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Biosynthesis, Characterization and Antibacterial Activity of Silver and Gold
Nanoparticles from the Leaf and Bark extracts of Zanthoxylum Capense.

by

MBAVHALELO JADE NEPHAWE

Dissertation in fulfilment of the requirement for the degree

MASTER OF SCIENCE

in

NANOSCIENCE

in the

FACULTY OF SCIENCE

UNIVERSITY OF JOHANNESBURG

Supervisor : Dr. D.T. Ndinteh

Co-supervisors : Dr. V. Mavumengwana
: Dr. N. Niemann

2015
 DEDICATION

I dedicate this work to my husband Tshilangano Kenneth Tshikosi, for showing me the
meaning of determination and the value of patience! And for inspiring me to make the
very most of the chances life has granted me.

ii
 ACKNOWLEDGEMENTS

I would first of all like to thank the Lord for giving me the strength to reach for my goals,
endurance to complete my studies, guidance through my life, and for just being there on
the days that the sun didn’t shine.

In pursuit of this academic endeavour I feel that I have been especially fortunate as
inspiration, guidance, direction, co-operation, love and care all came in my way in
abundance and it seems almost an impossible task for me to acknowledge the same in
adequate terms.

I would like to thank my supervisors Dr D.T. Ndinteh, Dr V. Mavumengwana, and Dr N.

Niemann and for the continued support and guidance they provided and the knowledge
that they shared with me throughout my studies. Thank you for the invaluable
contributions to my project, as well as pointing me in the right direction when I needed it.
Thanks for all your effort and inputs in reviewing my work and giving feedback and
constructive criticism on how to best improve it. Most importantly, I would like to thank
them for dedicating their own personal time to make this project a completed success -
all is much appreciated.

Thank you to my friends and colleagues at the Department of Biotechnology and food
technology for their advices, assistance and encouragement. A special thanks to my dear
friends the Big Five for the moral support and encouragement; Tendani Sebola, Sharon
Pelo, Mbali Webb and Nkem Okerafor. You all mean more to me than I would ever be
able to say in words. Thanks to everyone else I shared the lab with and made my
experience at the lab a fruitful one.

I may be failing in my duties if I do not thank all my batch mates for their constant
encouragement. The 2014 NANO UJ group, you are more like brothers and sisters to me.
Thank you for all your support, kind words of encouragement and for providing me with a
social life. I am grateful and honoured to have you all in my live. Penny Mathumba,
Mandla Chabalala, Gauta (Au) Mathlou, Lerato Mabe, Mphoma Matseke, Lwazi and
Koloti. I will relish your memories for years to come.

iii
Cordial cooperation, friendly collaboration, fruitful advice and guidance were received
from many other persons from the start to the end of this piece of work. I am immensely
grateful to all of them and regret for my inability to mention everyone by name.

This project was made possible by the funding provided by the National Nanoscience
Postgraduate Teaching and Training Platform (NNPTTP) through the Department of
Science and Technology (DST).

Inadequate thought in terms of words and expression, this dissertation owes a lot to my
beloved family and husband for their support, suggestions, inspiration, encouragement &
good wishes for the success of my dissertation.

iv
 PRESENTATIONS

CONFERENCE AND SYMPOSIUM PRESENTATIONS

 Nephawe, M.J., Mavumengwana, V., Niemann, N., and Ndinteh, D.T, Biosynthesis
and Characterization of Silver and Gold Nanoparticles from the Leaf and bark
extracts of Zanthoxylum capense, UJ cross faculty postgraduate symposium, South
Africa (Johannesburg), 13 October 2015. Poster presentation.

 Nephawe, M.J., Mavumengwana, V., Niemann, N., Ndinteh, D.T., Biosynthesis and
Characterization of Silver and Gold Nanoparticles from the Leaf and Bark Extracts
of Zanthoxylum Capense, DST-Howard University Advance-It Women in Stem
Conference, South Africa (Johannesburg), 27-29 October, 2015. Oral presentation.

