATPase : Adenosinetriphosphatase
BEA : Benzene/ Ethyl acetate/ Ammonia
BHA : Butylated Hydroxy Anisole
BHT : Butylated Hydroxy Toluene
CD3OD : Deuterated methanol
CDCl3 : Deuterated chloroform
CEF : Chloroform/ Ethyl acetate/ Formic acid
CFU : Colony Formation Unit
CGs : Cardiac Glycosides
CL : Chemiluminescence
COSY : Correlated SpectroscopY
CTs : Condensed Tannins
CUPRAC : Cupric Reducing Antioxidant Capacity
CxTs : Complex Tannins d : doublet
xix dd : doublet of doublet
DDMP : 2,3-Dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one
DMADP : Dimethylallyl Diphosphate
DMSO : Dimethyl Sulfoxide
DNA : Deoxyribonucleic Acid
DPPH : 2, 2-diphenyl-1-picrylhydrazyl
EMW : Ethyl acetate/ Methanol/ Water
ESI : Electrospray Ionization
F-C : Folin-Ciocalteu
FRAP : Ferric Reducing Antioxidant Power
GC-MS : Gas Chromatography-Mass Spectroscopy
HClO : Hydrochlorous acid
HHDP : Hexahydroxydiphenic
HIV/AIDS : Human Immunodeficiency Virus/Acquired Immuno Deficiency Syndrome
HO. : Hydroxyl radical
HSQC : Heteronuclear Single Quantum Coherence
HTs : Hydrolysable Tannins
ICF : Information Consensus Factor
INT : p-Iodonitrotetrazolium chloride
IPP : Isopentenyl Pyrophosphate
IUPAC : International Union of Pure and Applied Chemistry
LC-MS : Liquid Chromatography-Mass Spectroscopy
LDL : Low Density Lipoprotein
xx m : multiplet
MBC : Minimum Bactericidal Concentration
MHB : Mueller Hinton Broth
MHz : Mega Hertz
MIAs : Monoterpenes Indole Alkaloids
MIC : Minimum Inhibitory Concentration
MREC : Medunsa Research Committee
NHLS : National Health Laboratory Service
NICD : National Institute of Communicable Diseases
NMR : Nuclear Magnetic Resonance
NO. : Nitrogen monoxide n.d : not dated
ORAC : Oxygen Radical Absorbance Capacity
PCL : Photochemoluminescence
PGE2 : Prostaglandin E2
PMs : Plants Metabolites
Ppm : Part per million
PTLC : Preparative Thin Layer Chromatogram
Rf : Retention factors
RNA : Ribonucleic Acid
ROO. : Peroxyl free radical
ROS : Reactive Oxygen Species rpm : rotation per minute
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SANBI : South African National Biodiversity Institute
SARs : Structure Activity Relationships
SD/STD : Standard
SMU : Sefako Makgatho Health Sciences University
TEAC : Trolox Equivalent Antioxidant Capacity
TLC : Thin Layer Chromatography
TM : Traditional Medicine
TOCSY : Total Correlated SpectroscopY
TOSC : Total Oxidant Scavenging Capacity
TPC : Total Phenolic Content
TRAP : Total Radical-Trapping Antioxidant Parameter
TUT : Tshwane University of Technology
UV : Ultra-Violet
WHO : World Health Organization
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RESEARCH OUTPUT
M.T Olivier, F.M Muganza, L.J Shai, S.S Gololo, L.D Nemutavhanani (2017). Phytochemical screening, antioxidant and antibacterial activities of ethanol extract of Asparagus suaveolens aerial parts. South African Journal of Botany 108: 41 – 46
Mutendela Tabize Olivier; Freddy Munyololo Muganza; Sechene Stanley Gololo and Leshweni
Jeremia Shai (2016). In vitro Antigonorrhea activity of the aerial part of Asparagus suaveolens n-hexane fraction and palmitone as a bioactive compound. Natural products communications
11(9): 1319 –1321
CONFERENCES
M.T. Olivier; L.J. Shai; F.M. Muganza and S.S. Gololo, Isolation and characterisation of antioxidant compounds from Asparagus suaveolens South African chemical institute (SACI)
National symposium, November 2015, Durban, South Africa, Poster
M.T. Olivier; S.M. Ndlovu; L.J. Shai; and S.S. Gololo (2013). Isolation and characterisation of antioxidant and antibacterial compounds from the aerial part of Asparagus suaveolens. Research week at the University of Limpopo (Medunsa campus) from the 9th to the 11th September 2014.
Oral presentation.
M.T. Olivier; S.M. Ndlovu; L.J. Shai; and S.S. Gololo (2013). Isolation and characterisation of antioxidant and antibacterial compounds from the aerial part of Asparagus suaveolens. South
African chemical institute (SACI) (young chemists’ symposium), September 2013, University of Johannesburg, South Africa, Oral presentation
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ABSTRACT
Worldwide, people have relied on plants for maintenance of health and treatment of diseases.
Asparagus suaveolens, a plant species that belongs to the family Asparagaceae, has been used since ancient times as medicinal plant for treating epilepsy, complications emanating from infectious diseases (e.g., gonorrhoea) and many others. Despite the wide traditional usage of
Asparagus suaveolens for medicinal purposes, no studies have been reported on the isolation of bioactive compounds from its solvent extracts in South Africa and/or anywhere else.
The aerial parts of Asparagus suaveolens were collected from Bolahlakgomo village in
Zebediela sub-region of Limpopo province, South Africa. The ethanol extract of the aerial parts was fractionated into three fractions (n-hexane, chloroform and butan-1-ol fractions) using liquid-liquid fractionation procedure. Fractions were then subjected to screening for phytochemical composition using standard test methods for presence of different classes of metabolites, as well as for antioxidant activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay and antibacterial activity against four bacterial strains (Staphylococcus aureus ATCC
29213, Enterococcus faecaelis ATCC 29212, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853) using bioautographic procedure. Micro-dilution method was also used to determine the minimum inhibitory concentration (MIC) of fractions against four bacterial strains and clinical isolates of Neisseria gonorrhoea designated F, G, K, L, M, N, O and P.
Bioassay-guided fractionation was thereafter conducted to isolate compounds with antioxidant and antibacterial activities.
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The isolated compounds were finally subjected to quantitative antioxidant (DPPH scavenging) and antibacterial activity test (N. gonorrhoea), as well as structural elucidation.
A comprehensive fingerprint of phytochemical content of the fractions of the ethanol extract of
Asparagus suaveolens was established. In the n-hexane fraction, the following metabolites were present: alkaloids, terpenoids and steroids. Glycosides and proteins were in low amounts.
Whereas coumarins, reducing sugar, carbohydrates, anthraquinones, flavonoids, saponins and tannins were absent. In the chloroform fraction, traces of the following metabolites were present: alkaloids, flavonoids, glycosides, anthraquinones, terpenoids and reducing sugar; while all other tested metabolites were absent. Saponins and proteins, in butan-1-ol fraction fractions were present. Flavonoids, glycosides and reducing sugars were found in reasonable amount according to the phytochemical test results. Only coumarins were in lower amounts while the other tested metabolites were absent. All fractions were found to have antioxidant activity, as shown by reduction of DPPH. The chloroform fraction showed the highest DPPH inhibition strength
(71.4%) followed by butan-1-ol fraction (69.4%) and lastly n-hexane fraction (35.4%) at a concentration of 2.5 mg/ml. The IC50 of n-hexane fraction was higher than 2.5 mg/ml used as the highest concentration of all fractions. The chloroform and butan-1-ol fractions had IC50 values of
0.37 and 0.42 mg/ml, respectively. The bioautographic antibacterial activity of fractions against four bacterial strains (S. aureus, E. coli, E. faecalis and P. aeruginosa) showed negative results on the Thin layer chromatography (TLC) plates and had recorded high minimum inhibitory concentration (MIC) values when their quantitative antibacterial test were performed. However, good MIC and the minimum bactericidal concentration (MBC) values were recorded against clinical isolates of Neisseria gonorrhoea identified as F, G, K, L, M, N, O, and P. n-Hexane
xxv fraction showed higher MIC values followed by chloroform and finally butan-1-ol. This pattern was also observed for the MBC values of fractions.
The bioassay-guided fractionation of different fractions resulted in the isolation of five compounds. Three of these compounds were fully characterized using NMR, GC-MS and LC-
MS techniques. Compound 1 was identified as 16-hentriacontanone (palmitone); compound 2 was identified as n-heptacosanol; compound 4 seems to be a novel compound and the proposed name is 6-[3,4-Dihy-droxy-6-methyl-5-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2- yloxy)-tetrahydro-pyran-2-ylmethyl]-3-(2’,4’-dihydroxy phenyl)-5,7-dihydroxy-chromen-4-one.
The known biological activities of compound 1 and 2 justify the usage of Asparagus suaveolens extracts in traditional medicine. Compound 1 showed weak DPPH inhibition (26.2%) with a
IC50 value of >2.5 at 2.5 mg/ml of fraction compared to that of the n-hexane fraction (35.4%) from which it was isolated. For Compounds 4 and 5, their DPPH inhibition at 2.5 mg/ml of fraction were not very different to that of the butan-1-ol fraction from which they were isolated with IC50 values of 0.45 and 0.41 mg/ml respectively. Compounds 1, 4 and 5 showed good antibacterial activity against clinical isolates of Neisseria gonorrhoea with lower MIC and MBC values than fractions from which they were isolated.
Furthermore, the findings of the current study justify the usage of Asparagus suaveolens by traditional healers as anti-epileptic in Northern Lesotho and anti-Neisseria gonorrhoea in the
Limpopo province in South Africa.
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CHAPTER 1
INTRODUCTION, BACKGROUND, RESEARCH PROBLEM AND AIM OF STUDY
1.1. Introduction and Background
The present dissertation reports on the isolation and characterisation of the antioxidant and antibacterial compounds from the aerial parts of Asparagus suaveolens. The aerial parts of
Asparagus suaveolens are widely used by traditional healers of Bolahlakgomo village in
Zebediela sub-region, Limpopo province (South Africa) to treat infectious diseases, in particular sexually transmitted infections. Van Wyk et al. (2009) classified Asparagus suaveolens among
South Africa’s indigenous medicinal plants and as such, the informant consensus factor (ICF) of
Asparagus suaveolens was not determined before conducting this research project.
The continuous usage of traditional medicine (TM) in the world cannot be attributed to only cultural and poverty reasons, but to the inefficiency of many of the existing commercialised drugs as well (Tenover, 2006). Many of the current drugs are unable to eradicate, reduce, and/or inhibit some illnesses that affect humankind such as HIV/aids, cancer, diabetes, tuberculosis and many other diseases. This can be explained by two factors: firstly, the presence of new illnesses caused by unknown bacteria and viruses; and secondly, these microorganisms (bacteria and virus) are developing resistance in respect to existing drugs (Van Wyk, et al., 2009). In order to address this situation adequately, it is important to seek alternative sources of drugs from nature.
1
The Southern African region is exceptionally rich in plant diversity with approximately 30,000 species of flowering plants (Tenover, 2006). Only 10% of these plant species are used in traditional medicine for about 80% of the population in the region. The region also contains a huge number of native Asparagus species (Norup et al., 2015), and most of them are used as traditional medicine, diet and ornament since ancient time (Van Wyk et al., 2009).
The present research aimed on the isolation and characterisation of the chemical constituents with inherent biological activities from the aerial part of Asparagus suaveolens native to South
Africa. Besides its usage as antiepileptic, Asparagus suaveolens is widely used in traditional medicine for treatment of microbial infections as well as in improving men’s libido in Limpopo province (South Africa) (Gololo, undocumented source). Furthermore, Asparagus suaveolens is used to treat livestock diseases including cattle sore, urine infection, red water umbilical cord infection, general ailments and as after-birth treatment (Dold and Cocks 2001).
1.2. Study problem
Previous studies have indicated that plant species belonging to Asparagus genus have biological activities of medicinal importance. In this regard, Jäger et al (2005) reported the screening of the antiepileptic property of Asparagus suaveolens. Later on, several authors (Mashele and
Kolesnikova, 2010; Mashele and Fuku, 2011; Fuku et al., 2013) have reported the anticancer, antioxidant and antibacterial activities of Asparagus laricinus and Asparagus africanus.
However, regardless of the extensive traditional usage and identification of Asparagus species
2 for medicinal purposes, less is reported on the isolation of bioactive compounds from the extracts of Asparagus species in general and in particular, Asparagus suaveolens found in South Africa.
1.3. Research question
Several medicinal plants are used by villagers of Bolahlakgomo village to treat bacterial infections, particularly sexually transmitted diseases. The scientific validation of the efficacy of these medicinal plants is lacking. It is therefore the aim of this study to answer the following questions:
1. Do the extracts of Asparagus suaveolens possess any bioactive compound(s) with
antioxidant and/or antibacterial activities?
2. Are the observed activities, if any, sufficient to afford scientific endorsement for the
continual usage of these plant species in traditional medicine?
1.4. Purpose of the study
1.4.1. Aim of the study
The aim of this study was twofold:
1. To evaluate the antibacterial and antioxidant activities of the aerial part of extracts of
Asparagus suaveolens.
2. and to isolate and characterise compounds with antioxidant and/or antibacterial activities
from the aerial part extracts of Asparagus suaveolens.
3
1.4.2. Objectives of the study
The aims of the study were achieved through the following objectives:
Screening of the aerial part extracts of Asparagus suaveolens for biological activities
(antioxidant and antibacterial).
Isolation of the bioactive compounds from the aerial part extracts of Asparagus
suaveolens.
Structural identification of the isolated compounds and comparison of antioxidant and
antibacterial activities of isolated compounds to those of the fractions from the ethanolic
aerial parts extracts of Asparagus suaveolens.
Comparison of the antioxidant and antibacterial activities of the isolated compounds to
those of drugs in the market.
1.5. Scope of the study
This study comprises of six chapters and their content is as follows:
Chapter one: provides the introduction and background, study problem, research question
as well as the purpose and the scope of the project.
Chapter two: gives a comprehensive detail on the literature review.
Chapter three: outlines the research methodology.
Chapter four: presents the results and their discussions.
Chapter five; presents the conclusion and recommendations.
4
Chapter six; includes bibliography of the work cited, supplementary pages including
spectra of isolated compounds and the approval certificate of the research project by the
Medunsa Research Ethic Committee (MREC).
5
CHAPTER 2
LITTERATURE REVIEW
2.1. Introduction
The literature review is based on publications written or translated into English, spanning the period from 1966 to 2015. The literature review is arranged into the following themes: An overview on traditional medicine, medicinal plants, plants metabolites, antioxidant and antibacterial activities, bio-guided isolation, overview on Asparagus species and the presentation of Asparagus suaveolens as medicinal plant under study.
2.2. Overview on traditional medicine
The relationship between humans and animals with plants has its origin from the beginning of life. Plants supply oxygen, medication, food, shelter, and transport for humankind. In the world, human beings belong to different cultures; fortunately, all these cultures have similar needs in nature (Gurib-Fakim, 2006; Mamedov, 2012). Even animals like primates (chimpanzee, gorillas and monkeys) use nature to satisfy their health needs. These animals consume plants with different medicinal properties such as anti-inflammatory, immunostimulant, anti-diarrheal, digestive aids, analgesic and antimicrobial activities, as well as fertility regulators when feeling discomfort in their systems (Halberstein, 2005; Godfraind, 2010).
6
Therefore, every given group or population sharing the same culture has traditional knowledge related to the health of its people and animals (Loundou, 2008). Traditional medicine is defined as “health practices, approaches, knowledge and beliefs incorporating plants, animals and/or mineral-based medicines, spiritual therapies, manual techniques and exercises, applied singularly, or in combination in the maintenance of health, as well as to prevent, diagnose or to treat physical and mental illnesses with objectives of maintaining the well-being of human and animals. This knowledge is transmitted either verbally or in writing from generation to generation” (Karou et al., 2007; Giovannini et al., 2011).
Traditional medicine was practised and reported by great civilizations of ancient times such as the North Africans, Chinese, Indians, Greeks, Mesopotamians and many others (Philipson,
2001). Earliest writings from these civilizations with reference to healing herbs and foods indicate a pre-historic origin of medicinal plants (Loundou, 2008). Archaeological evidence shows that as far as Palaeolithic age about 60,000 years back from now, as found from fossil studies, people were using plants for their primary health care (Rout et al., 2009).
2.3. Medicinal plants
Medicinal plants are defined in different ways by different authors. Fellows (1991) defined medicinal plants as “all plants that indicate that they contain a substance or substances which modulate beneficially the physiology of sick mammals, and that it has been used by man for that purpose”. In the same year, Farnsworth and Soejarto (1991) also identified medicinal plants as
“all higher plants that have been alleged to have medicinal properties that have effects related to
7 health or which have been proven to be useful as drugs by western standards, or which contain constituents that are used as drugs”. Adhikari et al (2012) classified medicinal plants as “Any plant with one or more of its organs containing properties that can be used for therapeutic purposes or which can be used as precursors for the synthesis of various drugs”.
Some of these plants are still used like previously prescribed in ancient times as oils (Gurib-
Fakim, 2006). For example oils of Cedrus species, Cupressusse mpervirens, Papaver somniferum and Commiphora species. Others have been subjects of research that led to isolation of biologically active compounds and discovery of drugs such as morphine, quinine, menthol, digoxin, aspirin, cocaine, codeine, digitoxin, (+)-catechin 5-gallate, isoimperatorin, and many others (Rates, 2001; Balunas and Kinghorn, 2005; Bero et al., 2009). Plant compounds are subdivided into two major categories based on their molecular weight (Liu, 2011) and their role in plants (Agostini-Costa et al., 2012). These two categories are referred to as primary and secondary metabolites (Rao and Savithramma, 2012).
2.4. Plant metabolites (PMs)
Primary metabolites play a role of providing nutrients to plants and therefore responsible for their growth and developments (Liu, 2011). They are essential for the well-being and the existence of plants and they include polysaccharides, proteins, lipids, and nucleic acids
(Agostini-Costa et al., 2012). Secondary metabolites play a defensive role against herbivores and pests, rather than being involved in plant growth. Plant metabolites are discussed below in detail.
8
2.4.1. Secondary metabolites
Secondary metabolites consist of organic compounds formed from secondary metabolism in plants and their molecular weight are often less than 2000 atomic mass unit (amu) (Liu, 2011).
These metabolites play a different role including being chemical messengers, plant defenders against herbivores, microorganisms and occur differently in plants according to species
(Agostini-Costa et al., 2012). They are called non-essential to the existence of plants but show various range of biological activities that are potential leads to drug discovery (Rao and
Savithramma, 2012). Their activities including antioxidant and antibacterial are mostly from the presence of a hydroxyl group –(OH), amino group –(NH2), thiol group –(SH), aromatic ring and the unsaturated chains in their structures (Edreva, 2008). Secondary metabolites are classified according to their structures and their behaviour into alkaloids, flavonoids, coumarins, lignans, quinones, terpenoids, and many others (Agostini-Costa et al., 2012). These secondary metabolites are discussed below.
2.4.2. Classes of different secondary metabolites in plants
2.4.2.1. Flavonoids
Flavonoids are polyphenolic secondary metabolites present in plants parts such as fruits, vegetables, grains, barks, roots stems and flowers; as well as plant derived products such as tea and wine. Being the largest class of polyphenolic compounds, more than 4000 flavonoids have been identified and many others are still being discovered (Nijveldt et al., 2001). All flavonoids
9 have the skeletal structures that contain two aromatic rings (A, B) bonded to each other by a three carbon heterocyclic pyran ring (C); with the exception of chalcones which have only two rings (A and B) and a pseudo ring (Cushnie and Lamb, 2005).
Flavonoids are classified according to different substituents on the pyran ring, absence or presence of double bonds on ring C, number of hydroxyls on A and B rings and carbonyl group attached to C4 of ring C. The classes of simple flavonoids include flavones, flavanones, flavonols, isoflavones, isoflavanones, chalcones, anthocyanins, flavanols and flavans (Cushnie and Lamb, 2005; Kumar and Pandey, 2013), whose structures are shown in Figure 2.1 below.