 Nephawe, M.J., Ndinteh, D.T., Mavumengwana, V., Niemann, N., Biosynthesis and
Antibacterial Activity of Silver and Gold Nanoparticles from aqueous extracts of
zanthoxylum capense, 42nd National Convention of the South African Chemical
Institute, South Africa (Durban), 29 November - 4 December 2015. Oral
presentation.

v
 ABSTRACT

The biosynthesis of nanoparticles has many advantages over tedious, expensive and toxic
physical and chemical methods of synthesis. Plants are stocked with valuable metabolites
that are capable of reducing metal salts to form nanoparticles. In this study, aqueous leaf
and bark extracts of Zanthoxylum capense were reacted with AgNO3 and HAuCl4 to
determine the plants reducing abilities and hence synthesis of Ag and Au nanoparticles
capabilities. The goal was to develop a reliable, eco-friendly and easy process for the
synthesis of silver and gold nanoparticles using extracts of medicinal plant Zanthoxylum
capense.

Characterization of the nanoparticles formed by the aqueous extracts was performed

using Ultraviolet visible (UV-vis) spectroscopy, Dynamic light scattering, Fourier
transforms infrared spectroscopy (FTIR), Transmission electron microscope (TEM).
Nanoparticles were characterised by measuring their relevant physicochemical
properties. Among the determined properties are size, shape, zeta potential and surface
charge. UV-vis spectrophotometry was used as a confirmatory as well as a characterizing
tool. Phytochemical tests revealed that the leaf and bark extracts of the plant contained
“alkaloids, sterols, terpenoids, flavonoids, steroids, phlabotannins and reducing sugars”
which were linked as potential reducing agents.

Synthesized nanoparticles were confirmed by the change of colour of gold and silver and
growth of nanoparticles were monitored by surface Plasmon behaviour using UV-Vis
Spectroscopy. Water soluble biomolecules present in the plant were responsible for the
conversion of silver and gold ions to Ag-NPs and Au-NPs. UV-Vis Spectrum of synthesized
Ag-NPs and Au-NPs exhibited peaks at 419 and 567 nm corresponding to its surface
plasmon absorption. Transmission electron microscopy showed polydispersed Ag-NPs
ranging from 2-30 nm and Au-NPs ranging from 5-45 nm. The FT-IR results indicate the
presence of different functional groups present in the biomolecule capping the
nanoparticles.

vi
The disk diffusion assay showed Au/Ag nanoparticles to have enhanced activity against
bacteria K. oxycota, B. subtilis, S. epidermidis, P. vulgaris, M. smegmatis and S. aureus
with zones of clearance of 4.1 mm, 4.2 mm, 4.3 mm, 5.8 mm, 6 mm, and 7.5 mm
respectively. The antimicrobial activities of the plant as well as Au-NPs were tested using
the minimum inhibitory concentrations (MIC) assay against a panel of microorganisms.
The highest sensitivities observed for the Au-NPs were that against S. aeruginosa, M.
smegmatis and B. cereus with a MIC value of 2, 1 and 2 mg/ml.

The size and shape of NPs are the keys to their biomedical properties. Green synthesis of
NPs is a feasible way for the future. This study showed that NPs can be synthesized very
easily. The phytochemicals present in Z. capense extracts reduce the silver and gold ions
into metallic nanoparticles. This strategy reduces the cost of production and the
environmental impact.

vii
 TABLE OF CONTENTS

Section Page

Affidavit ................................................................................................................... i

Dedication ............................................................................................................... ii

Acknowledgements ................................................................................................ iii

Presentations ......................................................................................................... v

Abstract .................................................................................................................. vi

Table of contents .................................................................................................. viii

List of figures .......................................................................................................... xi

List of schematics ................................................................................................. xiii

List of tables’ ........................................................................................................ xiv

List of abbreviations .............................................................................................. xv

List of Units........................................................................................................... xvi

CHAPTER ONE ............................................................................................................................... 1