3' 3' 3' 2' 4' 2' 4' 2' 4' B 8 1 B 8 1 B 8 1 O 2 5' O 5' O 2 5' 7 7 7 A C 6' A C 2 6' A C 6' 3 6 6 3 6 3 5 4 5 4 5 4 OH (1) Flavone (2) Flavanone (3) Flavonol O O O
3' 3' 2' 4' 8 1 2' 4' O 2 8 B 7 2 5' 8 1 B 7 O 2 5' 6 3 2' A 6' 7 3' 6 A C 6' 5 4 3 5 4 OH 6 3 O 4' 5 4 6' O (7) Chalcone (8) Anthocyanin (9) Isoflavanone 5'
Figure 2.1: Structures of different classes of flavonoids. Flavonoids have attracted interest from phytochemical researchers because of their biological activities that are beneficial to human health. They are well known for their antioxidant, antibacterial, antiviral, anti-allergic, antiplatelet and anti-inflammatory activities. They have also been reported to possess other activities such as antitumor, antimutagenic, prevention of ulcer,
10 coronary heart diseases, strokes, and many others (Benaventente-Garcia et al., 1997; Cowan,
1999; Prasain et al., 2004; Tapas et al., 2008; Agrawal, 2011; Sandhar et al., 2011; Liu, 2011;
Okuda and Ito, 2011). Flavonoids are also known for reducing blood-lipid and glucose in human being (Ghasemzadeh and Ghasemzadeh, 2011). As early mentioned, the focus of this study will only be on antioxidant and antibacterial properties of the plant investigated.
The antioxidant activity of flavonoids (phenolic derivatives) is influenced by the number and the positions of the hydroxyl groups on the rings of the backbone of their structures (see Figure 2.2 for an example of a flavonoid compound). The hydroxyl groups of flavonoids are used to deactivate excessive free radical in the body system (Kumar and Pandey, 2013).
Contribute to OH antioxidant potency 3' 4' OH B OH 7 O 2 A C
5 4 3 OH Increases the antioxidant Increase antioxidant OH O activity activity Facilitate electron delocalization (10)
Figure 2.2: The structure of quercetin, a flavonoid showing the antioxidant potency spots.
According to Pourmorad et al (2006); Balasundram et al (2006); and Bubols et al (2013), the above structure shows the substation patterns that have reference on the antioxidant activity of flavonoids which are:
11 i. Hydroxyl groups on C3’ and C4’ in ring (B): these hydroxyl groups have potential of reacting
with free radical and stabilise them. They are also considered targets for chelation of
transitional metals. ii. The hydroxyl groups at C3, C5 and C7 in rings (A) and (C) have a potential of reacting with
radicals, hence increase the antioxidant activity. iii. The double bond between C2 and C3 in ring (C) seems to facilitate the delocalisation of
electrons by connecting ring (B) to 4-oxo group of C4 in ring (A).
Previous studies showed that flavonoids are more effective in stabilizing peroxyl free radical
(ROO.) than the common synthetic antioxidants such as Butylated Hydroxy Toluene (BHT) and
Butylated Hydroxy Anisole (BHA) (Procházková et al., 2011; Pounrmorad et al., 2006). One molecule of flavonoid is able to stabilize two peroxyl free radicals as demonstrated in the reaction scheme below (Figure. 2.3).
OH OH O OH O O
CH3O O CH3O O CH3O O
ROO ROO OH O OH O OH O (11) (12) (13)
Figure 2.3: Reaction scheme showing how flavonoids reacting with free radical.
It was proved that the role of flavonoids in plant is to protects them against potential infection from microorganisms and aggression from insects/animals (Cushnie and Lamb, 2005; Bagla,
2014). Hence, flavonoids contribute to the plants’ antibacterial activity. This activity might be
12 due to their ability to form complexes with extracellular and soluble proteins and to form other complexes with cellular walls through nonspecific forces such as hydrogen bonding as well as by covalent bond formation (Kumar and Pandey, 2013). Lipophilic properties of flavonoids might disturb the membrane of microorganism, resulting in its growth inhibition or death (Cowan,
1999). The (B) ring of flavonoids, particularly flavones might form hydrogen bonds with nucleic acids, which can lead to inhibition of DNA and RNA syntheses in bacteria (Kumar and Pandey,
2013).
It was also reported that the presence of hydroxyl groups on the (B) ring decreases the activity of flavonoids against microorganisms compared to their homologues without or with less number of hydroxyl groups on (B) ring (Singh, et al., 2014). This has been the subject of discussion because other authors reported that the more the number of hydroxyl groups on flavonoids, the greater the activity of flavonoids against microorganism (Cowan, 1999). The structure activity relationships
(SARs) of flavonoids were studied (Mughal et al., 2006) and it was established that the replacement of hydroxyl groups –(OH) with methoxy group –(OCH3) decreased the antibacterial activity of flavones in particular. However, the replacement of one hydroxyl group by methoxy and another by nitro –(NO2) group restored the antibacterial activity of flavonoids in general. In addition, the replacement of oxygen with sulphur or nitrogen at C4 increases the activity of flavonoids. When 4-thioflavonoids and 4-iminoflavonoids contain fluorine attached at C4’ in (B) ring, the activity still exists. This activity decreases when the halogen attached on the (A) ring is less electronegative.
13
2.4.2.2. Tannins
The word “tannin” is derived from “tinning” which describes the process of transforming raw hides or skins into durable, non-putrescible leathers by using plant extracts (Kuete, 2013). This process was known since ancient time, but at the time, people were ignorant about the mechanism of that transformation. Tannins belong to the group of polyphenolic compounds as flavonoids, and might occur in any part of vascular plant (seed, fruits, leaves, wood, barks and roots), with molecular weight of 300 to 20000 amu (Khanbabaee and van Ree, 2001).
Based on different units that build up tannins structures as well as the different fragments produced after their treatment with water, tannases or with other enzymes, tannins are classified into three major categories namely hydrolysable, condensed and complex tannins (Kuete, 2013):
i. Hydrolysable tannins (HTs) consist of central carbohydrate moiety esterified with phenolic
acids mainly gallic acid and hexahydroxydiphenic (HHDP) acid. The hydrolysable tannins are
themselves subdivided in to gallotannins and ellagitannins (Chung et al., 1998).
Gallotannins consist of tannins in which the central carbohydrate is esterified by gallic
acid. Their hydrolysis by acids, bases or certain enzymes yield glucose and gallic acid.
Ellagitannins consist of esterified gallic acids bonded to the central carbohydrate, and the
gallic acids C-C coupled to each other. Their hydrolysis yields ellagic acid collected as
hexahydroxydiphenic acid.
14 ii. Condensed tannins (CTs) composed of flavan-3-ol nuclei (catechins). They are currently
referred to as proanthocyanidins or broadly as polyflavonoids (Bruyne et al., 1999).
iii. Complex tannins (CxTs) are referred to as tannins in which ellagic acid and poly-flavonoids
moieties are bonded to the same carbohydrate unit (Khanbabaee and van Ree, 2001). The
carbohydrate moiety might be cyclic or acyclic.
As flavonoids, tannins exhibit a range of pharmacological activities including antioxidant antibacterial and antiyeast activities (Kuete, 2013). In addition, tannins are reported to possess hepatotoxic, antimutagenic, anticarcinogenic, immunomodulator and antiviral activities (Chung et al., 1998). According to De Bruyne et al (1999), condensed tannins have shown anti- inflammatory, vascular and cardiac effects, anti-ulcer activity, pulmonary inflammation and anti- diarrhoeal activity.
Previous research findings demonstrated that one molecule of condensed tannin is able to stabilize eight peroxyl free radicals, whereas one molecule of ascorbic acid can only stabilizes one and tocopherol two (Bruyne, 1999). The higher the degree of polymerization the more radicals scavenged per molecule. Hence, antioxidant activity of tannins is directly proportional to the molecular weight.
15
OH O RO OH HO OR O O O HCOO OH RO RO HO HO COOH OH R = Gallic acid HO HO HO OH OH OH (14) Gallic acid (15) Hexahydroxydiphenic acid (16) Gallotannin (hydrolysable tannin)
OH OH O HO OH OR O HO O HO O OH O OR HO OH O OH RO HO HO O HO OH OH HO OH (17) Ellagitannins (hydrolysable tannin) O OH (18) Condensed tannin
OH
OH OH OH OH OR HO HO OH HO OH O O O OH HO OH RO O OH O O O HO O OR HO O O O OH RO OH HO HO OH OH HO OH (19) Complex tannins (20)
Figure 2.4: Structures illustrating hydrolysable, condensed and complex tannins subclasses.
16
Tannins have been reported to be bactericidal or bacteriostatic (Chung et al., 1998). Generally, the antibacterial activity of condensed tannins is proportional to the number of flavanol units
(Bruyne, 1999). If the number of flavanol increases, the activity increases. For gallotannins, the antimicrobial activity is associated with the presence of ester linkage between gallic acid and carbohydrate. The complexation of tannins with microbial enzymes such as cellulase, pectinase, xylanase, peroxidase, lactase and glycosyl transferase increase their antibacterial activity
(Bruyne, 1999). Tannins can form complexes with proteins through hydrogen bonding, hydrophobic effects as well as by covalent bond formation (Cowan, 1999). However, with these properties, tannins are able to deactivate microbial adhesions enzymes and can envelope transport proteins. The formation of chelate between tannins and metal ions such as ferric (Fe3+) or cupric (Cu2+) ion, reduce the availability of metal ion for microorganisms, hence it increases their antibacterial activity (Chung et al., 1998).
2.4.2.3. Coumarins
The name coumarin is derived from “coumarouna”, vernacular name of Tonka bean (dripteryx odorata), a South American plant from which the first simple coumarin was isolated (Lacy and
O’Kennedy, 2004; Jain and Joshi, 2012; Kuete, 2013). Coumarins are compounds consisting of benzopyrone which is a combination of benzene and α-pyrone rings. Huge amount of coumarins are found in green vegetables, but some of them are also present in fungi and bacteria
(Montagner, 2008). They are well known for their pleasant vanilla-like odour (Hoult and Paya,
1996).
17
Coumarins include a group of secondary metabolites present in almost all higher plants with the structure derived from benzopyran subdivided into: i. α-Benzopyrone (20) consisting of coumarins. ii. γ-Benzopyrone (21) in which flavonoids are major components.
O
O O O (21) Benzopyrone (22) Benzopyrone
Figure 2.5: Structures showing different classes of coumarins (Lacy and O’Kennedy, 2004).
More than 1300 coumarins have been isolated from green plants, fungi and bacteria (Mirunalini and Krishnaveni, 2011). Although, their role in plants is still unclear, coumarins are suspected of being used by plants as defenders against external predators as well as growth inhibitors (anti- auxins), control of respiration and photosynthesis (Grazul and Budzisz, 2009; Kai et al., 2008;
Liu, 2011). Coumarins are classified into five subgroups (Hoult and Paya, 1996; Lacy and
O’Kennedy, 2004; Borges, 2005; Mantagner et al., 2008; Grazul and Budzisz, 2009; Kai et al.,
2008; Liu, 2011; Jain and Joshi, 2012 and Venugopala et al., 2013):
1. Simple coumarins (22) consist of two rings. A pyrone ring is attached to benzenic ring from
position 5.
2. Isocoumarins (23) consist of two rings (pyrone ring and phenyl ring). The pyrone is attached
to phenyl ring from position 3.
18
3. Pyranocoumarins (24) belong to the class of coumarins containing an additional pyran ring
system attached to the phenyl ring.
4. Furanocoumarins (25) consist of additional furan ring system attached to the phenyl ring
system of coumarins
5. Dimeric, trimeric coumarins (polycoumarins) (26, 27): refer to those containing two, three or
many coumarins units.
O O O O O O O O O O
(23) Coumarins (24) Isocoumarins (25) Pyranocoumarins (26) Furanocoumarins
RO O O O
RO O O O O O O O O O O (28) Trimeric coumarin (27) Dimeric coumarin
Figure 2.6: Chemical structures showing different subclasses of coumarins.
Coumarins have demonstrated biological activities that include antiviral (anti-HIV), antitumor, anti-hypertension, anti-inflammatory, anti-osteoporosis, pain reliever, anti-allergic, hepatoprotective, antithrombotic, anticarcinogenic and prevention of asthma (Kai et al, 2008;
Grazul and Budzisz, 2009; Venugopala et al., 2013). Many coumarins derivatives are used as anticoagulant (Liu, 2011). Other coumarins and their derivatives have antioxidant activity
(Kontogiorgis and Hadjipavlou-Litina, 2004). Coumarins have shown almost similar biological activities to flavonoids (Montagner, 2008; Grazul and Budzisz, 2009).
19
Many, if not all coumarins, especially those with a hydroxyl group, are able to scavenge a
. - number of reactive oxygen species (ROS) such as superoxide radical ( O2 ), hydroxyl radical
(HO.), hypochlorous acid (HClO). They also participate in the process of injury repair caused by these reactive species by stopping lipid peroxidation process (Kontogiorgis and Hadjipavlou-
Litina, 2004; Mirunalini and Krishnaveni, 2011). Furthermore, the antioxidant activity of coumarins can be explained in the same way as that of flavonoids, due the similarity in their structures (Bubols et al., 2013). The structure below (Figure 2.7) shows the possible positions of hydroxyl groups, which are likely to increase the antioxidant activity of coumarins.
7-OH and 8-OH increase the activity OH 8 1 6-OH and 7-OH HO 7 O 2 O increases the activity 3 If phenyl group is attached 6 to C3 as a substituent, the 6-CH O substituents HO 5 4 3 activity increases decreases the activity OH 5-OH increase the activity (29)
Figure 2.7: Structure-activity relationship of coumarins in respect to antioxidant activity, taken from Bubols et al (2013).
The substitution of one of the following groups –OCH3 or –OR in coumarins structure is likely to decrease their antioxidant activity. The presence of benzoic rings at ortho position to each other contributes strongly to antioxidant scavenging properties of coumarins. In general, the antioxidant activity of coumarins and their derivatives is based on their structures (Mirunalini and Krishnaveni, 2011).
20
According to some studies, several coumarins have antimicrobial activity against many fungi,
Gram–positive bacteria and viruses. However, coumarins are well known for their anti- inflammatory, antithrombotic and vasodilatory activities (Cowan, 1999). Mirunalini and
Krishnaveni (2011) reported that coumarins containing a free hydroxyl group at C6 possess antifungal activity whereas those with free hydroxyl group at C7, showed antibacterial activity.
2.4.2.4. Lignans
This naturally occurring category of secondary metabolites belongs also to phenolic compounds.
Previously, they were known as secondary metabolites made up of phenylpropanoid dimer in which the monomer (phenylpropane) (C6–C3) units were linked by a β-β’ bond (C8–C8’) (Moss,
2000; Suzuki and Umezawa, 2007; Sainvitu et al., 2012; Cunha et al., 2012; Kuete, 2013).
Lignans are largely found in higher plants (vascular plants) especially in African plants from
Piper (Peperaceae) and Zanthoxylum (Rutaceae) genera but also in plant species belonging to the families Asteraceae, Acanthaceae, Oleaceae, Apocynaceae, Lauraceae, and Myristicaceae
(Kuete, 2013).
(30) Phenylpropane (C6-C3 unit) (31) C8-C8' Lignan
Figure 2.8: Structure of the phenylpropane and its C8–C8’ dimer (phenylpropanoid).
21
With the discovery of “neoligans”, the definition of lignans was extended to all naturally occurring compounds with low molecular weight produced from the oxidative coupling of hydroxyphenylpropene units other than C8–C8’ (Cunha et al., 2012; Kuete, 2013).
After long discussion concerning their classification, lignans were classified into many subgroups such as furofurans, furans, dibenzylbutanes, dibenzylbutyrolactol, dibenzylbutyrolactones, aryltetralins, arylnaphthalene, dibenzocyclooctadienes (Suzuki and
Umezawa, 2007; Marcotullio et al., 2014).
O O O O
(35) (34) O (33) OH
(32) Furofuran Furans
OH O O OH OH O
(36D) ibenzylbutan-9,9'-diol (37D) ibenzylbutane (38) Dibenzylbutyrolactol (39) Dibenzylbutyrolactone
OH O O OH
O O O (40) (41)
(43) (44) O Aryltetralins (42) Aryl naphthalene Dibenzocyclooctadienes
Figure 2.9: Chemical structures illustrating different subgroups of lignans.
22
When the two monomers (C6–C3) units of phenylpropane are attached to each other through other carbon–carbon bonds than C8–C8’, the compound belong to the category of neolignans.
9' 1' 9 1' 9' 1 1' 9 9' 4' 8 3' 3 3' 1 3 6 1 (45) (46) (47)
Figure 2.10: Chemical structures of different neolignans as lignan subclass.
Neolignans are subdivided into different subgroups considering the type of bridge that links the two C6–C3 units and the number of units within the compound. When the two units are linked from ether functional group, the compound belongs to oxyneolignan (Moss, 2000). In case that the compound contains more than two C6–C3 units, it belongs to sesquineolignan and dineolignan.
O O O
(48) Oxyneolignan (49) Sesquineolignan
O
O O O
O (50) Dineolignan
Figure 2.11: Chemical structures showing subclass of neolignans (oxyneolignan, sesquineo- lignan and dineolignan).
23
Norlignans are also considered as subgroup of lignans. The structure of norlignans dimers consist of phenylpropane (C6–C3) and phenylethane (C6–C2) units.
O
1' 8' 1 7
(52) O O (51) (53) (54) 9 O Norlignans
Figure 2.12: Structures of Norlignans as subclass of lignans.
Lignans play a major role in plant growth and defence against insects (Harmatha and Dinan,
2003). Generally, subgroup lignans are optically active compounds, which mean they may exist as enantiomers (Moss, 2000; Saarinen et al., 2002). This might make their isolation very difficult if they coexist. Because of their high structural diversity, lignans have manifestly and extraordinary a range of medicinal properties. Lignans are well known for their anticancer, antiviral, antioxidant, anti-inflammatory, antimicrobial, immunosuppressive, osteoporosis prevention and hepatoprotective activities (Wróbel et al., 2010; Liu, 2011; Cunha et al., 2012).
Lignans with hydroxyl groups on the benzene ring have potential antioxidant properties. This activity depends on the steric hindrance on the structure of the lignans (Eklund et al., 2005). In addition, the phenolic moieties present in lignans stabilise free radicals through resonance. Some of phenolic derivatives are able to bind some transition metal like iron (Fe) and form chelates.
This mechanism prevents formation of hydroxyl radicals and lipid peroxidation. (Kuete, 2013).
24
O O O O
O R RH O O O
(56) (57) (58) (55)
OH R O O O O O
O O
(59) (60)
RO O
Figure 2.13: Reaction scheme of lignans reacting with hydroxyl free radicals, taken from Eklund et al (2005).
The increasing number of substituents such as hydroxyl group, benzoyl, tigloyl, angeloyl, or exocyclic methylene groups in the cyclooctadiene ring and methylendioxyarylor methyl groups of the phenolic rings might influence the antioxidant activity of lignans (Choi et al., 2006).
Lignans with hydroxyl group show more antibacterial activity (Kumar and Singh, 2014).
Furthermore, lignans (lactones with carbonyl group at C9, bearing two methylendioxyaryl groups on the aromatic rings) showed antimicrobial activity (Cunha et al., 2012). This activity can increase with the introduction of polar substituent such as nitro group to the aromatic rings of the lignan.
25
2.4.2.5. Quinones
Quinones are secondary metabolites with a structure containing at least one benzoquinone (two carbonyl functional groups attached to a benzene) unit (Kontogiorgis and Hadjipavlou-Litina,
2004). Quinones are principally isolated from vascular plants, and animal kingdom, but also they occur as pigments in bacteria and fungi (Liu, 2011). Quinones can be derived from oxidation of hydroquinones (Kuete, 2013).
Quinones structures consist of an aromatic cyclic di-one (di-ketone) system. Based on their structure, quinones are classified as benzoquinones, naphthoquinone, phenathraquinones, anthraquinone and polyquinones (Kuete, 2013).