GENERAL INTRODUCTION ......................................................................................................... 1
1.0 Background ................................................................................................................................ 1
1.1 Aims and Objectives ............................................................................................................. 3
1.1.1 Aim ................................................................................................................................... 3
1.1.2 Objectives ....................................................................................................................... 3
CHAPTER TWO .............................................................................................................................. 4
LITERATURE REVIEW .................................................................................................................. 4
2.1 Background Information on medicinal plants ....................................................................... 4
2.1.1 Traditional Medicine .......................................................................................................... 5
2.1.2 Traditional Medicine in South Africa ............................................................................... 5
2.2 The Plant Family Rutaceae ..................................................................................................... 6
2.2.1 The Genus Zanthoxylum .................................................................................................. 7
2.3 The Plant Zanthoxylum Capense (Thunb.) Harv. ................................................................ 7
viii
 2.3.1 Botanical Description 0f Zanthoxylum Capense ........................................................... 7
2.3.2 Plant Distribution................................................................................................................ 8
2.3.3 Zanthoxylum capense Traditional Medicinal uses........................................................ 9
2.3.4 Chemical constituents of Zanthoxylum capense ........................................................ 11
2.4 Nanotechnology ...................................................................................................................... 13
2.5 Nanoparticles ........................................................................................................................... 14
2.6 Classification of nanoparticles .............................................................................................. 15
2.6.1 Silver Nanoparticles ........................................................................................................ 15
2.6.2 Gold Nanoparticles .......................................................................................................... 17
2.7 Methods for Nanoparticle Synthesis .................................................................................... 18
2.7.1 Physical Synthesis .......................................................................................................... 21
2.7.2 Chemical Synthesis ......................................................................................................... 21
2.7.3 Biosynthesis ..................................................................................................................... 22
2.8 Size and Shape of Nanoparticles ......................................................................................... 25
2.9 Antibacterial Activity of Nanoparticles ................................................................................. 27
CHAPTER THREE ........................................................................................................................ 30
EXPERIMENTAL METHODOLOGY .......................................................................................... 30
3.1 Reagents and chemicals ....................................................................................................... 30
3.2 Collection and Preparation of Plant Materials .................................................................... 30
3.2.1 Preparation of Leaf Extracts .......................................................................................... 30
3.2.2 Preparation of 0.1 M NaAuCl4 solution ........................................................................ 30
3.3 Phytochemical Screening of Plant Extracts ........................................................................ 31
3.3.1 Terpenoids (Salkowski’s Test)....................................................................................... 31
3.3.2 Steroids Test .................................................................................................................... 31
3.3.3 Alkaloids (Dragendroff’s Test) ....................................................................................... 31
3.3.4 Flavonoids Test................................................................................................................ 31
3.3.5 Tannins.............................................................................................................................. 32
3.3.6 Phlobatannins .................................................................................................................. 32
3.3.7 Reducing Sugars ............................................................................................................. 32
3.4 Biosynthesis of Silver and Gold Nanoparticles from Leaf and Bark Extracts of
Zanthoxylum capense ................................................................................................................... 32
3.4.1 Synthesis of Silver Nanoparticles ................................................................................. 32
3.4.2 Synthesis of Gold Nanoparticles ................................................................................... 32
3.5 Characterization of the synthesized silver and gold Nanoparticles................................. 33
3.5.1 UV-visible Spectra Analysis ........................................................................................... 33

ix
 3.5.2 Transmission Electron Microscopy (TEM) Analysis ................................................... 33
3.5.3 Fourier-Transformation Infrared (FTIR) Spectroscopic Analysis ............................. 33
3.5.3 Dynamic light scattering (DLS) (Zetasizer) .................................................................. 34
3.6 Antibacterial Activity ............................................................................................................... 34
3.6.1 Disk-Diffusion Antibacterial Assay ................................................................................ 35
3.6.2 Minimum inhibitory concentration assay (MIC) ........................................................... 36
3.6.2.1 Preparation of resazurin solution ........................................................................... 36
3.6.2.2 Antibacterial screening ............................................................................................ 37
CHAPTER FOUR .......................................................................................................................... 39
RESULTS AND DISCUSSION .................................................................................................... 39
4.1 Phytochemical screening of plant extracts ......................................................................... 39
4.2 Biosynthesis of Silver And Gold Nanoparticles from Aqueous Extracts......................... 41
4.3 Characterization Results and Analysis ................................................................................ 43
4.3.1 UV-Vis spectra analysis .................................................................................................. 43
4.2.2 FT-IR spectra analysis .................................................................................................... 48
4.2.4 Transmission Electron Microscopy (TEM) analysis ............................................. 51
4.2.5 Particle size and potential charge analysis.................................................................. 54
4.2.5.1 Nano particle size analysis ..................................................................................... 54
4.2.5.2 Zeta potential analysis ............................................................................................. 58
4.3 Antibacterial activity of Zanthoxylum capense leave and bark extracts, (Nanoparticles)
.......................................................................................................................................................... 61
4.3.1 Disk Diffusion Antibacterial Assay ................................................................................ 61
4.3.2 Minimum Inhibitory Concentration Assay .................................................................... 63
CHAPTER FIVE............................................................................................................................. 65
CONCLUSSION AND RECOMENDATIONS ............................................................................ 65
REFERENCES .............................................................................................................................. 67

x
 LIST OF FIGURES

Figure Description Page

Figure A2.1: Leaflets of Z. capense (S.F. van Vuuren)…………………………………………….………...9

Figure A2.2: Geographical distribution of Z. capense in South Africa (SANB…….…….…….….9

Figure 2.3: Chemical structures of the alkaloids 1 − 9 isolated from Zanthoxylum

capense (Luo et al, 2012; Luo et al, 2013)…….…………………………………………….11

Figure 2.4: Chemical structures of the lignans 10 − 12 and neolignans 13 – 14

isolated from Zanthoxylum capense (Luo et al, 2012; Luo et al,
2013)..............................................................................................................12

Figure 2.5: Chemical structures of N-isobutyl-(2E, 4E)-2, 4-tetradecadienamide (15) and

lupeol (16) isolated from Zanthoxylum capense (Luo et al, 2012; Luo et al,
2013)..............................................................................................................12

Figure 2.6: Different approaches of synthesis of metal nanoparticles…………………………….18

Figure 2.7: Protocols employed for synthesis of nanoparticles (a) bottom to top approach
and (b) top to bottom approach………………………………………………………………..19

Figure 2.8: Mechanisms of toxicity of Ag NPs on bacterial cell (Hajipour et al.,

2012)……………………………………………………………………………………………….………..28

Figure 3.1: Layout of 96-well test plate showing wells for testing activity of selected plant
extracts on individual test bacteria i.e. 2 types of bacteria per plate ( • and o
)(green and blue), d.H2O control wells (- )(orange).…………..………………………38

Figure 4.1: Positive colour change results for (a) shows the colour change before and after
the reduction of Ag+ into Ag-NPs by Z. capense aqueous leaf extract reaction
with 0.5 mM solution of AgNO3 and (b) Z. capense aqueous leaf extract with
0.5 mM HAuCl4 (Au-NPs) ……………………………………………..…………………………….. 43

xi
Figure 4.2: UV-visible absorption spectra of representative Silver nanoparticles
synthesized using leaf and Bark extracts of Zanthoxylum capense (0.5Mm of
AgNO3)…………………………………………………………………………………………………………45
Figure 4.3: UV-visible absorption spectra of representative gold nanoparticles synthesized
using the using leaf and Bark extracts of Zanthoxylum capense (0.5Mm of
HAuCL4)……………………………….……………………………………………………………………….47

Figure 4.4: FTIR spectra of a) Zanthoxylum bark extract b) synthesized Au nanoparticles

and c) synthesized Ag nanoparticles…………………………………………………………….48

Figure 4.5: Representative TEM image and corresponding size distribution histogram for
Ag NPs (0.5mM)…………………………………………..……………………………..……………….51

Figure 4.6: Representative TEM image and corresponding size distribution histogram for
the Au NPs (0.5mM)…………..………………………………………………………….…………….53

Figure 4.7: Size distribution graph of the hydrodynamic size of the silver nanoparticles
treated with aqueous Leaf extract of Zanthoxylum capense……………..….………54

Figure 4.8: Size distribution graph of the hydrodynamic size of the silver nanoparticles
treated with aqueous bark extract of Zanthoxylum capense………………………..55

Figure 4.9: Size distribution graph of the hydrodynamic size of the gold nanoparticles
treated with aqueous Leaf extract of Zanthoxylum capense.…….……….…………56

Figure 4.10: Size distribution graph of the hydrodynamic size of the gold nanoparticles
treated with aqueous bark extract of Zanthoxylum capense…………....…….…57

Figure 4.11: Zeta potential distribution graph illustrating the zeta potential of Z. capense
aqueous leaf and bark silver nanoparticles (0.5mM)……………….…….…….…….58