O O O O O
O O O (61) Benzoquinone (62) Naphthoquinone (63) Phenathraquinone (64) Anthraquinone
Figure 2.14: Chemical structures representing different classes of quinones.
Quinones are very important class of secondary metabolites due to the number of biological activities they exhibit including neurological, antiplasmodial, antioxidant, trypanocidal, anti-
HIV, antibacterial, antitumor, and inhibition of Prostaglandin E2 (PGE2) biosynthesis and anti- cardiovascular (Kuete, 2013). Previous research on quinones proved that the above biological activities are related to the redox character of their carbonyl functional group (Eklund et al.,
2005; Choi et al., 2006; Kumar and Singh, 2014).
26
The hydrogen atoms present in hydroquinone are essential in radical polymerisation reactions to terminate the chain reaction by generating quinones. Generally, the movement of two hydrogen atoms from hydroquinones (diphenol) leads to a conversion into quinones (diketone). The two newly formed ketones of phenanthraquinone can thereafter stop lipid peroxidation (Kuete, 2013).
O H O O
R R
(65) (66) (67) H O H O O
L L
O O O OL (68) O (69) OL (70) OL (71) OL
Figure 2.15: Reaction scheme representing the antioxidant properties of hydroquinone and quinones.
Being aromatic and containing two ketone groups, these two characteristics might be the origin of higher antibacterial activity exhibited by quinones. Their greater activity against microorganism can also be justified by their possibility to complex with amino acids from proteins; which often lead to the inactivation of protein (Cowan, 1999). In particular, naphthoquinones are well known for their antibacterial and antifungal activities. These quinones subclass seem to be toxic against all living organisms owing to their supposed mechanism of action (Kuete, 2013).
27
2.4.2.6. Terpenoids
Terpenoids, sometimes called isoterpenoids refer to terpenes, are the largest and structurally diverse class of secondary metabolites in plants. They are built up from isoprene units connected with head-to-tail fashion, known as Ruzicka’s rule (Barkovich and Liao, 2001). This rule has exceptions or difficulties when the number of carbon is higher than 25 (Liu, 2011). Terpenoids are classified according to the number of isoprene units that are in the compound. The following table contains different classes of terpenoids:
Table 2.1: Different classes of terpenoids (Wagner and Elmadfa, 2003; Liu, 2011).
Terpenoids Number of carbon atoms Number of isoprene units
Monoterpene 10 2
Sesquiterpene 15 3
Diterpene 20 4
Triterpene 30 6
Tetraterpene 40 8
Polyterpene >40 >8
Although isoprene unit is considered as the starting of all terpenoids, it was reported that it is not involved in the formation of these compounds and not even present in plants (Dewick, 2002).
However, it was proved that isopentyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) might be considered as only biosynthetic precursors for all terpenoids
(Mahmoud and Croteau, 2002; Aharoni et al., 2005; Goto et al, 2010; Nagegowda, 2010).
28
Head Tail
(72) Isoprene unit (73)
O
(74) (75) (76) (77) (78)
Monoterpenoids
(81) (80) (79)
Sesquioterpenoids
Diterpenoids (83) (82)
(84) Tetraterpenoid ( -Carotene)
Figure 2.16: Chemical structures illustrating different classes of terpenoids.
Terpenoids are very important in human because of their biological activities that include anticancer, antiparasitic, antimicrobial, antioxidant, antiallergenic, antispasmodic, antihyperglycemic, anti-inflammatory, chemotherapeutic, anti-ulcer, anticarcinogenic, antimalarial, hepaticidal, diuretic, and immunomodulatory. Terpenoids are used as skin
29 penetration enhancers, natural insecticides in storing agricultural products (Aharoni et al., 2005;
Ajikumar et al., 2008).
Monoterpenes, sesquiterpenes as well as diterpenes, especially those with hydroxyl groups and double bond carbon–carbon (conjugated to each other and possessing allylic hydrogens) possess antioxidant activity (Wojtunik et al., 2014). Hence, they are able to stop or reduce the formation of radicals in cells. Monoterpenes with allylic hydrogen show higher radical scavenging activity than those without allylic hydrogen and conjugated system toward 2,2-diphenyl-1-picrylhydrazyl
(DPPH) radical scavenging method.
CH2 CH CH R O2N O2N H (88)
NN NO2 CH2 CH CH R NNH NO2 (86)
O2N O2N (87 (89) (85) CH2 CH CH R
Figure 2.17: Reaction scheme showing how DPPH radical abstracts the allylic hydrogen from terpenes.
The antioxidant potency of tetraterpenoids depends on the number of conjugated carbon–carbon double bonds as well as the functional groups within the structure (Wagner and Elmadfa, 2003).
Monoterpenes and sesquiterpenes are major components in essential oil (Bakkali et al., 2007;
Chamorro et al., 2012). They have a range of biological activities, including antibacterial. In general, essential oils (terpenoids) are able to interact with lipids from the cell membrane and
30 mitochondria of bacteria because of their hydrophobic properties and that leads to the disturbance of the structure of membrane and mitochondria (Burt, 2004). Bacteria will continue to lose more molecules and ions and this will lead to bacterial death. Furthermore, the presence of hydroxyl groups in phenolic terpenoids is responsible for the destabilisation of bacterial cytoplasmic membrane (Ultee et al., 2002).These phytochemicals can act also as proton exchangers by reducing the pH gradient across the cytoplasmic membrane. Terpenoids with
- acetate (CH3COO ) and alkenyl (CH3CHCH-) groups had demonstrated a high antibacterial activity than those with alkyl groups (Burt, 2004).
2.4.2.7. Saponins
The word “saponin” took its origin from Latin word “sapo” which can be translated in English
“soap”. Saponins belong to a class of secondary metabolites occurring mainly in plant kingdom
(Vincken et al., 2007) whereby about 500 genera of plants contain saponins (Negi et al., 2013) and in a wide range of foods including Asparagus, beans, blackberries, peas, potatoes, sugar beet and tea (Sparg et al., 2004).
Saponins belong to the vast group of glycosides, which consists of sugar moiety unit(s) linked to a nonpolar steroidal aglycone or to triterpene. The non-saccharide aglycone part of a saponin molecule is called genin or sapogenin. According to Sparg et al (2004), using genin part of saponins as reference, saponins are divided into three subclasses: i. Triterpenes glycosides, which are the most common and occur mainly in the dicotyledonous
angiosperm.
31 ii. Steroid glycosides, which are exclusively present in the monocotyledonous angiosperms. iii. Steroid alkaloid glycosides (steroid amine), which are classified by a haemolytic properties
that are generally attributed to the interaction between the saponins and the sterols of
erythrocyte membrane.
H O N
O O
HO HO HO (90) Triterpene class (91) Steroid class (92) Steroid alkaloid class
Figure 2.18: Skeletal structures of different groups of saponins using genin part as reference.
Saponins may contain one or two sugar nucleus, hence they are called monodesmosidic (sugar chain is attached at C3) and bidesmosidic (sugar chain is attached to C3 and C28).
COOH 28 COO 28 Glc
3 Glc O 3 (93) Monodesmosidic Glc O (94) Bidesmosidic
Figure 2.19: Structure of Monodesmosidic and Bidesmosidic saponins.
32
However, some plants such as Lurerne, Acacia auriculiformis, Astragalus cephalotes var brevicalyx and many others contain tridesmosidic saponins (Vincken et al, 2007) like
Cephalotoside A isolated from Astragalus cephalotes var brevicalyx (Çalis et al., 1998).
O OH O HO OH
OH (95) Cephalotoside A O
O OH HO O HO O OH OH HO OH OH
Figure 2.20: Chemical structure of Cephalotoside A, a tridemosidic saponin.
Saponins possess a number of properties including that of being able to lower the surface tension and produce foam when dissolved in water (Thakur et al., 2011). This property makes difference in between saponins and others glycosides groups. Saponins are also characterised by a haemolytic action (Sparg et al., 2004), generally precipitate by cholesterol in alcoholic solution and the most important is that saponins are toxic to cold-blooded animals (fish). Hence, they have been the subject of extensive research for their insecticidal, antibiotic, fungicidal and number of pharmacological properties. Despite this property, saponins oral toxicity is very low to mammals (Dini et al., 2001). Beside the above properties, saponins showed important pharmacological properties including, molluscicidal activity, anti-inflammatory, antifungal/antiyeast, antibacterial, antitumor activity and cytotoxic, antiviral (Sparg et al., 2004), antioxidant, immunostimulant, anti-hepatotoxic, anti-carcinogenic, anti-diarrheal, anti- ulcerogenic, anti-oxytoxic, hypochlolesterolemic, anti-coagulant, hepatoprotective,
33 hypoglycemic, neuroprotective, inhibition of dental caries and platelet aggregation (Negi et al.,
2013). Other reports on pharmacological properties of saponins suggested that, saponins play a role in stimulation of luteinizing hormone release leading to abortifacient properties, immunomodulatory potential via cytokine interplay and adjuvant properties for vaccines as immunostimulatory complexes (Thakur et al., 2011).
Previous researchers reported that 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP saponin) can stop the free radical chain reaction by reacting with hydroxyperoxide (Yoshiki et al., 1998). The reaction product yields hydroxyperoxide intermediate for which the activity is affected by aglycone structure of the saponin. The same study reported that, the reducing power of soya bean saponins increases with the addition of benzoquinone structure. Unfortunately, the ideal cannot be generalise to all saponins because the antioxidant activity was not from the common part of all saponin. Hence, more investigations need to be conducted in this regards to shed more clarity on the antioxidant property of saponins.
O O OH OH OOH O O O CH3 O CH3
. O2 R R (96) DDMP saponin (97) Dihydroxyperoxide
Figure 2.21: Reaction scheme of saponin with superoxide free radical.
34
Saponins with three branched chain sugar moieties but without any oxygen functionalities at C2 and C12 exhibit antiyeast activities. Nevertheless, those with 2β-hydroxyl groups or keto groups at C12 exhibit a very low or no activity at all, and the same was observed for saponins with two sugar moieties (Miyakoshi et al., 2000; Francis et al., 2002). Furthermore, the aglycone part of saponins does not show antiyeast activity. Hence, the antiyeast property of saponins depends on number of sugar moiety and oxygen at C2 and C12. Wina et al (2005) reported that saponins could kill or damage protozoa by forming complexes with sterols in the protozoal membrane surface; the membranes become impaired and eventually disintegrate.
2.4.2.8. Cardiac glycosides
This naturally plant occurring secondary metabolites name is derived from the impact that they have made on the heart functions (Hallböök et al., 2011; Liu, 2011). Since ancient time until now, cardiac glycosides (CGs) have potential in treatment of heart related pathologies; they occur in limited number of plants families and from some frogs species (Winnicka et al., 2006).
The general structures of cardiac glycosides consist of linked sugar moiety at C3 and a lactone ring at C17 (Pongrakhananon, 2013).
O O O Lactone ring O
17 Ring of 17 C D Steroid core C D
A B 3 3 A B O O
Sugar Sugar (99) Bufadienolides (98) Cardenolides
Figure 2.22: Structures showing the two classes of cardiac glycosides.
35
Based on the type of lactone ring, cardiac glycosides (CGs) are divided into two major subclasses viz cardenolides (carrying five membered unsaturated heterocyclic lactone ring at position C-17) and bufadienolides (carrying six membered unsaturated heterocyclic lactone ring at position C-17) (Pongrakhananon, 2013; Wink, 2010).
Cardiac glycosides are called cardiotonics, which are used in treatment of congestive heart failure and cardiac arrhythmias such as atrial fibrillation (Prassas and Diamandis, 2008;
Pongrakhananon, 2013). Cardiac glycosides increase the force of heart contraction without increasing the consumption of oxygen by inhibiting membrane-bound sodium-potassium- activated Adenosinetriphosphatase (Na+/K+-ATPAase) (Prassas and Diamandis, 2008; Joy and
Alam, 2012). They are able to suppress the active counter-transportation of Na+ and K+ ions within cell membrane that increases the intracellular Na+ ion concentration and at the same time, the concentration of intracellular K+ ion decreases. Hence, the cardiac concentration will increase. Recently, cardiac glycosides were reported for their anticancer (Newman et al., 2008) and cytotoxicity (Kumar et al., 2013) biological activities.
Diaz et al (1997) have reported about epidemiologic studies that proved an inverse link between coronary artery disease and the intake of antioxidant especially vitamin E and C supplementary agent which can reduce morbidity and mortality caused by these diseases. This means, introduction of theses antioxidant (Vitamin C and E) into low-density lipoprotein (LDL), inhibit oxidation leading to lipid oxidation protection. In other words, incorporation of antioxidant into vascular cells may reduce the oxidation of LDL, which may result into less monocyte adhesion, less foam cell formation, less cytotoxicity to vascular cells, and therefore improvement of vascular function (Cherubini et al., 2005).
36
Cardiac glycosides do not have any direct effect on bacteria. Nonetheless, it was reported that, digoxin, the most widely used cardiac glycoside, undergoes significant metabolism to a cardio inactive metabolite where its lactone ring is reduced (Saha et al., 1983). This reduction reaction of digoxin appears to be facilitated within the human gastrointestinal tract by Eubacterium lentum, a common anaerobic intestinal flora.
2.4.2.9. Alkaloids
Alkaloids are the largest nitrogen containing secondary metabolites occurring in microorganisms, animals, and mainly in higher plants, and some time in lower plants as free compounds or as salts (Liu, 2011; Kuete, 2013). Alkaloids are naturally basic (alkali-like), but this property depends on the presence and the location of other functional groups within the molecule. Sometime alkaloids, especially 1,2-dehydro pyrrolizidine ester alkaloids are toxic to animals when absorbed (Edgar and Wiedenfeld, 2011).
Alkaloids play a defensive role against herbivores and pathogens agents to plants (Facchini,
2001). A number of amino acids including tyrosine, phenylalanine, anthranilic acid, tryptophan/tryptomine ornithine/ arginine, lysine, histidine and nicotinic acid have been found to be precursors of majority of alkaloids (Ziegler and Facchini, 2008; Evans, 2009). However, some others use purines as precursors (Wink, 2010).
Using the skeletal structure classification, alkaloids are classified into two major categories:
(i) non-heterocyclic alkaloids (atypical alkaloids, protoalkaloids or biological amines) and
37
(ii) heterocyclic alkaloids (typical alkaloids) which are further divided into 14 groups according
to their ring structures such as Pyrrol and Pyrrolidine, Pyrrolizidine, Pyridine and Piperidine,
Tropane, Quinoline, Isoquinoline, Aporphine, Norlupinane, Indole, Indolizine, Imidazole,
Purine, Steroid, and Terpenoid (Evans, 2009; Saxena et al., 2013).
CH3O (100) N (103) (101) (102) CH3O CH2 CH2 NH2 N N N
CH3O H H H
NH (108) N N N N (104) (105) (106) (107) H
N N N (109) (110) N NH (111) (112) NH NH N + (113) N (114)
Figure 2.23: Some chemical structures of alkaloids isolated from medicinal plants.
Alkaloids are pharmaceutically very important because of their biological activities such as antibacterial, antimalarial, analgesia, anaesthesia, anticancer, cardiant, antihypertension, cholinomimericactior, releasing cought, spasmolysis, vasodilatation, anti-arrhythmic and anti- asthma (Facchini, 2001; Liu, 2011).
Miliam et al (2012) reported that, the halogenation of synthetic alkaloids containing phenolic substituents displayed more antioxidative activity than those with methoxyl groups, and also
38 some aporphine and phenanthrene alkaloids, might weakly increase their antioxidant activities in free cell systems. Rau et al (2014) reported that, among the whole alkaloid classes, monoterpenes indole alkaloids (MIAs) are widely studied because of their potential pharmaceutical properties. The same study concluded that, the presence of rings, double bonds, amines as well as the presence of glycosylation, might likely affect the efficiency of their antioxidant activities. The antioxidant activity of benzylisoquinoline alkaloids depends on the presence of conjugated π electrons systems. The allylic or phenolic hydrogens are ready to be removed in order to stabilise free radicals, which might be delocalised further in the case of allylic hydrogen; and formation of keto group for the case in phenolic hydrogens (Cassels et al.,
1995). Alkaloids isolated from Fumaria capreolata and Fumaria bastardii plants such as protopine, cryptonine, stylopine, fumariline, phtalidiisoquinoline, fumaritine, fumarafine, and dehydrobenzophenanthridine have antioxidant properties (Sen et al., 2010).
Alkaloids isolated from plants and showing the antibacterial activity have been found more effective against Gram-positive bacteria than Gram-negative bacteria (Pfoze et al., 2005). This might be explained by the presence of extra outer membrane in their cell walls which do not allow the penetration of alkaloids compounds into bacterial cells for Gram-negative bacteria. For instance, among alkaloids, Indoloquinones display many pharmacological properties, including antimicrobial (Karou et al., 2005).
Furthermore, previous researchers found that the presence of lipophilic character in alkaloids molecules might facilitate the permeability of the membranes of the microorganisms and thereby inhibit their growth (Salih et al., 2011; Li et al., 2014). Methylenedioxy group on the phenolic
39 part of an alkaloid is likely to increase the antimicrobial activity of that alkaloid compared to that of methoxy group (Gu et al., 2014).
2.4.3. Biological activity of secondary metabolites
Jackson et al (2007) defined a biological activity as a specific ability or capacity of a particular metabolite to achieve a defined biological effect. Therefore, each microorganism/virus might have a specific biological activity to which it responds. Hence, there is unlimited number of biological activities against microorganism/virus such as antioxidant, antibacterial, anti- inflammatory, antidiabetes, anticancer, antituberculosis, anti-allergic, antiplatelet, antitumor, antiplasmodial and many others. In this study, antioxidant and antibacterial activities of the ethanolic extract of the aerial part of Asparagus suaveolens are investigated.
2.4.3.1. Antioxidants
An antioxidant is defined as a substance that is able to terminate, inhibit or delay the oxidation of molecules such as proteins, lipids, nucleic acids, and carbohydrates by preventing the initiation and propagation steps of oxidation chain reactions (Antolovich et al., 2002). Antioxidants can repair damages done to body cells, which might be the source of human discomfort such as aging, coronary artery diseases, degenerative cancer. and many other diseases. Hence, they
. - . remove free radical intermediates such as superoxide anion ( O2 ), hydroxyl radical (HO ), peroxyl free radical (ROO.), (Nitrogen monoxide (NO.) and many other free radicals and reactive species, which are able to damage other chemical species in living system if their generation is
40 not controlled (Kim et al., 2011). These reactive species are generated within or outside the organism, hence some are called endogenous free radicals and others are called exogenous free radicals (Vallyathan and Shi, 1997; Sen et al., 2010; Kabel. 2014).
There are many way in which an antioxidant activity can be experimentally evaluated: Oxygen
Radical Absorbance Capacity (ORAC), Total Radical-Trapping Antioxidant Parameter (TRAP),
2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay, Total Oxidant Scavenging Capacity (TOSC), 2,2'- azino-bis(3-ethylbenzothiazoline)-6-sulphonic acid (ABTS) radical cation inhibition activity,
Total phenolic content (TPC), Chemiluminescence (CL), Photochemiluminescence (PCL),
Croton or β-Carotene Bleaching by LOO., Low-Density Lipoprotein (LDL) Oxidation, Ferric
Reducing Antioxidant Power (FRAP), Copper Reduction Assay (CUPRAC, AOP-90), Folin-
Ciocalteu (F-C) AOC or Total Phenolics assay, Trolox equivalent antioxidant capacity (TEAC) and many more (Prior et al., 2005). Beside all these quantitative antioxidant screening, there is a quick and accurate qualitative antioxidant assay called bio-autography antioxidant assay. Bio- autography assay and 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay were used in this work due to their simplicity, time management and low cost.
2.4.3.2. Antibacterial
The word antibacterial refers to an agent or chemical substance that kills (antibactericidal), destroys, suppresses or inhibits (antibacteostatic) the growth of bacteria with a minimum damage to the host. Antimicrobial activity is used when the compound is interfering with the growth,
41 progression of microorganisms such as viruses, fungi, as well as bacteria. In this study, the word microorganism will refer to bacteria.