Figure 4.12: Zeta potential distribution graphs illustrating the zeta potential of Z. capense
aqueous leaf and bark gold nanoparticles (0.5mM)………………………..………….59

xii
 LIST OF SCHEMES

Schematics Description Page

Scheme 4.1: Possible mechanisms for the green synthesis of silver and gold
nanoparticle using plants extracts…………………………………………………………...41

xiii
 LIST OF TABLES

Table Description Page

Table 2.1: Taxonomy of the Rutaceae family……………………………………………..……………………6

Table 2.2: Ethnomedicinal use of Zanthoxylum capense……………….………..…………..…………10

Table 2.3: Summary of advantages and disadvantages associated with each of the
synthesis methods………………………………..……………….……………………………………….20

Table 2.4: Leaf extracts used to synthesize Ag and Au NPs and the resultant shapes and
size ranges……………………………………………………………………………….………………….…25

Table 2.5: A comparison of the advantages and disadvantages of antimicrobial NPs Vs free
antimicrobial agents, (Huh and Kwon, 2011)……….…………………….……………………29

Table 4.1: Phytochemical compounds present in leaf and bark extracts of Zanthoxylum
capense……………………………………………………………………………………….……….………..39

Table 4.2: Results of colour change with 2 ml aqueous leaf and bark extracts when added
to (0.5 mM) AgNO3 and HAuCl4 solutions at 25°C………………………………..…………42

Table 4.3: Synthesized silver and gold nanoparticles from aqueous extracts of the plant Z.
capense, size, PDI, and zeta potential…………………….…………………………….…….…..56

Table: 4.4: A table showing the stability of the NPs according to the potential charge.
………..………………………………………….………………………………………………..…….....……..60

Table 4.5: Antibacterial activity from the aqueous leaf and bark extracts of Z. capense
and aqueous nanoparticles………………………………………………………………..…………61

Table 4.6: Minimum Inhibitory Concentration results of Methanol leaves extract and Au-
NPs synthesized from aqueous leaf extracts of Z. capense…………….………...……64

xiv
 LIST OF ABBREVIATIONS

Ag-NPs : Silver nanoparticles

Ag : Silver

AgNO3 : Nitrate

Au : Gold

CSIR : Council for Scientific Industrial Research

DMSO : Dimethylsulfoxide

HAuCl4 : Hydrochloroauric Acid/ Gold Chloride

MHB : Mueller Hinton Broth

MIC : Minimum Inhibitory Concentration

NNS : National Nanotechnology Strategy

NPs : Nanoparticles

UV-Vis : Ultra violet – visible spectroscopy

SPR : Surface Plasmon Resonance

TEM : Transmission Electron Microscopy

USD : United States of America Dollar

Au-NPs : Gold nanoparticles

FT-IR : Fourier transform-infrared spectroscopy

DNA : Deoxyribonucleic acid

PCR : Polymerase Chain Reaction

xv
 LIST OF UNITS

L : Litre
g : Gram
mM : Milli molar
mV : Millivolts
V : Volume
% : Percentage
hr(s) : Hour(s)
μL : Microliter
min : Minute
nm : Nanometre
˚C : Degree Celsius
μg/kg : Microgram/kilogram
TM : Traditional Medicine
ml : Millilitre
μm : Micrometre
w/v : Weight/volume
μg : Microgram
nM : Nano molar
sec(s) : Second(s)

xvi
 CHAPTER ONE

GENERAL INTRODUCTION

1.0 Background

Nanotechnology involves the manipulation of matter on an atomic and molecular scale

which results in materials that have at least one dimension in the 1-100 nm range (Allhoff
et al., 2010). Materials at nano-scale have properties that differ to their bulk
counterparts. The morphology, size and shape of nanoparticles (NPs), are the key players
in nanotechnology that can be controlled by changing certain parameters in their
synthesis procedures. These characteristics determine the properties of NPs and in turn
their potential applications (Mohanpuria et al., 2008). The electrical and optical
properties of silver (Ag) and gold (Au) NPs have been utilized in various biological
applications (Chamakura et al., 2011). Ag and Au NPs are applied in various products such
as shampoo, shoes and cosmetic products; along with medical and pharmaceutical
applications. Silver nanocrystalline particles are also known to have antimicrobial activity.
They have been used in catalysis, in micro-electronics and other therapeutic applications
(Jain et al., 2007). Au nanoparticles have shown applications mainly in diagnostics and
drug delivery systems (Song and Kim, 2009).