According to their mode of action, bacteria are known as saprophytic (no virulent) and pathogenic. Saprophytic bacteria are biologically important for their biochemical action in digestion, recycling of the nutrients, nitrogen fixation, drug preparation and decomposition of dead organic matter (in metabolism) (Myrold and Posavatz, 2007; Khalil et al., 2014). A pathogenic bacterium consists of a small portion of bacteria species but is responsible for many of the infectious diseases (Baron, 1996).
The antibacterial assay, which is part of the antimicrobial susceptible testing (AMST) methods, is performed in many ways. Three of them have been widely used for quantitative purpose including disk diffusion method, dilution method and the combination of the two called diffusion and dilution method (Lalitha, 2012). However, there is a quick antibacterial qualitative method called antibacterial bio-autography method which aimed to localize qualitatively the antibacterial activity onto a TLC plate (Katerere and Luseba, 2010; Suleimen et al., 2010). Dilution method has been considered as the reference of in vitro method for all AMST. This method has also been used where equivoque or unreliable results from disk diffusion methods as well as when the quantitative result is required (Wiegnad et al., 2008).
2.4.4. Bio-guided isolation of secondary metabolites
Bioactive secondary metabolites are considered as the origin of the production of pharmacological drugs from natural resources such as plants and some bacteria (Pieters and
42
Vlientrick, 2005). Previously, studies on natural products was about isolating compounds and establishing their chemical structures only. However, nowadays, the principal aim in compound isolation is the discovery of new drugs, which can be able to be used in medicine modern. Thus, isolation of organic compounds from plants should be associated to a biological activity
(Katerere and Luseba, 2010). Hence, in the current study, antioxidant and antibacterial activities were considered. The isolation driven by biological activity is called bioassay guided fractionation (Pieters and Vliendtrick, 2005). Every step of the isolation procedure is monitored through a bioassay, which may be qualitative or quantitative.
2.5. Asparagus species: Overview
Asparagus, a Greek word which means “stalk’’ or ‘’shoot’’, plant species previously belonged to the subfamily of Asparagae in the family of Liliaceae (Mashele and Fuku, 2011; Kole, 2011;
Ntsoelinyane and Mashele, 2014). There after Fellingham and Meyer (1995) classified all
Asparagus in one genus, without subgenera. By then, the genus consisted of about 100 to 300 monoecious (having male and female reproductive organs in separate flowers on the same plant) and bisexual species (Dinan et al., 2001; Madhavana et al., 2009). Recent research findings on
Asparagus indicate the existence of 120 to 400 species distributed into two genera including
Asparagus and Hemiphylacus (Kole, 2011; Norup et al., 2015). Hemiphylacus consists only of five species and all have been found exclusively in Mexico. Asparagus subgenera is therefore, divided into three including Asparagus, Protoasparagus and Myrsiphyllum, all originate from
Eastern Mediterranean region, Asia and Africa, especially in Southern Africa region, where more than half of them are native. Asparagus can be herbaceous, perennial, tender woody shrubs or as
43 vines; usually with thorny and spindle-sharped roots and they can grow from 20 cm to more than
5 m in length (Kole, 2011).
Asparagus species are used as ornamental, medicinal and diet plants (Kole, 2011; Štajner et al.,
2002; Benincasa and Tei, 2007). As the Table 2.2 indicates, Asparagus are mostly considered as medicinal plants. This is due to the phytochemical constituents that include steroids, saponins, sapogenins saccharides, acetylenic compounds, sulphur containing compounds and many other bioactive secondary metabolites present in their different parts (Goyal et al., 2003; Madhavana et al., 2009; Van Wyk et al., 2009; Raval et al.,2012). These different ingredients present in
Asparagus species have multiple biological properties including anti-ulcer, antioxidant, antitumor, immune-stimulant, immune-adjuvant (boost the amount of antibodies), anti- inflammatory, antibacterial and many others (Van Wyk et al., 2009).
The first scientific research on Asparagus had started by a mathematician and physician Scottish
John Arbuthnot who had published a book in 1731 (Mitchell, 2001) in which he indicated that
Asparagus officinalis consumption was responsible for foetid smell of urine. Thereafter, it was found that, Asparagusic acid (1,2-dithiolane-4-carboxilic acid) and its derivatives were responsible for urine foetid smell from person who ingested Asparagus officinalis (Sun et al.,
2010). Thereafter, many metabolites with different biological activities had been isolated from
Asparagus species such as flavonoids, saponins, steroids and many others.
44
Table 2.2: Some of Asparagus plants and their usages (Fouche et al., 2008; Van Wyk et al., 2009; Kubota et al., 2012).
Asparagus usages
Ornamental Medicinal Diet
Asparagus asparagoides Asparagus adscendens Asparagus maritimus
Asparagus densiflorus Asparagus aethiopicus Asparagus acutifolius
Asparagus myriocladus Asparagus asparagoides Asparagus albus
Asparagus plumosus Asparagus capensis Asparagus officinalis
Asparagus retrofractus Asparagus exuvialis
Asparagus sperngeri Asparagus falcatus
Asparagus virgatus Asparagus laricinus
Asparagus plumosus
Asparagus retrofractus
Asparagus setaceus
Asparagus stipulaceus
Asparagus struatus
Asparagus suaveolens
Asparagus subulatus
Asparagus transvaalensis
Asparagus verticillatus
45
2.6. Plant under current study: Asparagus suaveolens
2.6.1. Discovery of Asparagus suaveolens
Asparagus suaveolens (Burch) was collected for the first time by William John Burchell in 1822 and it was named in memory of its first collector (Jessop, 1966). Later on in 1983, Obermeyer
Anna Amelia collected a similar plant, which he called Protoasparagus Suaveolens (Oberm)
(Fellingham and Meyer, 1995). Thereafter, it was found that these two species were similar and identical. Taxonomists decided to keep both names; they are, therefore synonym (Wiegand,
2006). In South Africa, the plant is called Bush veld Asparagus or wild Asparagus (English),
Katdoring (Afrikaans) and Mvane (Xhosa) (Global plants n.d).
2.6.2. Taxonomy of Asparagus suaveolens
After a long discussion on the classification of Asparagaceae family, taxonomists agreed that, the family consisted by one genus with two subgenera including Asparagus and Hemiphylacus.
Asparagus suaveolens belong to that of Asparagus. According to sv.wikipedia (n.d), the following taxonomy appears to be accepted by everyone.
Kingdom: Plantea Class: Liliopsida Genus: Asparagus
Subkingdom: Tracheobionta Order: Asparagales Species: suaveolens
Phylum: Tracheophyta Family: Asparagaceae
46
2.6.3. Distribution of Asparagus suaveolens
Asparagus suaveolens is a wild plant native of Eastern and Southern African regions, but widely distributed in South Africa, Botswana, Swaziland, Malawi, Namibia, Lesotho, Zimbabwe,
Mozambique, Tanzania and Kenya (Useful tropical plants n.d). In South Africa, the plant is spread in all nine provinces (Eastern Cape, Free State, Gauteng, Kwazulu-Natal, Limpopo,
Northern Cape, Mpumalanga, Northern Cape and Western Cape). It is called iMvane (siXhosa),
Kadoring in Afrikaans, Lesitwane (Setswana) or wild Asparagus in English (Dold and Cocks,
2001; Red list of South African Plants n.d).
Figure 2.24: Asparagus suaveolens. Photograph taken near Bolahlakgomo village, Limpopo Province, South Africa (November, 2013).
47
2.7.4. Botany of Asparagus suaveolens
Asparagus suaveolens is a thorny straight sometime woody shrub with small leaves, which remain in winter, shining pale brown stems, with white flowers, which occur in autumn and early summer with red fruit or black berry (Jessop, 1966). Asparagus suaveolens can grow up to 1 m tall but for some cases it can go to 1.5 m in the rocky grassland (savannah) (Van Wyk et al.,
2009).
2.6.5. Ethnomedicinal usages of Asparagus suaveolens
i. Roots of Asparagus suaveolens are the most used part for different traditional medicinal
purposes including treatment of sexually transmitted infections, in particular gonorrhoea, as
well as in improving men’s libido (Gololo, personal communication). ii. Leaves of Asparagus suaveolens are used as traditional medicine for treating epilepsy in
Northern Sotho (Van Wyk et al., 2009). Epileptic patient inhaled the smoke from burned
material. iii. According to exhaustive investigation conducted by Rasethe et al, (2013), the community Ga-
sekgopo in Limpopo province, South Africa, uses Asparagus suaveolens (sephatlalatsa) as
traditional medicine.
Beside the use of Asparagus suaveolens species in human traditional medicine, it has also been used to treat diseases in livestock.
48 i. For example, roots of Asparagus suaveolens are used to treat cows after giving birth. Roots
are boiled then about 750 ml aqueous extract is given every morning to the cow that gave
birth (Dold and Coks, 2001; McGaw and Eloff, 2008). ii. The Tswana speaking community from Madikwe in North West province, South Africa, uses
Asparagus Suaveolens to treat cattle sore, urine infection, red water umbilical cord infection
and general ailments (Van der Merwe et al., 2001).
The information provided above on the traditional usage of Asparagus suaveolens were the only onces found in the literature in our possession.
49
CHAPTER 3
MATERIAL AND METHODS
3.1. Instrumentations and apparatus
The following instruments and material were used: weighing balance (Sartorius, CPA225D,
Germany), Retsch (100) grinding/milling machine (Germany), Shaker (Labotec 262, South
Africa), UV lamp, Spectroline, ENF-280C/FE, USA), UV-Vis spectrophotometer (Jenway 7300,
United Kingdom), Rotary evaporator (Buchi R-200, Labotec, South Africa), Nuclear Magnetic
Resonance (NMR 400 MHZ Oxford), Liquid Chromatography- Mass Spectrometer (LC-MS,
Thermo scientific, USA), Gas Chromatography-Mass Spectrometer (GC-MS, Shimadzu QP
2010SE, Japan), Incubator (orbital shaker incubator, Lasec SA, Taiwan), 96-well microtiter
TM TM plates (Thermo scientific, Denmark), CO2 incubator (Fisher scientific Isotemp CO2 incubator, 13-25526, USA), water bath (Nüve NB20, Turkey), petri dish containing agar media, micropipettes, pipette tips; glass ware and Thin layer chromatography.
3.2. Solvents and chemicals
The following solvents and chemicals were used: n-hexane, chloroform, acetone, methanol, butan-1-ol, formic acid, ethyl acetate, benzene, dichloromethane, ammonium (25%), 2,2- diphenyl-1-1picrylhydrazyl (DPPH), L-ascorbic acid, gentamicin, amoxicillin, Meyer’s reagent, ferric chloride, hydrochloric acid, ammonia (25%), sulphuric acid, sodium hydroxide, acetic
50 acid, Molisch reagent, Fehling’s solutions (A,B) copper (II) sulphate, butylated hydroxytoluene
(BHT), magnesium balls and p-iodonitrotetrazolium chloride (INT).
3.3. Collection, identification and preparation of plant material
3.3.1. Collection of the plant material
The aerial parts of Asparagus suaveolens were collected at the beginning of summer as advised by Liu (2011) at Bolahlakgomo village (situated at -24.451218o South latitude, 29.326738o East longitude and 922 meters above sea level) in Lepelle-Nkumpi Municipality of the Limpopo province, South Africa.
Figure 3.1: Maps showing sample collection side, near Bolahlakgomo village in Lepelle- Nkumpi Municipality of the Limpopo province, South Africa (Google Maps n.d).
51
3.3.2. Identification of the plant
The collected aerial part of Asparagus suaveolens was sent to the South African National
Biodiversity (SANBI), Pretoria herbarium, Gauteng Province, South Africa; for identification.
The specimen of the plant was subjected to a botanical identification procedure. Resulting in discovery of Asparagus suaveolens voucher in the South African National Biodiversity Institute
(SANBI), classified as medicinal plant under the specimen number PREART 0001903 (Global plants n.d).
3.3.3. Preparation of plant material
After collection, the aerial part of Asparagus suaveolens was cut into small pieces and dried at room temperature. Thereafter, dried plant material was ground to powder using Retsch (mils SM
100) grinding machine (Germany). The resultant powder was then kept in the dark until further usage.
3.4. Extraction
Five hundred grams (500 g) of powered plant material was macerated at room temperature with
5000 ml of ethanol for 24 hours on a shaker at 150 rpm. The process was repeated three times using same volume of fresh ethanol solvent with the same plant material. The mixture was allowed to settle down before being filtered using Whiteman No. 1 filter paper. The solvent was
52 then evaporated using rotary evaporator under reduced pressure at 20 to 400C. The remaining solvent extract mixture was transferred into a pre-weighed beaker, and then ventilated to dryness.
The percentage yield (% in w/w) of ethanol extract was calculated using the formula below:
Mass of extract Percentage yield (w/w) = X 100 Mass of the plant material
The solid of ethanol extract was kept in the dark at the room temperature til the future usage.
3.5. Fractionation of the aerial part ethanol extract of Asparagus suaveolens
The ethanol extract was re-dissolved using a mixture of methanol and water (9:1, v/v) and then fractionated in a separating funnel (liquid-liquid separation method) with n-hexane (extracting nonpolar compounds), followed by chloroform (extracting intermediate compounds) and butan-
1-ol (extracting polar compounds) respectively according to Liu, (2011). The procedure was repeated several times with each solvent until 90 to 95% of soluble parts have been extracted.
Thereafter, solvents were evaporated using rotary evaporator under reduced pressure at 20 to
400C and ventilated as described in Section 3.4. Liquid-liquid extraction was carried out according to the following flow chart.
53
Ethanol Extract
Suspended in Methanol/Water (9:1)
n-Hexane Fraction MeOH/H2O Layer
Evaporation of MeOH
Chloroform Fraction Aqueous Layer
Adds Butan-1-ol
Butan-1-ol Fraction Water Fraction
Figure 3.2: Schematic representation of the liquid-liquid fractionation of ethanolic aerial part extract of Asparagus suaveolens using different organic solvents.
The mass of each fraction (n-hexane, chloroform and butan-1-ol) was recorded, then kept into in the dark until further usage and the water fraction was discarded.
3.6. Phytochemical analysis of the fractions
Qualitative phytochemical composition of the fractions from the ethanol extract of the aerial part of Asparagus suaveolens was determined as described by Usman et al (2009); Zohra et al
(2012); Joseph et al (2013) with minor modifications.
54
3.6.1. Alkaloids
About 2.0 mg of the fraction was treated with Mayer’s reagent: solution made from 5.00 g of potassium iodide (KI) and 1.36 g of mercuric chloride (HgCl2) in 100.00 ml of water.The appearance of precipitates in the mixture indicated the presence of alkaloids in the fraction.
3.6.2. Tannins
The qualitative analysis of tannins was conducted by dissolving 0.5 g of each fraction with 20 ml of distilled water in a test tube and the mixture was then filtered. To the filtrate, few drops of
0.1% of Ferric chloride (FeCl3) were added. The appearance of a blue black colouration confirmed the presence of tannins in the fractions.
3.6.3. Saponins
Small amount of each solid fraction (n-hexane, chloroform and butan-1-ol) was boiled together with 20 ml of distilled water over a water bath and filtered. About 10 ml of each fraction filtrate was mixed with 5 ml of distilled water in a test tube and vigorously shaken. The formation of a persistent froth indicated the presence of saponins.
3.6.4. Flavonoids (Shindo’s test)
Each fraction was mixed with small quantity of magnesium balls and few drops of a concentrated HCl was added to the mixture before being heated for five minutes over a boiling
55 water bath. The appearance of orange red colour confirmed the presence of flavonoids.
3.6.5. Cardiac glycosides
About 5 ml of each aqueous fraction solution was mixed with 2 ml of glacial acetic acid containing 1 drop of Ferric chloride (FeCl3). The above mixture was carefully added to 1 ml of concentrated H2SO4. The appearance of brown rings indicated the presence of the cardiac glycosides in the fraction.
3.6.6. Anthraquinones
Presence of anthraquinones in each fraction was determined by dissolving small amount of each fraction with 10 ml of chloroform and the mixture was shaken before being filtered. Thereafter, 5 ml of 10% solution of NH3 was added to the filtrate. The appearance of pink/red colour indicated the presence of anthraquinones.
3.6.7. Terpenoids
Each fraction was mixed with 2 ml of chloroform and then 3 ml of concentrated H2SO4 was carefully added to form a layer. The appearance of reddish brown colouration of the interface show positive result for the presence of terpenoids in the fraction.
56
3.8.8. Steroids
The test was performed by mixing 0.2 g of each fraction with 10 ml of chloroform. After filtration, 2 ml of the resulting solution was mixed with 2 ml of acetic acid before carefully introducing concentrated sulphuric acid (H2SO4) in few drops and the resultant solution was cooled on ice. The change in colour from violet to a dark green colour indicated the presence of steroids.
3.6.9. Reducing sugars
About 0.5 g of each fraction was dissolved using distilled water and then filtered. The filtrates were heated over a boiling water bath and then mixed with 5 ml of Fehling’s solution A and B at equal volume. The appearance of brick red precipitate indicated the presence of reducing sugars.
3.6.10. Proteins
Two milliliters (2 ml) of each aqueous fraction solution of each fraction was transferred into test tubes. Three drops of 10% sodium hydroxide (NaOH) solution were added into the test tube, thereafter 3 to 6 drops of 0.5% copper (II) sulphate (CuSO4) solution were also added. The positive test was indicated by the colour change of copper sulphate, from blue to violet colour.
57
3.6.11. Coumarins
Few amounts of each fraction were dissolved in 1 to 2 ml of hot distilled water, and then equally divided into two test tubes. One was considered as control, and in the other, 0.5 ml of 10%
NH4OH was added. Two drops were spotted on filter paper and examined under UV light. The presence of coumarins was indicated by intense fluorence.
3.7. Screening for biological activity
3.7.1. Antioxidant screening
3.7.1.1. Bio-autographic antioxidant assay
The bio-autography antioxidant assay was carried out using the protocol as previously reported by Masoko et al. (2010) and Koto-te-Nyiwa et al. (2014) with minor modifications. Briefly 100
µl of each fraction solution (n-hexane, chloroform and butan-1-ol) (125 mg/ml) was loaded onto thin layer chromatography (TLC) plates. TLC plates were developed in a tank using three different solvent system viz. benzene, ethyl acetate, ammonia (BEA, 18:2:0.8, v/v/v); chloroform, ethyl acetate, formic acid (CEF, 10:8:2, v/v/v) and ethyl acetate, methanol, water
(EMW, 10:1.35:1, v/v/v). TLC plates were then allowed to dry before being sprayed with 0.2% methanolic solution of 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical. The appearance of yellow spots indicated a positive result of antioxidant activity by the constituents of the tested fractions.
58
3.7.1.2. DPPH free radical scavenging assay
The eassay was performed according to the pre-established procedure as used by Moyo et al.
(2012), with minor modifications. One milliliter (1 ml) of a freshly prepared methanolic solution of DPPH (0.2 mM) was mixed with 1 ml of aqueous fraction solution (n-hexane, chloroform or butan-1-ol) (0.5-2.5 mg/ml). The mixture was vortexed and left in the dark for 30 minutes before its absorbance was measured spectrophotometrically at 517 nm. Ascorbic acid and butylated hydroxytouene (BHT) solutions were used as standards. The radical scavenging activity was expressed as percentage DPPH inhibition calculated using the following equation.
(Abs control - Abs sample ) % of DPPH inhibition = X 100 Abs control
Where, Abssample is the absorbance reading of the extracts or the standard sample with DPPH and
Abscontrol the absorbance of DPPH without extracts or standard.
3.7.2. Antibacterial assay
3.7.2.1. Bio-autography antibacterial assay
Four general bacterial strains; Staphylococcus aureus (ATCC 29213), Escherichia coli (ATCC
25922), Enterococcus faecalis (ATCC 29212) and Pseudopodium aeruginosa (ATCC 27853) were used to carry out this test. This antibacterial bio-autography assay was performed according to the method used by Suleiman et al (2010) with slight modifications. Hundred microliters (100
µl) of fraction solution (n-hexane, chloroform or butan-1-ol) (125 mg/ml) were loaded onto TLC
59 plate, and then developed using solvent systems as described in Section 3.7.1.1 and dried. The plates were thereafter sprayed until wet with actively growing bacterial strains suspended in
Mueller Hinton Broth (MHB) with an estimated density of 5 x 105CFU/ml. The plates were incubated for 24 hours at 370C before being sprayed with solution of p-iodonitrotetrazolium chloride (INT) (2 mg/ml). TLC plates were further incubated for 1 hour at the same conditions.
The violet zones colour on the TLC indicated the bacteria growth while white or clear spots on the TLC plates indicated the bacterial growth inhibition.
3.7.2.2. Minimum inhibitory concentration (MIC)
The MICs of each fraction were determined using the same bacterial strains as in Section
3.7.2.1 and eight Neisseria gonorrhoeal strains used by WHO as reference strain for global quality assurance and quality control of gonococcal antimicrobial resistance testing identified as
F, G, K, L, M, N, O and P (Unemo et al., 2008). The experimental procedure was adopted from
Eloff (1998) with slight modification. Briefly, 100 µl of MHB was pipetted into 96-wells plate.
Thereafter, 100 µl of each fraction solution (n-hexane, chloroform or butan-1-ol) (125 mg/ml) prepared in water was added to first wells followed by a twofold serial dilution. Forty microliters
(40 µl) of an overnight bacterial suspension were added to each well. The 96-wells plate was then sealed and then incubated for 18 to 24 hours at 370C for the general bacteria and for 18 to
O 24 hours at 37 C in the presence of 10% of CO2 for the specific bacteria. After incubation, 40 µl of p-iodonitrotetrazolium chloride (INT) (2 mg/ml) was added and then the plate further incubated for about 1 hour at the similar condition. Gentamicin and Amoxicilin solutions were used as positive control. Wells that showed pink colour indicated bacteria growth, but those that
60 displayed yellow colour or colourless indicated no bacteria growth inhibition (formation of formazan). The MICs were recorded as the lowest concentrations that inhibited visible bacteria growth.
3.7.2.3. Minimal bactericidal concentration (MBC)
The minimum bactericidal concentration (MBC) of each fraction (n-hexane, chloroform and butan-1-ol) were determined using the same bacteria (N. gonorrhoea) as in Section 3.7.2.2. The experiment was performed using the protocol as reported by Bär et al (2009) with slight modifications.
Hundred microliters (100 µl) of Mueller Hinton broth was introduced into each well, thereafter
100 µl of n-hexane, chloroform or butan-1-ol fraction solution with the same concentration as in
Section 3.7.1.1 was pipetted into first wells, followed by a two-fold serial microdilution before
40 µl of the actively growing bacterial strains suspended in MHB were added into each well. The microtiter plate was incubated for 24 hours. Thereafter, 40 µl was pipetted-out from each well and sported onto the solid medium nutrient agar disks. The disks were then incubated at the similar condition as microtiter plates. The minimum bactericidal concentrations (MBC) were recorded as the lowest concentration that inhibited visible bacterial growth on the agar medium.
3.8. Bio-guided isolation of antioxidant and antibacterial compounds
Every step of the isolation was monitored by a qualitative antioxidant and antibacterial activity as described in the previous sections. This isolation procedure was accomplished by using
61 column chromatographic (19 mm x 61 cm) method and the preparatory thin layer chromatography in some instances. All three fractions (n-hexane, chloroform and butan-1-ol) were considered for bio-guided antioxidant isolation because they showed some positive results on the preliminary qualitative antioxidant and antibacterial tests.
3.8.1. n-Hexane fraction
The column (19 mm x 61 cm) was packed with a mixture of silica gel 60 and n-hexane: dichloromethane (9:1, v/v) and allowed to settle overnight before the sample was loaded. The sample (4.3 g) was dissolved into the solvent system then, filtered before being loaded into the packed column. The solvent mixture was slowly added into the column while the different fractions were collected drop wise into different test tubes. Antioxidant activity was constantly performed on the collected fractions. Those with similar profiles, based on TLC profile, were combined together into one pre-weighed beaker. Two compounds (1 and 2) were obtained from this fraction. Compound 1 (1.5 g) was collected as white amorphous powder and compound 2
(0.3 g) as cream white powder.
3.8.2. Chloroform fraction
A mass of 80 g of silica 60 was mixed with the mobile phase, n-hexane and ethyl acetate (9:1, v/v). The column was packed following the same procedure as described in Section 3.8.1. A mass of 2.7 g of chloroform fraction was dissolved into a small amount of solvent system before being loaded onto a packed column. During the elution process, the mobile phase ratio was
62 modified by increasing its polarity to (7:3 v/v); and after to (5:5 v/v). Compound 3 (0.005 g) was collected in test tubes 54 to 59 as gray powder.
3.8.3. Butan-1-ol fraction
A column (19 mm x 61 cm) was packed using chloroform, ethyl acetate and methanol (6:3:1, v/v/v) as solvent system. Due to the higher polarity of the targeted compounds from this fraction, a short column (50 g of silica gel 60) was used. An amount of 4.3 g from butan-1-ol fraction was dissolved into a small volume of solvent system then, filtered before being loaded into a packed column. Compound 4 (1.7 g) was eluted between test tube numbers 67 to 83 as yellow powder after drying the solvent system. In order to elute the second targeted compound (compound 5), the polarity of the solvent system (chloroform, ethyl acetate and methanol) was increased by modifying its ratio to 2:6:2 (v/v/v). This eluted the whole remaining impurity after the elution of compound 4. Because of higher polarity, compound 5 was eluted as impure from the column by using 100 % methanol. A mass of 1.9 g was collected from methanol was loaded onto a preparative thin layer chromatography plate (PTLC) before being developed into EMW (40:5:5, v/v/v) mobile phase. The part of PTLC containing compound 5 was scraped, dissolved in methanol then filtered. Compound 5 (0.976 g) was collected as yellow powder.
3.9. Structure elucidation of isolated compounds
The structure elucidations of isolated compounds were accomplished by usage of the following analytical instruments: Nuclear Magnetic Resonance (NMR 300 MHz Oxford and NMR 400
63
MHz Varian), Gas Chromatography/Mass Spectrometry (GC-MS, Shimadzu QP 2010SE, Japan) and Liquid chromatography/Mass Spectrometry (LC-MS, Thermo Scientific LCQ Deca XP,
USA).
3.9.1. Nuclear magnetic resonance
The NMR spectral data of the isolated compounds were obtained using varian 400 MHz NMR and Oxford 300 MHz NMR instruments from Tshwane University of Technology (TUT) and
Sefako Makgatho Health Sciences University (SMU), respectively. From these instruments, 1H
NMR, 13C NMR and two dimensional spectral (COSY, HSQC, and TOCSY) data were collected.
3.9.2. Gas Chromatography-Mass Spectrometry
The purity and mass of compounds 1 and 2, were determine using Gas Chromatography-Mass
Spectrometry (Shimadzu GC-MS QP2010, Japan) connected to auto-sampler. The capillary column InertCap 5MS/SiL (5% Phenyl and 95% methylpolysilarylene) with a diameter of 0.25 mm, length of 30.0 mm and a film thickness of 0.25 µm was used. Helium (99.99%) was used as carrier gas. Small amount of samples were dissolved into chloroform before 0.2 µl being injected into the instrument. The oven temperature was set to an initial temperature of 500C and hold at that temperature for 1 min. Thereafter, the temperature was changed to 1800C, 2400C, 2800C and
3000C with a rate of 200C/min and kept at each temperature for 5 min. The injection inlet
64 temperature was set at 2900C and the total run time was of 33.50 min. The range of the mass spectral scanned was between m/z = 50 to 700.
3.9.3. Liquid chromatography-Mass spectrometry (LC-MS)
The purity test of compound 4 was done by LC-MS (thermos scientific, USA) connected to an auto-sampler. The mobile phase carrying the sample was in the ratio of one is to one (1:1, v/v) of water and dimethyl sulfoxide (DMSO). The hypersil ODS (250 x 4.6; 5 µm) column was used.
The ionization (positive mode) of the sample was done by using electrospray ionization (ESI) and the single quadrupole was used for mass analysis. Five microliter (5 µl) of the mixture was injected into the instrument generating two spectra, one showing the compound resolution (LC) and the other one showing the fragmentation pattern of the sample (MS).
65
Powdered plant material
Extraction with Ethanol
Ethanol Extract
Liquid-Liquid Extraction
Hexane Fraction Chloroform Fraction Butan-1-ol Fraction
Phytochemical analysis Antioxidant screening Antibacterial screening Bio-guided Isolation 1. column --Terpenoids -- Bio-autography -- Bio-autography 2. TLC -- Flavonoids -- MIC by micro-dilution -- DPPH scavenging 3. Prep.TLC -- Alkaloids -- MBC -- NMR -- Coumarins -- MBC/MIC Pure compounds -- GC-MS -- Tannins S E -- LC-MS -- Anthraquinones Biological -- Proteins activity test -- Saponins -- Glycosides -- Steroids -- Carbohydrates -- Reducing sugar Antioxidant screening Antibacterial screening
-- MIC by micro-dilution -- DPPH scavenging -- MBC -- MBC/MIC
Figure 3.3: Sketch summarizing the entire methodology involved in the current research project (SE: Structure Elucidation).
66
CHAPTER 4
RESULTS AND DISCUSSIONS
4.1. Extraction and fractionation for qualitative and quantitative screening
The powdered aerial part of Asparagus suaveolens was extracted using ethanol solvent (95% absolute) which has an ability to extract both polar and nonpolar compounds. A mass of 500 g of powdered plant material was macerated into solvent for 24 hours, constantly shacked at 150 rpm.
Ethanol has extracted 34.567 g (w/w), equivalent of 6.913%. This extraction was followed by liquid-liquid fractionation of the ethanolic extract using n-hexane, chloroform and butan-1-ol, which provided the following results:
14 12.526 g (w/w)
12 Mass
10 %
8.678 g (w/w) 8
6 4.856 g (w/w) 4
Mass and % yield % and Mass 2.505 % 1.735 % 2 0.971 %
0 n-hexane Chloroform Butan-1-ol Solvent Fractions
Figure 4.1: Graph showing percentage yield and mass recovered when the ethanolic extract from aerial part of Asparagus suaveolens was fractionated using n-hexane, chloroform and butan-1-ol.
67
The results from liquid-liquid fractionation indicated that, n-hexane extracted the highest amount
(12.526 g w/w) from the ethanolic extract followed by butan-1-ol (8.678 g w/w) and chloroform
(4.856 g w/w). The results thus suggest that the aerial part of Asparagus suaveolens contained more of nonpolar compounds followed by polar compounds and moderate compounds which were extracted by chloroform.
4.1.1. Phytochemical analysis of solvent fractions
The qualitative phytochemical analysis gives the estimated information on the presence or absence of different classes of secondary metabolites in each solvent fraction (n-hexane, chloroform and butan-1-ol). These fractions were subjected to a range of phytochemical tests, and the results are given in Table 4.1
Alkaloids, terpenoids and steroids are moderately present in n-hexane fraction whereas, glycosides and proteins were at lower concentration. All other tested metabolites were found to be absent in n-hexane fraction. Chloroform fraction indicated the low presence of alkaloids, flavonoids, glycosides, anthraquinones, terpenoids and reducing sugar, whereas other tested metabolites classes were found to be absent. The presence of flavonoids, glycosides and reducing sugar appear to increase from n-hexane to butan-1-ol. Proteins and saponins showed a strong presence in butan-1-ol fraction. Flavonoids, glycosides and reducing sugar where moderately present in butan-1-ol fraction. This justified the polar nature of butan-1-ol fraction.
68
Table 4.1: Qualitative phytochemical analysis of n-hexane, chloroform and butan-1-ol fractions from aerial part of Asparagus suaveolens.
Solvent fractions Phytochemical test ______n-Hexane Chloroform Butan-1-ol
Alkaloids ++ + -
Tannins - - +
Saponins - - +++
Flavonoids - + ++
Glycosides + + ++
Anthraquinones - + -
Terpenoids ++ + -
Steroids ++ + +
Carbohydrates - - -
Reducing sugar - + ++
Proteins + - +++
Coumarins - - +
+++ = strong presence; ++ = moderate presence; + = low presence; - = negative
In addition, coumarins and tannins were present at lower quantity while the remaining metabolites were negatively tested in butan-1-ol fraction. Carbohydrates are reported to be absent in all three fractions. The presence of alkaloids in n-hexane might increase its antioxidant activity if alkaloids involved contain allylic or phenolic hydrogens ready to stabilize free radicals
(Cassels et al., 1995). Alkaloids also have been reported to inhibit growth of more Gram-positive than Gram-negative due to extra shell that limits the penetration of the alkaloids into the cell
69 membrane of bacteria (Karou et al., 2005). This is justified by the presence of the extra outer phospholipidic membrane that makes the cell wall of Gram-negative impermeable. The presence of phenolic derivatives such as flavonoids and coumarins are known for their antioxidant properties (Mirunalini and Krishnaveni, 2011; Kumar and Pandey, 2013).
According to previous studies on Asparagus species, Karmarkar et al (2012) reported the absence of steroids but presence of flavonoids, saponins, tannins and steroids in ethanolic (80%) extract of the whole aerial part of Asparagus racemosus plant. In the same year, Ravinshankar et al (2012) have reported the presence of steroids and flavonoids in Asparagus racemosus roots when extracted with ethanol (95%). One year later, Jayashree et al (2013) reported a negative result of steroids whereas the positive results of flavonoids, glycosides and terpenoids when
Asparagus racemosus roots were extracted with methanol. Ntsoelinyane and Mashele (2014) reported the presence of steroids, flavonoids, reducing sugar and terpenoids in aqueous extracts of Asparagus laricinus leaves while its stems showed negative results.
4.1.2. Antioxidant screening of n-hexane, chloroform and butan-1-ol fractions
4.1.2.1. Bio-autography antioxidant assay of n-hexane, chloroform and butan-1-ol fractions
Bio-autography antioxidant assay is a quick method to detect antioxidant activity of an extract or a compound after being spotted onto a TLC, developed into different mobile phase such as ethyl acetate, methanol and water (EMW, v/v/v); benzene, ethanol and ammonia (BEA, v/v/v); and chloroform, ethanol, formic acid (CEF, v/v/v). Thereafter, the dried TLC plates were sprayed
70 with 0.2% solution of DPPH free radical. The reduction of DPPH in presence of an antioxidant agent changes its colour from purple-red to yellow (Figure 4.2).
BEA
CEF
EMW
Ethanol extract n-Hexane Chloroform Butan-1-ol
Figure 4.2: TLC plates on which 100 µl of fraction solution (125 mg/ml) were spotted, developed into BEA, CEF and EMW solvent systems, dried before being sprayed with DPPH free radical solution (0.2%) showing yellow spots which confirming the presence of antioxidant activity into n-hexane, chloroform and butan-1-ol fraction extracted from aerial part of Asparagus suaveolens.
71
Observing the TLC plates, yellow spots in the middle of purple-red colour indicate the presence of antioxidant activity, which explains the reduction of DPPH free radical. From n-hexane fraction until the butan-1-ol fraction, yellow spots are visible, allowing us to conclude that all fractions contain antioxidant compounds. However, the difference lies in the intensity and the number of yellow spots between the fractions. On one hand, there are fractions that contain many yellow spots and on the other hand, there are the fractions that contain intense yellow colour. The assumption is that the antioxidant compounds in these fractions might depend on the number and the intensity of visible yellow spots on TLC plates after being sprayed with a methanolic solution of DPPH. Therefore, if there are more yellow spots on the TLC it means that there is more antioxidant compounds in the fraction and if there is intense spot it also means that the antioxidant compounds present were more reduced by DPPH.
n-Hexane is a nonpolar solvent used, in most cases, in the extraction of less polar compounds from a mixture. Looking at the results of the TLC plates in Figure 4.2, the n-hexane fraction shows the presence of yellow color, but less dense than in other fractions. These might give some information on its antioxidant character. Given the assumption made in the previous paragraph, the n-hexane fraction would contain less compounds exhibiting antioxidant activity. Therefore, we can conclude that the ethanolic extracts of the aerial part of Asparagus suaveolens contain less non-polar compounds possessing an antioxidant activity.
Chloroform and butan-1-ol fractions appear to have more compounds with antioxidant activity.
This can be observed by an increase of the number of yellow spots on TLC plates as shown in
Figure 4.2. Also, the intensity of yellow colour of chloroform and butan-1ol fractions increases
72 when compared to that of n-hexane. The weak antioxidant character of n-hexane fraction as well as the strong antioxidant character of chloroform and butan-1-ol fraction of ethanolic extracts of aerial parts of Asparagus suaveolens might be explained by the presence of derivatives of essential oils (for n-hexane), phenolics and polyphenolics (for chloroform and butan-1-ol) as reported by Ajikumar et al. (2008) and Ignat et al.(2011). Therefore, the antioxidant activity from n-hexane fraction might be originated from derivatives of essential oils. The activity from chloroform and butan-1-ol, might be due to the presence of phenolic (flavonoids, coumarins and others) and polyphenolic (tannins and others) derivatives.
4.1.2.2. Quantitative DPPH free radical scavenging assay
The antioxidant activity of the fractions (n-hexane, chloroform and butan-1-ol) was determined by their capacity of decolourising the purple-red colour of DPPH to yellow. The mixture of fraction solutions and DPPH was incubated in the dark for 30 minutes before the absorbance at
517 nm was measured using a spectrophotometer. Ascorbic acid and BHT methanolic solutions were used as standard and methanolic solution of DPPH free radical as control. Results of this assay are displayed in the following graph.
73
120 n-Hexane
100 Chloroform Butan-1-ol 80
Asc. Acid (Std) 60 BHT (Std)
at 517 nm 517 at 40
20
DPPH activity scavegenign dadical DPPH 0 0 0.5 1 1.5 2 2.5 Concentration of fractions mg/ml
Figure 4.3: DPPH free radical scavenging of n-hexane, chloroform and butan-1-ol fractions. Fraction solution was mixed with a DPPH solution then incubated in the dark for about 30 minutes to 1 hour before the absorbance can be measured using spectrophotometer at 517 nm
Among all fractions, chloroform fraction inhibited more DPPH free radical with the highest percentage of 71.409% at 2.5 mg/ml of concentration, followed by butan-1-ol fraction with
69.400% and the last by n-hexane fraction with 35.413%. This indicates that, chloroform fraction contains compounds with higher antioxidant activity, allowing them to donate hydrogen to
DPPH free radical than compounds from butan-1-ol and compounds from n-hexane. The synergetic effect among compounds with antioxidant activity from chloroform fraction might be at the origin of the higher antioxidant activity observed.
The half-maximal inhibitory concentration (IC50) is the concentration that gives the 50% of inhibition. The IC50 of all fractions were determined. The IC50 of n-hexane was higher than the highest used concentration (>2.5 mg/ml). For chloroform fraction, the IC50 = 0.37 mg/ml. The
74 butan-1-ol fraction exhibited an IC50 value of 0.42 mg/ml. The lower the IC50, the stronger the antioxidant activity. Hence, chloroform appears to show higher antioxidant activity followed by butan-1-ol and n-hexane fraction. The standards show strong antioxidant activity with IC50 of
0.25 mg/ml and 0.27 mg/ml for BHT and Ascorbic acid respectively in respect to all fractions.
But their IC50 values are very closer to each other.
These results confirm the assumption made on the proportionality of the intensity of yellow colour and the antioxidant activity of n-hexane fraction compared with that of chloroform in
Section 4.1.2.1. Classes, number of secondary metabolites and the position of hydroxyl group on these secondary metabolites might be at the origin of these differences. Although chloroform fraction indicated the higher percentage of inhibition among all fractions, standards used
(Ascorbic acid and BHT) have shown the highest percentage DPPH scavenging than all fractions. BHT showed higher antioxidant capacity with the inhibition of 99.872% at a concentration of 2.5 mg/ml than ascorbic acid with 99.426%.