Nanoparticles have been produced by physical and chemical methods; however, there is a
need for environmentally safe, low-cost, rapid, less laborious, easily scaled-up synthesis
methods. The chemical processes that are used to synthesize NPs are expensive and also
lead to toxic chemicals on the surface of the NPs that may have an adverse effect in
medical applications. Biosynthesis has thus solved this problem by eliminating the need
for high pressure, energy or toxic chemicals and is an area of research that is becoming
quite popular.

The ongoing emergence of multi drug resistant bacteria and the infections caused by
them is on the rise. This is alarming and a global threat. The overuse or misuse of
antibiotics has reduced their efficacy and correspondingly bacterial resistance increased.

1
The lower effectiveness of antibiotics causes thousands of deaths worldwide (Engler et
al., 2012). Antimicrobial resistance has a significant negative impact on the outcome of
treatment therapy and increase the risk of cross infections in hospitals. Multi drug
resistant pathogens cause many problematic and challenging infections, for eg. Gram
positive Staphylococcus aureus has evolved from penicillin resistant phenotypes into a
methicillin resistant strain (MRSA), which has become a global epidemic (Engler et al.,
2012; Patel et al., 20120).

Medicinal plants give us hope of treating such multi drug resistant infections since nature
is the only source to provide a variety of chemical compounds that can be used for new
drug discovery (Chanda and Rakholiya, 2011). A number of secondary metabolites like
phenols, flavonoids, glycosides, alkaloids, saponins, triterpenes, etc. produced by plants
are pharmacologically active. The added advantage of using natural products
therapeutically is that they are safe, economical and often cause lesser side effects. The
plant extracts can be used singly or in combination with antibiotics or other plant extracts
or some chemicals i.e. combination therapy. This was the next approach to combat the
multidrug resistant bacteria. This combination therapy or synergistic therapy proved quite
successful (Chanda and Rakholiya, 2011; Rakholiya and Chanda, 2012).

Increasing resistance against antibiotics is a burning health problem. So there is an urgent

and dire need to improve the existing drugs or find new, novel strategies to overcome this
problem. Reducing the particle size is an efficient and reliable tool to endeavor. The
therapeutic applicability of silver and medicinal plants in treating bacterial infections is
already well known (Gopinath et al., 2010; Chanda et al., 2013). Electric colloids of silver
became the mainstay of antimicrobial therapy in the first part of the 20th Century until
the introduction of antibiotics in the early 1940s, historically; silver has been a major
therapeutic agent in medicine, especially in infectious disease (Alexander, 2009).Recently,
synthesis of nano particles (NPs) with the help of medicinal plants is attempted; the
reduction of silver and gold to nano size is accomplished by the secondary metabolites
present in the medicinal plants. Nano particles exhibit completely new or improved
properties as compared to the larger particles of the bulk material that they are made up
of (van den Wildenberg, 2005).

2
1.1 Aims and Objectives

1.1.1 Aim

This study primarily aims to investigate the synthesis and characterization of Silver and
Gold Nanoparticles from leaf and bark extracts of Zanthoxylum capense, determine their
antibacterial activity on pathogenic bacteria.

1.1.2 Objectives

In order to meet the project aim, the following objectives were addressed:

1. To synthesize Ag and Au NPs from aqueous leaf and bark extracts of Zanthoxylum
capense using HAuCl4 and AgNO3 respectively

2. To characterize the synthesized Ag and Au NPs by UV-vis spectrophotometry,

TEM and FTIR and Zetasizer

3. To identify the nature of the phytochemicals responsible for the reduction of

AgNO3 and HAuCl4 respectively.

4. To investigate the antibacterial activity of the Ag and Au NPs against selected

sensitive and resistant pathogenic bacteria.

3
 CHAPTER TWO
LITERATURE REVIEW

2.1 Background Information on medicinal plants

Throughout the generations, human beings have relied on nature (especially plants) for
their basic needs including food and medicines (Gurib, 2006). The majority of these plants
are higher plants which include flowering plants and the conifers (Gurib, 2006). They
dominate the vegetation of the land in the world and are the main source of food for
animals and people. Thus plant resources offer a variety of products to humans and
animals. There are many reasons for the wide spread use of these plants and these
include their inherent and distinct chemical and biological properties as well their
availability (De Wet et al., 2005, Fennel et al., 2004).