4.1.3. Antibacterial activity
4.1.3.1. Antibacterial bio-autography assay
After incubation of 20 to 24 hours, no fractions (n-hexane, chloroform and butan-1-ol) showed clear visible inhibition spots for all four general bacteria used for bio-autography antibacterial screening (Figure 4.4). These results might be explained by many factors such as: the aerial parts of Asparagus suaveolens do not possess compounds with antibacterial activity
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E. coli S. Aureus A B
P. aeruginosa E. faecalis C D
Ethanol n-Hexane Chloroform Butan-1-ol Ethanol n-Hexane Chloroform Butan-1-ol
Figure 4.4: TLC plates developed into EMW solvent system then sprayed with actively growing bacterial strains suspension, than sprayed with p- iodonitrotetrazolium chloride (INT) showing antibacterial results. The presence of purple-reddish colour on the TLC explained that the microorganisms are able to reduce INT.
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The other reason could be that, the concentration of fraction used was not sufficient to inhibit the bacterial growth. Teka et al. (2015) reported negative results when leaves material (512 µg/ml) of Asparagus africanus were tested againt S. aureus (ATCC 29213) and E. coli (ATCC 25922) using the broth microdilution method in 96-well microplates.
When Ntsoelinyane and Mashele (2014) tested stems and leaves of Asparagus raricinus against
S. aureus and others, they observed no bacterial inhibition for stem parts but a significant inhibition was observed for leaves when using agar dilution method. It was also reported that, the ethanolic extract of stem bark and leaves of Asparagus flagellaris failed to inhibit the growth of
P. aeruginosa and S. aureus, while only inhibiting growth of E. coli, at 400 mg/ml using disc diffusion method (Mshelia et al., 2008).
4.1.3.2. Minimum inhibitory concentration (MIC)
4.1.3.2.1. Minimum inhibitory concentration (MIC) of general bacteria
After analysing the results in Section 4.1.3.1 the plant fractions were subjected to quantitative analysis. The four common nosocomial bacteria were used for the quantitative antibacterial test using micro dilution method to determine the minimum inhibition concentration of plant fractions (n-hexane, chloroform and butan-1-ol). Results of these tests are displayed in Table
4.2.
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Considering S aureus, n-hexane shows a lower MIC value (15.625 mg/ml), followed by that of chloroform and butan-1-ol. E. coli appears to be more resistant to all fractions with a MIC of
62.500 mg/ml for chloroform and butan-1-ol, but with a MIC higher than that for n-hexane.
E. faecalis showed the lowest MIC of 6.510 mg/ml for all fractions. This showed that in average, n-hexane presented the higher antibacterial activity compare to the two other fractions. This antibacterial character of n-hexane might be due to potential presence of essential oils in it. P. aeruginosa showed a lower MIC of 62.5 with n-hexane and higher MIC of 15.625 against chloroform.
Table 4.2: MIC results of different fractions (n-hexane, chloroform and butan-1-ol) against selected general bacteria
Fractions (mg/ml) Microorganisms______n-Hexane Chloroform Butan-1-ol Gentamicin Amoxicillin
S. aureus 15.625 31.250 31.250 1.228 1.658
E. coli >62.500 62.500 62.500 1.453 2.104
E. faecalis 6.510 6.510 6.510 2.012 1.597
P. aeruginosa 62.500 15.625 31.250 0.934 1.681
> = The MIC is higher than that in well No. 1 containing a concentration of 62.5 mg/ml
In comparison with the previous studies on the same species, Madikizela and colleagues (2013) reported the MIC of Asparagus africanus and Asparagus falcatus leaves against S. aureus were in the range of 1.56 to 12.50 mg/ml when extracted with petrol ether, dichroromethane, 80% of ethanol and water. Furthermore, there was no inhibition when Asparagus africanus leaves extracts (512 μg/ml or lower) were to be subjected to antibacterial test using E. coli, P. aeruginosa, and S. aureus (Teka. et al., 2015). Whereas, when ethanolic extract of Asparagus
78 flagellaris was screened for its antibacterial activity, the inhibition occurs from 400 mg/ml and higher for E. coli. For S. aureus and P aeruginosa, no visible inhibition was observed at the concentration up to 1000 mg/ml (Mshelia at al., 2008). Despite the resistance of all four general microorganisms in respect to solvent fractions, the two used standards (gentamicin and amoxicillin) demonstrated a higher inhibition with values ranging from 0.934 to 2.104 mg/ml.
4.1.3.2.2. Minimum inhibitory concentration (MIC) of fractions against specific bacteria (Neisseria gonorrhoea)
After observing resistance of all four general bacteria in respect to solvent fractions, these solvent fractions (n-hexane, chloroform and butan-1-ol) were thereafter tested for their in vitro antibacterial activity against a number of specific bacterial strains for which the Asparagus suaveolens extracts have been used for by traditional healers (Gololo, undocumented source).
Eight Neisseria gonorrhoea strains used by WHO (2008) as reference strain for global quality assurance and quality control of gonococcal antimicrobial resistance testing identified as F, G, K,
L, M, N, O and P (Unemo et al., 2008). National Health Laboratory Service (NHLS), division of the National Institute for Communicable Diseases (NICD) Edenvale, Gauteng (South Africa), donated these N. gonorrhoea strains.
The microdilution method was used to evaluate the efficiency of solvent fractions against these clinical isolates N. gonorrhoea through their MIC values. Gentamicin and Amoxicillin were again used as positive control (standards). The results are displayed in Figure 4.5.
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From all three solvent fractions, n-hexane showed lower MIC values (great antibacterial activity) against all seven N. gonorrhoea strains except for bacteria M where butan-1-ol fraction showed higher activity followed by n-hexane fraction. After the higher activity of n-hexane, chloroform came at second position except for strains M and N where chloroform has shown lower activities against both bacteria. Butan-1-ol fraction showed lower activity against all bacteria except for M where its activity is higher compared to those of n-hexane and chloroform. Comparing to the standards, the general perception is that Amoxicillin appears to have a lower MIC than
Gentamicin with exception of G, K, and P where it was found that Amoxicillin has higher MIC.
35 n-hexane 30 chloroform Butan-1-ol 25 Gentamicin (STD) Amoxicillin (STD) 20
15
MIC mg/ml MIC 10
5
0 F G K L M N O P N. gonorrhoea strains
Figure 4.5: MIC of eight clinical isolate, WHO (2008) N. gonorrhoea tested against three solvent fractions and two standards. Microtiter plate containing 100 µl of fraction, 100 µl of Moller Hinton Broth and 40 µl of 24 hours old bacterial suspensions were mixed then incubated for 18 to 24 hours in presence of 5 to 10% of CO2. A volume of 40 µl of INT was added to each well then incubated for 30 minutes to 1 hour in the similar conditions.
n-Hexane fraction recorded a stronger inhibition than that of Amoxicillin against F, O and P N. gonorrhoea. Also, the same n-hexane fraction appears to be more effective than standards
80 gentamicin against F, G, O, and P N. gonorrhoea. Chloroform and butan-1-ol presented lower activity (high MIC values) against all N. gonorrhoea strains. According to results reported by
Mshelia et al (2008) indicated that, when ethanolic extract of the aerial parts of Asparagus flagellaris was subjected to antibacterial screening, using N. gonorrhoea strains, the visible inhibition occurs from the concentration of 200 mg/ml. Hence, ethanolic extract from Asparagus suaveolens seems to be more effective than ethanolic extract from Asparagus flagellaris.
4.1.3.3. Minimum bactericidal concentration (MBC) of solvent fraction against specific bacteria (Neisseria gonorrhoea)
The minimum bactericidal concentration (MBC) was used to determine how effective the solvent fractions are able to kill N. gonorrhoea strains. After the incubation of microtitre plate containing the mixture of solvent fraction and N. gonorrhoea strains, 40 µl of it was spotted and sub- cultured into agar disks containing nutrient then incubated further at the same conditions as stated in Section 3.7.2.2. The highest dilution that gave no visible bacterial growth was considered as the MBC.
Observing the results in Figure 4.6, n-hexane fraction appears to present a strong MBC activity against all N. gonorrhoea strains (F, G, K, L, M, N, O and P) in comparison with all other fractions (chloroform and butan-1-ol). This might be due to the presence of essential oils, especially terpenoids in the fraction as demonstrated by the phytochemical analysis results in
Table 4.1. Terpenoids are able to interact with lipids from cell membrane and mitochondria of bacteria, which might lead to the disturbance of the structure of membrane and metochondria; generating the death of the bacteria (Burt, 2004). In addition, the bacterial cytoplasmic
81 membrane was also reported of being disturbed by the presence of terpenoids containing hydroxyl group (Ultee et al., 2002). Hence, the disturbance of cell membrane, cytoplasmic membrane as well as that of mitochondria of bacteria by the potential presence of terpenoids among other metabolites in n-hexane fraction might be at the origin of higher MBC activity observed. However, chloroform showed a strong activity against F, L, O and P N. gonorrhoea whereas butan-1-ol fraction showed strong activity against G, K, M and N N. gonorrhoea strains.
In all cases, standards used still showing higher activity compare to all fractions tested.
35 n-hexane chloroform 30 Butan-1-ol Gentamicin (STD) 25
Amoxicilin (STD)
20
15 MBC mg/mlMBC
10
5
0 F G K L M N O P N. gonorrhoea strains
Figure 4.6: Minimum bactericidal concentration (MBC) results of eight clinical isolate (WHO 2008 N. gonorrhoea) tested against solvent fractions (n-hexane, chloroform and butan-1-ol) then, subcultured into New York City Agar. Thereafter, incubated for 18 to 24 hours in presence of 5 to 10% of CO2, the highest dilution that gave the visible inhibition was considered as the MBC value.
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4.1.3.4. Ratio MBC/MIC of solvent fraction
The extraction of the ratio between MBC and MIC aimed to see which of the used solvent fractions showed bacteriostatic or bactericidal character. The results were extracted from Figure
4.5 and Figure 4.6.
When observing the Figure 4.7, it appears that n-hexane fraction showed a huge difference between the MIC and the MBC against the following N. gonorrhoea F, G, O and P, with values ranged between 2.059 to 36.790. Hence, n-hexane was considered as bacteriostatic against F, G,
O and P N. gonorrhoea. However, for the remaining strains, n-hexane fraction was considered as bactericidal due to the low MBC/MIC ratio.
n-Hexane 25.0 Chloroform Butan-1-ol Gentamicin (STD)
Amoxicillin (STD)
5.0 MBC/MIC
1.0 F G K L M N O P N. gonorrhoea strains
Figure 4.7: Results showing MBC/MIC ratio when solvent fractions (n-hexane, chloroform and butan-1-ol) where subjected to 8 WHO (2008), N. gonorrhoea using the micro-dilution method.
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French (2006) reported that an agent is usually considered as bactericidal if the MCI value is no more than four times the MIC this means, if MBC/MIC ratio is higher than four, the agent is considered as bacteriostatic). The killing of N. gonorrhoea by n-hexane fraction depends on the concentration of solvent fraction, especially that of n-hexane fraction. Chloroform fraction also showed significant differences between the MIC and the MBC values against F, L, M, and O N. gonorrhoea. Therefore, chloroform fraction might be considered as bacteriostatic to those N. gonorrhoea strains but bactericidal to G, K, N and P strains. For the case of butan-1-ol fraction, it can be considered as bacteriostatic against G, K, M and O strains, but bactericidal against F, L,
N and P strains.
The strong MIC and MBC values of n-hexane fraction might be due to the presence of essential oils (terpenoids) as well as that of alkaloids found present when discussing the phytochemical analysis in Section 4.1.1. The activity can be explained by the lipophilic character of alkaloid molecules which might facilitate the permeability of the membranes of the bacteria and thereby inhibit their growth (Salih et al., 2011; Li et al., 2014). Alternatively, the presence of methylenedioxy group on the phenolic part of an alkaloid might also increase its antimicrobial activity (Gu et al., 2014). Assuming that the activity is due to the presence of terpenoids; being the major component of essential oils, these metabolites might interact with lipids from cell membrane and mitochondria of bacteria because of their hydrophobic properties and that can lead to the disturbance of the structure of membrane and of mitochondria, resulting to the death of the bacteria (Burt, 2004).
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4.2. Isolation of active compounds from ethanolic fractions
Bio-guided isolation method was used to isolate antioxidant and antibacterial compounds from thet fractions of ethanolic aerial part extract of Asparagus suaveolens by using column chromatography. Each fraction (n-hexane, chloroform and butan-1-ol) was treated separately in order to isolate compounds with antibacterial and/or antioxidant activity.
4.2.1. n-Hexane fraction
After the packing of a column chromatographic (19 mm x 61cm) by using 70 g of silica gel 60,
2.3 g of n-hexane fraction were loaded in it. Using the solvent system of n-hexane and dichloromethane in the proportion of (9:1, v/v), two pure compounds were obtained. Identified as compound 1 and compound 2.
4.2.1.1. Compound 1
The column was eluted using n-hexane and dichloromethane (9:1, v/v) as mobile phase, leading to the collection of small fractions in different test tubes. From fraction 47 to fraction 55, led to the isolation of a pure compound identified as compound 1. After evaporation of solvent system, a mass of 1.5 g of pure compound 1 was collected and appeared as white amorphous powder.
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4.2.1.1.1. Structure elucidation of compound 1
The proposed structure of compound 1 was achieved by using some spectroscopic analysis methods such as NMR, FT-IR and GC-MS spectroscopy. The purity test and the molecular mass of compound 1 was achieved using GC-MS instrument. The result of GC experiment (compound
1 dissolved in chloroform) showed the presence of one signal with a retention time of 26.497 minutes. This indicates that, sample was pure enough to be taken to NMR for further analysis.
The expended mass spectrum (MS) of compound 1 showed a molecular ion signal at m/z
450.480 as presented in Figure 4.8. The chemical shifts of compound 1 are shown in Table 4.3
(1HNMR) and Table 4.4 (13CNMR). The NMR, COSY and TOCSY spectra of compound 1 are shown in Annexure 1.
Figure 4.8: GC-MS results of compound 1 isolated from n-hexane fraction using chloroform as blank.
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1 Table 4.3: H NMR (400 MHz) chemical shifts data of compound 1 dissolved in Chloroform- d3 (CDCl3).
Position of H Chemical shift in ppm Multiplicity (J in Hz) No. of H
1 and 31 0.86 Triplet (6.6) 2x3H
2-13 and 19-30 1.24 Multiplets 2x24H
14. 1.51 Doublet (6.6) 2x2H
15. 2.36 Doublet of doublet, dd (7.2, 7.8) 2x2H
16. - - -
13 Table 4.4: C NMR (100 MHz) chemical shifts data of compound 1 dissolved in Chloroform-d3 (CDCl3).
No. of carbon 13C chemical shift (ppm) No. of C Nature of the carbon
1 and 31 14.3 2 CH3
2 and 30 22.7 2 CH2
3 and 29 31.9 2 CH2
4 to 13 and 19 to 28 29.5 to 29.9 20 CH2
14 and 18 23.9 2 CH2
15 and 17 42.8 2 CH2
16 212.0 1 C=O
1 The H NMR (Annexure 1A) indicated the presence of methyl hydrogens (CH3-) signals at around 0.86 ppm and appears as a triplet with a coupling constants of 6.6 Hz. This peak is believed to be CH3- in the molecule. The signals at 1.24 ppm can be considered as multiplets, which account for 2x24Hs. These protons are supposed to be in a chain molecule. A signal at
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1.51 ppm is observed as doublet (d, J 6.6 Hz) and it is accounted for β-methylene hydrogens
(2x2Hs) to the carbonyl group within the molecule. The α-methylene groups signal (2x2Hs) are observed at 2.36 ppm as doublet of doublets (dd, J 7.2 &7.4 Hz).
13C NMR (Annexure 1B) shows an overall of 31 carbons in which one signal is at around 212.0 ppm and the other at around 14.3 ppm (2xCH3) and that is a characteristic of both carbonyl and methyl carbons respectively. The 212.0 ppm is a characteristic of ketone carbon; this suggests that in the unknown molecule there is a ketone functional group.
Looking at the two dimensional (2D) analysis (COSY) (Annexure 1C), there is correlation between protons present in this suggested molecule. The peak observed at 0.87 ppm on COSY couples with the peak at 1.24 ppm and the latter couples with a peak at 1.51 ppm and last, the peak at 1.51 ppm couples with the peak at 2.36 ppm. This confirmed that, carbons on the molecule are linear or a chain molecule.
When analysing the total correlated spectroscopy (TOSCY), its shows a long range coupling (3 to 4 bonds apart) between protons. There is a coupling correlation between H-15a,b and H-14a,b; also with H-13a,b (Annexure 1D).
There is an opinion that the unknown compound could be a symmetrical molecule. If the symmetrical molecule is considered, the mass obtained corresponds to the structure discussed above. Therefore, the suggested structure could be as depicted below.
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Ha Hb 15 14 aH Hb
Figure 4.9: Proposed structure of compound 1 (stereochemistry of the structure was ignored).
From all these data, it is confirmed that the unknown molecule could be a 31-carbon molecule in which the central carbon (C-16) is a carbonyl carbon with the molecular formula of C31H62O.
Mass spectroscopy (Figure 4.8) of compound 1 showed a molecular ion signal at m/z 450.480, which does correspond with the structure described above. Mass spectrum of compound 1 shows a number of signals corresponding to fragmentations of the molecule. Five major fragments were observed from the mass spectrum including m/z 43, m/z 57, m/z 71, m/z 239 (considered as base peak) and m/z 255. The three first fragments are often observed when the fragmentation involves an aliphatic ketone (Silverstein et al., 2014). The base peak (m/z 239) was observed after the cleavage of the C–C bond adjacent to the oxygen atom. The last signal (m/z 255) was the result of McLafferty's rearrangement on Cα–Cβ bond to carbonyl group. All these fragmentations are illustrated in the Figure 4.10 below.
Fragmentation from hydrocarbon chain m/z 57 + O C4H9
m/z 43 + m/z 71 C3H7 C H + 5 11
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Fragmentation leading to the base peak
O
O O
+ m/z 239, C16H31O , Base peak
McLafferty’s rearrangement H O
OH
OH
OH
+ m/z 255, C17H35O
Figure 4.10: GC-MS observed fragmentation of compound 1
According to the literature, compound 1 is identified by the International Union of Pure and
Applied Chemistry (IUPAC) as “16-hentriacontanone” and the common name of “palmitone”.
This compound was isolated for the first time from Neolitsea sericea leaves by Komae and
Hayashi (1971) and from leaves of Annona diversifolia by Ganzález-Trujano and colleagues
(2001). Palmitone (common name of hentriacontan-16-one) was considered as the first aliphatic ketone with anticonvulsant properties, which could be considered in the future as potential antiepileptic drug from natural source (Gonzalez-Trujano et al., 2001). The presence of palmitone in the aerial part of Asparagus suaveolens might be a contributing factor for usage of the species as an antiepileptic by traditional healers; usage confirmed by Jäger et al (2005).
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However, the literature in our possession shows that, palmitone (compound 1) was never reported of being isolated from Asparagus species in the world. Hence, this research project provides the first information on the isolation of palmitone (compound 1) from a native South
African Asparagus genus in particular and from the world in general. 1H and 13C NMR chemical shift of palmitone isolated from Annona diversifolia and this current work are displayed in the
Table 4.5 below.
Table 4.5: Chemical shifts of Palmitone according to Gonzalez-Trujano et al (2001) and this 1 13 current work both using CDCl3 as solvent for H (300MHz) and C (75 MHz) NMR, and its MS fragmentation patterns
1H shift(multiplicity) 13C shift,ppm(no.of carbon) m/z (rel. int)
1H G-T CW 13C G-T CW G-T / CW
+ H1 0.88(t,7.2) 0.86(3H, t, 6.6) C1 14.1(1xCH3) 14.3(1xCH3) M = 450(6)/450,
281(7)/281, H2-H13 1.25(br.) 1.24(12H,m) C2 22.7(1xCH2) 22.7(1xCH2) 239(100)/239,
C3 31.9(1xCH2) 31.9(1xCH2) 255(24)/255,
221(4)/221, C4-C13 29.3-29.7 29.5-29.9 183(7)/183, (10xCH2) (10xCH2) 156(5)/156,
125(9)/125, H14 1.55(m) 1.51(1H,d, 6.6) C14 23.9(1xCH2) 23.9(1xCH2) 96(14)/96,
H15 2.38(t,7.4) 2.36(1H, dd, C15 42.8(1xCH2) 42.8(1xCH2) 81(17)/81,
7.27&7.8) 57(9)/57
C16 211.7(1xC=O) 212.0(1xC=O)
G-T = Gonzalez-Trujano et al (2001) ; CW = Curent Work
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4.2.1.2 Compound 2
Compound 2 was isolated from n-hexane fraction in continuation of the column used to isolate compound 1 without changing the mobile phase (n-hexane and dichloromethane) (9:1, v/v).
Small fractions were collected in test tubes 83 to 110. After evaporation of the solvent system, a mass of 300 mg of pure compound, which physically appeared as cream white powder and named compound 2 was collected.
4.2.1.2.1 Structure elucidation of compound 2
NMR and GC-MS instrumental analysis were used to elucidate the structure of compound 2. The
NMR spectra (Annexures 2A, 2B, 2C and 2D) and chemical shifts (Table 4.6 and Table 4.7) provided information on number and position of proton and carbon atoms. Whereas the GC-MS
(Figure 4.11) provided the degree of purity, total mass as well as mass of different fragments of the molecule after evaporation.
When observing the GC-MS spectrum of compound 2, three peaks appeared with different retention time of 21.982, 24.253 and 26.805 minutes. Initially, it was thought that these results indicating a mixture of three different compounds. However, referring to the TLC plate fingerprint of compound 2, only one spot was observed. If the three different peaks represent three different compounds, their molecular mass could not be the same.
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A B
C D
Figure 4.11: GC-MS spectra of compound 2, isolated from n-hexane fraction using chloroform as blank showing three different retention times in A but with one fragmentation pattern shown in B, C and D.
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According to mass spectrum (MS) as displayed in Figure 4.11, all three eluted compounds possess a unique molecular ion signal of m/z 396. This might only be possible if all three compounds are isomers. The results reported by Abdel-Hay and colleagues in 2015 showed that the analysis by GC-MS of positional and constitutional isomers present in sample mixture might have different retention time. Furthermore, it was reported that capillary column might be used to separate different isomers present in the sample (Landowne and Lipsky, 1961). In this case, capillary column InertCap 5MS/SiL (5% Phenyl and 95% methylpolysilarylene) was used. This might confirm the hypothesis of which compound 2 consists of three isomers. Due to limited instrumental expertise to identify these three isomers, only the structure of isomer matching with the NMR spectra was elucidated.
The chemical shifts of 1H NMR (300 MHZ) and 13C NMR (75 MHz) of compound 2 are displayed in Table 4.6 and Table 4.7 in the following page.
Table 4.6: Chemical shift of 1H NMR (300 MHz) data of compound 2 dissolved in Chloroform- d3 (CDCl3)
Position of H Chemical shift in ppm Multiplicity (J in Hz) No. of H
1 3.64 Triplet (6.6) 2H 2.03 Singlet 1H from –OH 2 1.68 Mutltiplet 2H 3 to 25 1.25 Broad singlet 46H 26 1.32 Multiplet 2H 27 0.88 Triplet (6.6) 3H
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When analysing the 1H NMR (300MHz) spectrum (Annexure 2A) of compound 2, a triplet peak is observed from 3.64 ppm, which gives a coupling constant of 6.6 Hz. This proton is commonly observed at this chemical shift when bonded to carbon attached to a withdrawing (more electronegative) atom or group of atoms (C-O, C-N, C-X). A small peak appeared at around 2.03 ppm, characteristic of proton bonded to oxygen atom (-OH) a hydroxyl proton. From 1.56 to
1.68 ppm, there is an appearance of multiple peaks, which are characteristic of protons localised near the carbon containing the withdrawing group and proton on the carbon attached to methyl group. A broad singlet, huge peak appears at 1.25 ppm and these protons appear to be -CH2- in straight chain. Finally, another triplet occurred from 0.87 ppm with a coupling constant of 6.6
Hz. These protons are characteristic of methyl group.
13 Table 4.7: Chemical shift of C NMR (75 MHz) of compound 2 dissolved in Chloroform-d3 (CDCl3)
Position of C 13C chemical shift (ppm) No. of C Nature of the carbon
1 63.1 1 CH2-OH
2 32.8 2 CH2
25 31.9 2 CH2
4 – 24 29.3-29.7 19 CH2
3. 25.7 1 CH2
26. 22.6 1 CH2
27. 14.1 1 CH3
13C NMR (75 MHz) spectrum (Annexure 2B) of compound 2 showed a peak at 63.1 ppm which confirms the presence of the withdrawing group specially (C-OH) in the structure of compound
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2. At 32.8 ppm, a peak occurs which is characteristic of carbon atom at positon β from the carbon containing the withdrawing group. At 31.9 ppm, a single peak appears which might be allocated to the carbon atom at position γ from the methyl group. The carbon atoms in –CH2- in the straight chain appear from 29.7 to 29.3 ppm. The carbon at 25.7 ppm might be a –CH2- at position γ from the carbon attached to the withdrawing group and the other –CH2-, which appears 22.6 ppm appears to be that at position β from the methyl group. Finally, the peak at 14.1 ppm is characteristic of carbon from a methyl group.
Among the two-dimension of NMR of compound 2, only COSY (Annexure 2C) and HSQC
(Annexure 2D) were performed. From COSY NMR (300 MHz) of compound 2, there is a clear correlation between protons from the methyl group and those from the –CH2- in the straight chain, which confirming the aliphatic character of compound 2. Also from the HSQC data of the same compound 2, correlation could be made between carbons and hydrogens. The carbon at
14.1 ppm correlates with the protons at 0.88 ppm, which confirms the existence of a methyl group in the structure. In addition, the carbon at 63.1 ppm seems to correlate with protons at 3.64 ppm, which confirms also that the carbon carrying the withdraing group contains protons. With the help of the integration from 1H, 13C NMR (300MHz) and the GC-MS results, the proposed structure for compound 2 might contains 27 atoms of carbon, 56 protons and 1 atom of oxygen
(C27H56O). Compound 2 seems to be a primary alcohol.
OH 1
27
Figure 4.12: Proposed structure of compound 2 (stereochemistry of the structure was ignored).
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According to the IUPAC nomenclature, the name of compound 2 is n-heptacosanol (heptacosan-
1-ol). Chakraborty and colleagues (2012) have reported about the isolation of heptacosan-1-ol
from Acanthospermum hispidum plant. Because of the inaccessibility to their publication, the
chemical shift of proposed compound 2 were compared to those of octacosanol, primary linear
alcohol with 28 carbons isolated from Citrus jambahiri lush by Hamdan and El-shazly (2014).
The spectra were obtained with 300 MHz and 75 MHZ NMR; the results of their findings are
displayed in Table 4.8.
Table 4.8: Chemical shifts of octacosanol according to Hamdan and El-shazly (2014) and that of 1 n-heptacosanol reported in the current work both using CDCl3 as solvent for H (300MHz) and 13C (75 MHz) NMR, and its MS fragmentation patterns
1H shift (multiplicity) 13C shift,ppm (no.of carbon) m/z (rel. int)
1H H & E CW 13C H & E CW H & E / CW
+ H1 3.64(2H,t,6.6) 3.64(2H, t, 6.6) C1 63.1(CH2-OH) 63.1(CH2-OH) M =410(C28)/396 (C27), H2 1.56 (2H,m) 1.68(2H,m) C2 33.1(1xCH2) 32.8(1xCH2) 209/209, H3-H25 1.25(48H,br.s) 1.25(44H,br) C3 25.9(1xCH2) 25.7(1xCH2) 181/181, C4-C24 29.8-29.9 29.3-29.7 153/153, (23xCH2) (22xCH2) 125/125,
111/111, C25 32.21xCH2) 31.9(1xCH2) 97/97, H26 1.32 (2H,m) 1.32 (2H, m) C26 22.9 (1xCH2) 22.6 (1xCH2) 83/83,
H27 0.88(3H,t,6.0) 0.88 (3H,t,6.6) C27 14.3 (1xCH3) 14.1 (1xCH3) 57/57
H & E = Hamdan and El-shazly (2014); C.W = Curent work
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All aliphatic alcohols (from C20 to C36), including n-Heptacosanol, belongs to the category of policosanol compounds (Aicha Olfa Cherif, 2012). These compounds have been found active against the arteriosclerotic vascular, coronary heart diseases, as well as dermatologic diseases. In
2013, Awaad and colleagues have reported that n-heptacosanol was one of contributors of antiulcerative colitis activity when ethanolic extracts of aerial part of Euphorbia granuleta
Forssk had been tested.
Beside the spectrum of gas chromatography of compound 2, in which three signals were observed, the mass spectrum of the same compound showed the similarity in their fragmentation for all three different peaks as it is displayed in Figure 4.11. These testify, once again, the degree of similarity between the three peaks as seen in gas chromatogram. The Figure 4.13 below shows the possible fragmentations of compound 2 as it is shown by mass spectra.
+ + M - C20H41O + 2H + + + M - C18H37O + 2H M - C23H47O
OH
M - C H O+ + 2H+ + 23 47 M - C26H53 + + M - C19H39O + 2H
Figure 4.13: Possible fragmentation patterns of compound 2 as showed by the MS spectrum in Figure 4.1.
Only the fragment m/z 31 seems to be possible between carbons 1 and 2. Unfortunately, this fragment could not be seen because of the adjustment of the MS machine, which would only show the signals displayed from m/z 50 until m/z 700. All visible fragments were from the
98 hydrocarbon part. Being a long chain aliphatic alcohol, its fragmentation seems to take place as that of homologue hydrocarbon.
4.2.2. Chloroform fraction
Due to closeness of retention factors (Rf) values between spots on TLC from the chloroform fraction, a long packed column (19 mm x 61 cm) was used in order to increase the separation between them. Eighty grams (80 g) of silica gel was packed into a column after being mixed with the solvent system n-hexane and ethyl acetate in the ratio (9:1, v/v). A mass of 2.7 g of the fraction was loaded into a column. The ratio of the solvent system was changed from (9:1, v/v);
(7:3, v/v) and then to (5:5, v/v). Compound 3 was isolated from chloroform fraction using n- hexane and ethyl acetate (5:5, v/v) in test tube 101 to test tube 111. After evaporation of solvent, compound 3 was collected as a grey powder. The recovered mass of compound 3 was about 0.5 mg. Due to this poor recovery, compound 3 was not subjected to further characterisation.
4.2.3. Butan-1-ol fraction
Due to the higher polarity of this fraction and because the targeted compounds was highly polar, a short column (19 mm x 61 cm) made up with 50 g of silica 60 was packed using the mixture of chloroform, ethyl acetate and methanol (6:3:1, v/v/v) as solvent system. A mass of 4.3 g of solvent fraction was loaded into a previously packed column. Two compounds were eluted from this fraction (compounds 4 and compound 5).
99
4.2.3.1. Compound 4
Compound 4 was collected using the solvent mixture of chloroform, ethyl acetate and methanol
(6:3:1, v/v/v) from fractions 67 to 87 as yellow precipitates after the dryness of the solvent. A mass of 1.7 g of compound 4 was recovered during this process.
4.2.3.1.1. Structure elucidation of compound 4
Proton and carbon NMR (400MHz) and (100 MHz) spectra (Annexures 3A, 3B and 3C) were used to elucidate the structure of compound 4, which was collected as yellow powder. The purity and the mass of compound 4 was done using LC-MS instrument. The LC spectrum result
(Figure 4.14) showed the presence of one peak that confirmed the higher purity of compound 4 with a retention time of 3.599 minutes. The MS spectrum result (Figure 4.15) showed the base peak at m/z 303.00 together with others fragments.
100
Figure 4.14: Liquid chromatography spectrum of compound 4 dissolved in methanol with a retention time of 3.472 minutes.
101
Figure 4.15: Mass spectrometry spectrum of compound 4, dissolved in methanol with the base peak at m/z 303.0.
102
1 Table 4.9: Chemical shift of H NMR (400 MHz) data of compound 4 dissolved in methanol-d4 (CD3OD)
Position Chemical shift Multicity (J in Hz) No. of H
2 7.70 Singlet 1H
8 6.24 Singlet 1H
3’ 6.47 Singlet 1H
5’ and 6’ 6.93 Doublet (8.1) 2H
6 - Glucose
1’ 4.53 Singlet 1H
2’ 3.80 Multiplet 1H
3’ 3.75 Multiplet 1H
4’ 3.20 Mutliplet 1H
5’ 4.10 Multiplet 1H
6’ 3.70 Multiplet 2H
4’ - Rhamnose
1’’ 5.27 Singlet 1H
2’’ 3.80 Multiplet 1H
3’’ 3.76 Multiplet 1H
4’’ 3.48 Multiplet 1H
5’’ 4.53 Multiplet 1H
6’’ 1.06 Doublet (2.1, 3.9) 3H
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When observing 1H NMR spectrum of compound 4 (Annexure 3A) and Table 4.10 depicting the chemical shifts characteristics of the peaks are shown. 1H NMR spectrum displayed peaks that can be divided into different regions including aromatic protons from region around 6 ppm to 8 ppm. Protons from aromatic hydroxyl group (-OH) can be visible at around 5 ppm. A group of peaks between 3 ppm and 4 ppm that is common to protons from sugar moiety (Roslund et al.,
2011). Protons from alcohols groups (sugar parts) (-OH) are usually observed around 2 ppm and finally at around 1.2 ppm, methyl protons are observed. Signals at 4.5 ppm and at 5.12 ppm justify the presence of anomeric protons in the structure of compound 4. This confirms the presence of sugar moiety within the structure. Note that the stereochemistry of the anomeric protons were not part of the assignment maybe it can be considered in the near future.
Protons from aromatic region are subdivided into 3 categories. Single protons are justified by singlets peaks, which appear at 7.70 ppm (C2), 6.24 ppm (C8) and 6.47 ppm (C3’). At 6.93 ppm, appears a doublet with a coupling constant of 8.1 Hz. Finally, the other category consists of protons bonded to oxygen atom (-OH). The second part of protons consists of those appearing in sugar moiety. They are subdivided into two categories. The first category consists of protons attached to sugar carbons. Their peaks appear in the range from 3.20 ppm to 4.20 ppm and they are accounted as multiplets. At 1.06 ppm, appears a peak, which might be counted as doublet of doublets (dd) with coupling constant of 2.1 Hz and 3.9 Hz. The double of doublets might be the
CH3 of sugar moiety (rhamnose) connected to carbon 5 (C5) of that sugar. Peaks observed at 4.53 ppm and 5.25 ppm might be considered as anomeric protons from the two sugar moieties.
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Table 4.10: Chemical shifts of 13C NMR (100 MHz) data of compound 4 dissolved in Methanol- d4 (CD3OD)
No. of C 13C NMR (δ) No. of C Nature of the carbon
2 149.3 1 CH
3 122.0 1 C
4 178.5 1 C
5 145.6 1 C
6 135.4 1 C
7 157.5 1 C
8 94.5 1 CH
9 154.3 1 C
10 104.7 1 C
1’ 117.1 1 C
2’ 158.3 1 C
3’ 103.7 1 CH
4’ 165.7 1 C
5’ 115.8 1 C
6’ 122.8 1 CH
6 - Glucose
1’ 101.7 1 CH
2’ 75.1 1 CH
3’ 76.6 1 CH
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4’ 70.6 1 CH
5’ 68.8 1 CH
6’ 67.7 1 CH2
4 - Rhamnose
1’’ 99.6 1 CH
2’’ 71.3 1 CH
3’’ 71.8 1 CH
4’’ 73.3 1 CH
5’’ 77.7 1 CH
6’’ 17.9 1 CH3 ______
As for 1H NMR spectrum, 13C NMR spectrum depicting the chemical shifts characteristics of the peaks is represented in Annexure 3B and summarized in Table 4.11. 13C NMR spectrum is also divided into four groups. The first one consists of a unique peak appearing at around 178.5 ppm,
O which is in the domain of carbonyl group ( C ). The second group is that of aromatic carbons.
Fifteen carbons (15C) seem to be situated in aromatic region. The third group of carbon peaks consists of carbons in the range from 60 ppm to 80 ppm, which appears to belong to sugar part.
The last consists of carbon peak observed around 17.9 ppm, the observed peak is characteristic of methyl group (CH3) which seems to confirm the existence of methyl protons as suggested by
1H NMR. Hence, one sugar might contain a methyl group. The presence of peak between 99.6 ppm and 101.7 ppm, might also justify the presence of anomeric carbon atoms from sugar moiety.
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When observing the COSY NMR spectrum (Annexure 3C), protons from methyl group correlates with protons in the sugar part. In addition, it appears that protons from sugar parts are connected to each other. From the aromatic range, only two hydrogens from different carbons are related.
After an extensive analysis of all spectra, compound 4 was identified as isoflavonone with two sugar moiety (α-glucose at position 6 and β-rhamnose at position 4’). Isoflavonone part consist of hydroxyl group at C5, C7, C2’ and at C4’. The proposed structure of compound 4 is represented below (Figure 4.16) with a molecular mass of 610 g/mol and molecular formula of C27H30O16.
HO OH 7 O 6'' 2 OH 6' O 5'' 5' O 2' 1'' 1' O 6 4 O OH 5 OH OH OH OH OH O 4' OH
Figure 4.16: Proposed structure of compounds 4.
According to ChemDraw Ultra 7.0 software (http://chemdraw-ultra.software.informer.com/7.0/), the proposed IUPAC name of compound 4 is: 6-[3,4-Dihy-droxy-6-methyl-5-(3,4,5- trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yloxy)-tetrahydro-pyran-2-ylmethyl]-3-
(2’,4’-dihydroxy phenyl)-5,7-dihydroxy-chromen-4-one.
Looking at the mass spectrum of compound 4 (Figure 4.15), the base peak occurs at m/z 303.00.
This fragmentation seems to be that of isoflavonone part (m/z 302.00) without sugar moiety with
+ + one proton less m/z 303.00, (C15H10O7 + H ). Beside the base peak, other peaks were observed
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+ + + + + including m/z 136 (C8H8O2 ), 153 (C8H8O3 + H ) and m/z 465 (C21H20O12 + H ). The sugar part also seems to have a mass of about m/z 310.30. Hence, compound 4 might be fragmented into two parts showing the isoflavonone part only and hiding the sugar moiety part. More mass spectroscopy studies of compound 4 need to be conducted in order to find more possible fragmentation of the entire compound.
+ m/z 136, C8H8O HO O OH 7 6'' 2 OH 6' O 5'' 5' O 2' 1'' 1' O 6 4 O OH 5 OH OH OH OH OH O 4' OH
m/z 303, C H O + + H+ m/z 153, C H O ++ H+ m/z 465, C H O + + H+ 15 20 12 8 8 3 21 20 12
Figure 4.17: Proposed possible fragmentation of compound 4 according to mass spectrum.
4.2.3.2. Compound 5
After the collection of compound 4, the solvent system (chloroform, ethyl acetate and methanol) polarity was increased from (6:3:1, v/v/v) to (2:6:2, v/v/v) in order to elute the untargeted components. Due the higher polarity of the compound 5, 100% methanol was used to elute the fraction containing compound 5. The collected mass (1.9 g) of compound 5 was impure; hence it was subjected to the preparative thin layer chromatography using ethyl acetate methanol and water (40:5:5, v/v/v) as mobile phase.
The preparative TLC was run until the front solvent extrapolated to allow all impurity to be eluted and remained with compound 5 as pure as possible. A mass of 976 mg of compound 5
108 was collected as yellow powder. Unfortunately, due to lack of methanol deuterated solvent, compound 5 was not fully characterised. However, it was subjected to antibacterial and antioxidant quantitative analysis. The structure of compound 5 still to be determined.
Figure 4.18: Photographs showing the isolation process leading to compound 5.