In spite of the rapid developments in organic synthesis, medicinal plants and plant
extracts continue to play a significant role in modern medicine. The World Health
Organization estimates that 80% of the world’s population continues to rely mainly on
traditional medicines for their health care needs (Kamatenesi et al., 2007; WHO, 2002).

Higher plants also provide the major components in herbal medicines (Prozesky et al.,
2001). In South Africa, for example, there are over 30,000 species of higher plants and
about 3,000 of these species are used medicinally (Thring and Wehzi, 2006). These plant
medicines are thought to be relatively less toxic and have fewer or no side effects than
synthetic drugs thus they are believed to be safer (De Wet et al., 2005). Medicinal plants
are thus an important aspect of the daily lives of many people and an important part of
the South African cultural heritage (Van Wyk et al., 2002). Examples of a few well known
South African medicinal plants include the Cape aloes (Aloe ferox), buchu (Agathosma
betulina) and the devil’s claw (Harpagophytum procumbens). The contribution of plants
to medicine has led to the growing interest in natural and traditional medicines as a
source of new commercial products and this shows that medicinal plants are something
of the future, not only the past.

4
2.1.1 Traditional Medicine

Traditional medicine (TM) is the sum total of knowledge, skills and practices which are
based on beliefs and experiences indigenous to different cultures. The information on TM
is usually passed orally from one generation to the next, and this information has been
utilized in maintaining health, preventing, diagnosing and treating illnesses (WHO, 2003).

The use of indigenous medicine especially at the primary health care level is becoming a
top priority among developed and developing countries (McGaw et al., 2000). The revival
of interest in the use of phytomedicine is at a global level, as demonstrated in the annual
sales of herbal products worth over US$ 100 billion in the world. For instance, in 2002
more than 62% of Americans used contributing over US$ 20 billion to the industry. The
Eastern world is already well known for its adherence to herbal medicine. China and India
are two leading countries in this regard. Even in the Western world, popularity of
phytomedicine is increasing rapidly. Germany is the leading country in Europe followed by
France in the use of botanical supplements (Barnes et al., 2004; Gilani and Rahman,
2005). The increasing interest in the use of medicinal plants has prompted scientific
studies into natural products to determine whether their traditional uses are supported
with pharmacological effects or if their use is merely based on folklore (Sparg et al.,
2002).

2.1.2 Traditional Medicine in South Africa

African Traditional Medicine (ATM), as a major socio-cultural heritage, has been in

existence for thousands of years. This is the oldest and perhaps the most diverse of all
medical systems that was once believed to be primitive and strongly opposed during
colonial days and subsequently by medical practitioners (Elujoba et al., 2005; Gurib-
Fakim, 2006).

South Africa is renowned for its plant biodiversity. More plant species occur here than in
any other region of comparable size, making South Africa the richest country on earth in
terms of floral wealth (Van Jaarsveld, 2005). In South Africa, more than 60-80% of the
population relies mainly on traditional medicine for their primary health care needs
(Dauskardt, 1990). In most cases traditional healers are the first health care providers to

5
be consulted, especially in rural areas where the practice is deeply interwoven into the
cultural and spiritual life of the people (WHO, 2001). Traditional healers in South Africa
make use of a wide variety of plant species to treat different kinds of infectious diseases
which are prevalent in rural areas (Mcgaw et al., 2000). For instance, in KwaZulu-Natal,
more than 1032 plant species from over 147 families are used in traditional medicine
(Light et al., 2005). Most of the commonly utilized medicines in South Africa are still
derived from plants, many of which are sold in both the informal and commercial sectors
of the economy (Verschaeve et al., 2004).

2.2 The Plant Family Rutaceae

The Rutaceae family comprises almost 150 genera and 1,600 species of trees, shrubs, and
climbers distributed throughout the temperate and tropical regions of the world (Pallio et
al., 2008). The chief genera of this family are Citrus, Zanthoxylum, Ruta, Ptelea, Murraya
and Fortunella (Siddique et al., 2012). Most of the Rutaceae are aromatic plants whose
leaves, fruits or cotyledon in seeds contain a complex mixture of volatile aroma
compounds (Aziz et al., 2010).