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4.2.4. Summary of the isolation process
Ethanol Extract: 34.567 g
1. Suspended in Methanol:Water (9:1, v/v) 2. Addition of n Hexane
n-Hexane Fraction Methanol / water layer 12.526 g Column Chrom. Evaporation of methanol n-Hex:DCM (9:1, v/v) Aqueous residue
Addition of Chloroform Comp. 1 Comp. 2 1.5 g 300 mg
Chloroform Fraction Aqueous Layer 4.856 g Addition of Column Chrom. n-Hex: E.A Butan-1-ol (9:1, v/v); (7:3, v/v); (5:5, v/v)
Comp. 3 0.5 mg Butan-1-ol Fraction Water Fraction 8.678 g Column Chrom. Chlor:E.A:MeOH (6:3:1, v/v/v) Column Chr. 100 % MeOH
Comp.4 Comp. 5 PTLC Comp. 5 1.7 g 1.9 g E:M:W 976 mg (40:5:5, v/v/v)
Figure 4.19: The sketch showing the isolation process undertaken for the aerial part material of Asparagus suaveolens ethanolic extraction until pure compounds.
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4.3. Antioxidant and antibacterial activity of isolated compounds
Isolated compounds were analysed for antioxidant and antibacterial activities using the same procedure as described in Section 3.7.1.2, Section 3.7.2.2 and Section 3.7.2.3. However, compounds 2 and 3 were not object of these tests due to their lower recovery. Only compounds
1, 4 and 5 were subjected to these biological activities.
4.3.1. Screening of antioxidant activities of isolated compounds
From these results (Figure 4.20), it appears that compound 1 (Hentriocontan-16-one) from n- hexane fraction had low inhibition capacity. Compounds 4 and 5, both isolated from butan-1-ol, seem to have similar inhibition capacity in respect to DPPH free radical. However, compound 5 isolated also from butan-1-ol appears to inhibit strongly than compound 4 at low concentration but when the concentration increases, both compounds appear to have the same inhibition capacity. Comparing the inhibition of isolated compounds (compounds 1, 4 and 5) with those of fractions where they were isolated from, it appears that the inhibition strength of compound 1
(26.168% at 2.5 mg/ml) is lower than that of the parent fraction, n-hexane fraction (35.413% at
2.5 mg/ml). This difference might be explained by the synergetic effect of compounds being together for a better activity display. For butan-1-ol fraction, there is no huge difference between the inhibition values of the fraction (69.400% at 2.5 mg/ml) and those of compound 4 (67.094% at 2.5 mg/ml) and compound 5 (67.268% at 2.5 mg/ml). All standards (Ascorbic acid and BHT) have higher inhibition capacity than all the isolated compounds tested.
111
120
Comp. 1 100 Comp.4
80 Comp. 5
BHT (Std) 60 Asc. Acid (Std) 40
20
0 Radical scavenging activity at at scavenging Radical nm 517 activity
0 0.5 1 1.5 2 2.5 % Concentration mg/ml
Figure 4.20: DPPH free radical scavenging of compounds 1, 4 and 5 isolated from n-hexane and butan-1-ol fractions.
As for fractions, the IC50 of isolated compounds (compounds 1, 4 and 5) were determined. The
IC50 of compound 1 was higher (>2.5 mg/ml) than the concentration used. When comparing the percentage inhibition between the n-hexane fraction to that of compound 1 at 2.5 mg/ml, it appear that the fraction might have a lower IC50 than that of compound 1. This might be due to the synergetic aspect of compounds present in the extract. Compounds 4 and compound 5 show an IC50 of 0.45 mg/ml and 0.41mg/ml respectively. These values are very closer to that of butan-
1-ol extract (0.42 mg/ml), which means, the antioxidant activity observed in butan-1-ol might be from compounds 4 and 5.
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4.3.2. Minimum inhibitory concentration (MIC) of isolated compounds
The minimum inhibitory concentration (MIC) of isolated compounds against the WHO 2008 N. gonorrhoea (F, G, K, L, M, N, O and P) (Unemo et al., 2008) was determined as described in
Section 3.7.2.2. Results of these tests are displayed in Figure 4.21.
These results show that compound 5 appears to be effective against F, G, K and M strains than compound 1 and compound 4. Meanwhile, compound 1 showed a strong activity against L, O and P N. gonorrhoea strains. Compound 4 seems to show weak MIC compare to compound 1 and compound 5. However, compound 4 and compound 5 appear to have the same MIC value against N and P N gonorrhoea. The lowest antibacterial activity was observed when N. gonorrhoea L strain was subjected to compound 4. In some cases, isolated compounds have shown higher antibacterial activity than those of standards (Gentamicin and Amoxicillin). In that regard, compound 1 showed a strong activity against F and O gonorrhoeal strains than all standards. Compound 4 also showed strong activity than standards against F, G, N and O N. gonorrhoea strains. Compound 5 appears to show a strong antibacterial activity than both standards against F, G, N. gonorrhoea strains, higher than gentamicin against N. gonorrhoea K strain.
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30 Comp 1
Comp. 4 25 Comp. 5 20 Gentamicin (SD) Amoxicillin (SD) 15
10
5
MIC of Isolated compounds mg/ml of compounds MIC Isolated 0 F G K L M N O P WHO 2008 N. gonorrhoea strains
Figure 4.21: MIC of compounds (compound 1, 4, and 5) isolated from aerial part of Asparagus suaveolens tested against WHO 2008 N. gonorrhoea strains (F, G, K, L, M, N, O and P).
All isolated compounds react differently against different WHO 2008 N. gonorrhoea strains.
However, according to the results, all have shown a significant antibacterial activity, which might justify the use of Asparagus suaveolens crude extracts by traditional healers against gonorrhoeal infections. The presence of hydroxyl group on compound 4, characterised as isoflavonone derivative, justified the use of the plant against N gonorrhoea (Cowan, 1999). As for compound 1, the hydrophobic part of compound 1 (palmitone) can interacts with the non- polar part of lipids from membrane and mitochondria of the bacteria. Hence, the morphology of bacteria may be modified, which might lead to the bacterial death (Burt, 2004).
When comparing MIC results of isolated compounds to those of fractions, it appears that, the isolated compounds inhibited N. gonorrhoea strains more than the fractions in which they were
114 derived from. For example, N. gonorrhoea F, G, K, M, N and O present strong MIC when tested against isolated compounds. The tendency of Asparagus suaveolens antigonorrhoeal activity is that, the pure the compound, the more antigonorrhoeal activity. This favour the specificity activity than the synergetic effect of compounds in fractions.
4.3.3. Minimum bactericidal concentration (MBC) of isolated compounds
The minimum bactericidal concentrations (MBC) of the isolated compounds against the WHO
2008 N. gonorrhoea were determined according to the protocol described in Section 3.7.2.3.
Results of those tests are represented in Figure 4.22.
30 Comp. 1 comp. 4 25 comp. 5 Gentamicin (SD) 20 Amoxicillin (SD)
15
10
5
MBC of pure compounds mg/ml compounds of MBC pure 0 F G K L M N O P WHO, 2008 N. gonorrhoea strains
Figure 4.22: MBC results of compounds (compound 1, 4 and 5) isolated from aerial part of Asparagus sualveolens against WHO 2008 N. gonorrhoea (F, G, K, L, M, N, O and P).
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From the results, it appears that compound 5 showed higher MBC values against F, G and K N. gonorrhoea strains, whereas compound 1 showed strong activity against L, M, N, O and P N. gonorrhoea strains. Compound 4 and compound 5 seem to have the same MBC values against the N strain. The lowest MBC value was recorded with compound 4 against K N. gonorrhoea strains. Among all isolated compounds, only compound 5 showed strong activity against F strain compared to both standards. However, in respect to G strain, compound 5 showed a strong activity only against gentamicin (standard).
Compounds 1 and 4 have shown weak MBC activity against K, L, M and P strains with MBC values of 20.254, 18.563, 10.675, 13.457 mg/ml respectively for compound 1 and 24.689,
20.218, 11.925, 15.486 mg/ml respectively for compound 4. Compound 5 showed weak activity against bacterial strains L, M and P with MBC values of 21.548, 15.967 and 20.438 mg/ml respectively. N. gonorrhoea L, M and P strains seem to be resistant against all the isolated compounds (compounds 1, 4 and 5). When compared to standards used (gentamicin and amoxicillin), isolated compounds showed lower MBC activity against almost all N. gonorrhoea except for the case of compound 1 against F strain where the MBC activity of the compound greater than that of amoxicillin and plus minus equal to that of gentamicin. In addition, compound 5 has shown higher activity than both standards against F strain and higher MBC activity against amoxicillin. The use of Asparagus suaveolens against gonorrhoea infections by traditional healers might be justified by the presence of these metabolites, especially compound
1, which shows strong activity against WHO 2008 N. gonorrhoea strains and in some cases even stronger than the standards used (gentamicin and amoxicillin).
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4.3.4. MBC/MIC radio of isolated compounds
The ratio of MBC/MIC gives the information about the bacteriostatic and bactericidal character of the isolated compounds (French, 2006). The higher the ratio the more the compound is bacteriostatic and less bactericidal. However, the lower the ratio, the more the compound is bactericidal. Observing different ratios in respect to different N. gonorrhoea strains, it was revealed that compound 4 appears to be bacteriostatic with MBC/MIC ratio of 35.93 against N strain followed by compound 5 (34. 66) against the same strain. Compound 1 was considered as bacteriostatic against O N. gonorrhoea strain due the higher MBC/MIC ratio of 24.34.
Compound 5 also was found to be bacteriostatic against G N. gonorrhoea strain with MBC/MIC ratio of 14.11.
40 Comp. 1 35 Comp. 4 30 Comp. 5
Gentamicin (SD)
25 Amoxicillin (SD) 20
MBC/MIC 15
10
5
0 F G K L M N O P WHO, 2008 N. gonorrhoea strains
Figure 4.23: MBC/MIC ratio showing the bacteriostatic and bactericidal character of isolated compounds against the WHO 2008 N. gonorrhoea (F, G, K, L, M, N, O and P).
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The assumption is that, the killing of N. gonorrhoea G and N strains by compound 5, N. gonorrhoea N and N gonorrhoea O by compound 1 and compound 4 depends on their concentration. At low concentration, compounds (1, 4 and 5) are only able to inhibit the growth of G, N and O N. gonorrhoea strains. However, in the other cases, where the MBC/MIC is low, the killing of N. gonorrhoea is not depending on the concentration of compounds (French, 2006).
Hence, compounds that are considered to behave as bactericidal are as follow: compound 1 against F, G, K, M, N and P N. gonorrhoea strains, compound 4 against F, G, K, L, O and P N. gonorrhoea strains; and compound 5 against F, K, L, M, O and P N. gonorrhoea strains.
Generally, all the isolated compounds seem to react similarly against L, M and P N. gonorrhoea strains, and because of their low MBC/MIC ratio, they are bactericidal in respect to those strains.
French (2006) reported that the antibiotic (antibacterial agent) is considered as bactericidal when the MBC/MIC ratio is less than four. Gentamicin (standard) seems to show a bacteriostatic character in respect to N and O strains, whereas for the remaining N. gonorrhoea strains it might be classified as bactericidal. Amoxicillin standard in the other hand has been consistent with the bactericidal character except against N. gonorrhoea K strain.
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CHAPTER 5
CONCLUSION AND RECOMMENDATIONS
The usage of Asparagus suaveolens in traditional medicine for the treatment of several illnesses including epilepsy, sexually transmitted diseases and many others has its origin since ancient time. Since then, no scientific research finding has been reported on the plant’s biological activities or connecting its traditional usages to any of the biological activities. In addition, none of its components has ever been isolated anywhere in the world. Hence, the current study is the first reporting on Asparagus suaveolens, which has demonstrated the presence of antioxidant and antibacterial activities, and reporting on the isolation of secondary metabolites from of the plant’s aerial part (leaves, stems and barks combined).
By using ethanol as the extractive solvent, from 500 g of used powdered plant material, 34.567 g
(w/w) were recovered, about equivalent to 6.913%. The liquid-liquid separation of ethanolic extract resulted to three fractions including n-hexane, chloroform and butan-1-ol fraction, which has recovered the percentage of 2.50%, 0.971% and 1.735% respectively. The study managed to establish, for the first time ever, the phytochemical profile for n-hexane, chloroform and for the butan-1-ol fractions from ethanolic extracts. This analysis showed the presence of alkaloids, saponins, flavonoids, glycosides, anthraquinones, terpenoids, and steroids, reducing sugar, proteins and coumarins. The qualitative (bio autographic method) and quantitative (DPPH assay) antioxidant screening demonstrated that all fractions (n-hexane, chloroform and butan-1-ol) of the ethanolic extract of the aerial part of Asparagus suaveolens contained metabolites with
119 different degrees of antioxidant activity. Chloroform fraction and butan-1-ol fraction showed more antioxidant activity when compared to n-hexane fraction. An effective inhibition of DPPH free radical activity of 71.409% was observed for chloroform fraction followed by that of butan-
1-ol fraction 69.400% and finally the 35.413% inhibition for n-hexane fraction, all at a concentration of 2.5 mg/ml.
Among the four general bacteria, including S. aureus (ATCC 29213), E. coli (ATCC 25922), E. faecalis (ATCC 29212) and P. aeruginosa (ATCC 27853) used in this study against different ethanolic fractions (n-hexane, chloroform and butan-1-ol); E. coli (ATCC 25922) has shown high resistance in respect to all pre-cited fractions. By cons, all fractions (n-hexane, chloroform and butan-1-ol) have shown promising results against eight Neisseria gonorrhoeae strains (F, G,
K, L, M, N, O, P) collected from NHLS laboratory, Johannesburg in South Africa. In most of cases, the n-hexane fraction has shown a higher gonorrhoeal activity (lowest MIC value of 0.124 mg/ml against O strain), followed by chloroform fraction (with lowest MIC value of 5.173 mg/ml against F strain) and finally butan-1-ol fraction (with lowest MIC value of 6.348 mg/ml against O strain). In general, n-hexane fraction was the most bactericidal against the Neisseria gonorrhoea strains than chloroform and butan-1-ol fraction.
The isolation of compounds with antioxidant and/or antibacterial activities from all fractions (n- hexane, chloroform and butan-1-ol) using the bio-guided method has led to the identification of two compounds, among them compound 1 (palmitone) and compound 2 (n-heptacosanol); two unidentified pure compounds (compound 3 and 5) and the compound 4, which was assumed to be novel.
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Compound 1 (palmitone) was isolated as a white amorphous powder (1.5 g) from n-hexane fraction. When tested for antioxidant activity, palmitone showed a low inhibition of DPPH
(26.1% at 2.5 mg/ml), with an IC50 value higher than 2.5 mg/ml. However, palmitone has shown a promising antigonorrhoea activity, especially against F, O and P Neisseria gonorrhoea strains, with MIC values of 1.1 mg/ml, 0.1 mg/ml and 1.7 mg/ml respectively; which were significant when compared to those of the used standards (Gentamicin and Amoxicillin). Additionally, this is the first study, which highlights the presence of palmitone in Asparagus genus and demonstrates its in vitro antigonorrhea activity. Compound 2 (n-heptacosanol) was also isolated from n-hexane fraction as cream white powder (0.3 g). Due to its poor percentage of recovery, compound 2 has not been subjected to quantitative antioxidant assay as well as the antibacterial screening. Compound 3 was isolated as grey powder (0.5 mg) from chloroform fraction.
Compound 3 was unidentified due to its poor recovery percentage. The antioxidant and antibacterial screening were not done.
Compound 4 was isolated from butan-1-ol fraction as yellow powder (1.7 g). Compound 4 was identified as novel isoflavone compound with sugar moiety (α-glucose and β-rhamnose) on C6 of ring A. The molecular formula of compound 4 seems to be C27H30O16. The proposed IUPAC name of compound 4 is: 6-[3,4-Dihy-droxy-6-methyl-5-(3,4,5-trihydroxy-6-hydroxymethyl- tetrahydro-pyran-2-yloxy)tetrahydro-pyran-2-ylmethyl]-3-(2,4-dihydroxy-phenyl)-5,7- dihydroxy-chromen-4-one. When screened for antioxidant and antibacterial activity, compound 4 showed moderated activities of 67.1% at 2.5 mg/ml with an IC50 of 0.45 mg/ml and a MIC of
0.406 mg/ml, 0.650 mg/ml, 0.223 mg/ml and 0.650 mg/ml for F, G, N and O Neisseria gonorrhoeae respectively. The used standards have shown strong antioxidant activity than
121 compound 4. Compound 4 showed better activity against F, G, N and O Neisseria gonorrhea strains. Compound 5 was also isolated (976 mg) from butan-1-ol fraction as a yellow powder and tested for its antioxidant and antibacterial activities. Its antioxidant screened showed an inhibition of 69.4% at 2.5 mg/ml with IC50 value of 0.41 mg/ml, while its antibacterial activity showed the MIC of 0.325 mg/ml, 0.203 mg/ml and 0.244 mg/ml for F, G and N Neisseria gonorrhoeae strains respectively. The structure is yet to be elucidated.
In summary, the presence of palmitone in the aerial part of Asparagus suaveolens reveals a partial phytochemical responsible for the anticonvulsant property attributed to the plant in folk medicine from the Basotho tribe in the Kingdom of Lesotho. From the antibacterial activity, it was discovered that Asparagus suaveolens aerial part is a stronger antibacterial agent against
N.gonorrhoeae than the current used drugs (Gentamicin and Amoxicillin), especially against the
F, O and P strains, with palmitone as the bioactive compound. These observations justify its use as remedy in the traditional treatment of gonorrhoea infections by traditional healers in the
Limpopo Province, South Africa and may be used as an alternative medication against N. gonorrhoeae infections. We strongly recommend the expansion of this study to other biological assays such as anticancer, antituberculosis, anti-inflammatory, cytotoxicity, antidiabetes and many more; to reveal more mysteries.
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CHAPTER 6
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ANNEXURE 1
1 Annexure 1A: H NMR (400 MHz) of compound 1 dissolved in Chloroform-d3 (CDCl3) isolated from n-hexane fraction of ethanolic extract of aerial part of Asparagus suaveolens.
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13 Annexure 1B: C NMR (100 MHz) of compound 1 dissolved in Chloroform-d3 (CDCl3) isolated from n-hexane fraction of ethanolic extract of aerial part of Asparagus suaveolens.
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Annexure 1C: Two dimension COSY of compound 1 dissolved in Chloroform-d3 (CDCl3) isolated from n-hexane fraction of ethanolic extract of aerial part of Asparagus suaveolens
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Annexure 1D: Two dimension TOCSY of compound 1 dissolved in Chloroform-d3 (CDCl3) isolated from n-hexane fraction of ethanolic extract of aerial part of Asparagus suaveolens.
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ANNEXURE 2
1 Annexure 2A: H NMR (300 MHz) of compound 2 dissolved in Chloroform-d3 (CDCl3) isolated from n-hexane fraction of ethanolic extract of aerial part of Asparagus suaveolens (Photograph taken from NMR machine screen on the 18 January 2016).
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13 Annexure 2B: C NMR (75 MHz) of compound 2 dissolved in Chloroform-d3 (CDCl3) isolated from n-hexane fraction of ethanolic extract of aerial part of Asparagus suaveolens.
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Annexure 2C: Two dimensions COSY of compound 2 dissolved in Chloroform-d3 (CDCl3) isolated from n-hexane fraction of ethanolic extract of aerial part of Asparagus suaveolens.
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Annexure 2D: Two dimensions HSQC of compound 2 dissolved in Chloroform-d3 (CDCl3) isolated from n-hexane fraction of ethanolic extract of aerial part of Asparagus suaveolens.
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ANNEXURE 3
1 Annnexure 3A: H NMR (400 MHz) of compound 4 dissolved in methanol (CD3OD) isolated from butan-1-ol fraction of ethanolic extract of aerial part of Asparagus suaveolens.
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13 Annexure 3B: C NMR (100 MHz) of compound 4 dissolved in (CD3OD) isolated from butan-1-ol fraction of ethanolic extract of aerial part of Asparagus suaveolens.
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Annexure 3C: Two dimensions COSY of compound 4 dissolved in (CD3OD) isolated from butan-1-ol fraction of ethanolic extract of aerial parts of Asparagus suaveolens.
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ANNEXURE 4
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