Table 2.1: Taxonomy of the Rutaceae family

Kingdom Plantae Plants

Subkingdom Tracheobionta Vascular plants

Superdivision Spermatophyta Seed plants

Division Magnoliophyta Flowering plants

Class Magnoliopsida Dicotyledons

Subclass Rosidae

Order Sapindales

Family Rutaceae Rue family

6
A phytochemical survey of this family reveals many alkaloids, coumarins, flavonoids,
limonoids, and volatile oils (Lewis, 1983). Many of these compounds have been
associated with different biological activities, for example, antimicrobial (Aziz et al., 2010;
Ali et al., 2005), antidiarrhoeal (Mandal et al., 2010), anticholinesterasic (Cardoso-Lopes
et al., 2010, antileishmanial (Carlos, 2011), antiprotozoal (Severino et al., 2009), larvicidal
(Rajkumar and Jebanesan 2009; Emam et al., 2009) and antioxidant activities (Sun et al.,
2003; Wan et al., 2006). In family Rutaceae, the genus Zanthoxylum has been able to
provide a variety of secondary metabolites with interesting phytochemical and biological
activities (Patino, 2012).

2.2.1 The Genus Zanthoxylum

The genus Zanthoxylum comprises over 200 species distributed worldwide in the tropical
and temperate regions of North America, South America, Africa, Asia, and Australia (Ruth,
2011). In Southern Africa the family is represented by some 21 genera and approximately
270 species. The species in this genus are aromatic deciduous trees and shrubs (Negi,
2011). Members of Zathoxylum have been used in traditional medicines, perfumery and
the pharmaceutical industry (Patino, 2012). Various organs of this species are used to
treat a number of diseases, including dental caries, cardiac disease, respiratory disease,
stomach infections, and rheumatism (Mourg, 1998; Dhar, 1973).

2.3 The Plant Zanthoxylum Capense (Thunb.) Harv.

2.3.1 Botanical Description 0f Zanthoxylum Capense

Zanthoxylum capense (Thunb.) Harv. is known as small knobwood (English),

kleinperdepram (Afrikaans), monokwane (Sotho), umnungamabele (Zulu), Munungu
(venda) or umlungumabele (Xhosa) ((Smith, 1895; Hutchings et al., 1996; van Wyk et al.,
2009). It is a deciduous, aromatic and small multi-branched tree, usually about 5m in
height, but reaching 15m under favourable conditions. The young bark is smooth with
straight dark brown thorns and light to dark grey on mature branches, and the stems
have straight spines on scattered cone-shaped knobs.

The Small knobwood has glossy dark green leaves with clear gland dots in the scalloped
margin. They are borne in clusters on short side branches, unevenly compound with 4–8
7
pairs of leaflets plus a terminal one. They have a strong citrus smell when crushed. The
flowers have a sweet smell and are greenish white in colour, with 4 sepals and 4 petals.
The fruit is a round splitting capsule up to 5 mm in diameter, covered with glands, green,
turning red when ripe, splitting later to reveal a single black, oil-rich seed per capsule. The
wood is yellowish and fairly hard (Van Wyk and Gericke, 2000; Venter, 1996).

The tree bears fruit, which is said to contain oil, in clusters, which are small and orange
brown in colour and resemble oranges (Watt and Breyer-Brandwijk, 1962). The fruit is
acrid and tastes of lemon, and presents with a persistent burning sensation in the mouth
(Watt and Breyer-Brandwijk, 1962). When ripe, the fruits slit open and reveal shiny black
seeds inside. The flowers that are born are greenish-white and are inconspicuous (van
Wyk et al., 2009). Root and bark decoctions have a displeasing odour. (Watt and Breyer-
Brandwijk, 1962; Hutchings et al., 1996).

2.3.2 Plant Distribution

The Zanthoxylum capense (small knobwood) is distributed from Zimbabwe in the north,
South Africa and Mozambique. It is mostly found in dry to evergreen woodland and on
rocky hill slopes, but has adapted to a wide range of habitats. In South Africa it is found in
the Eastern Cape, Free State, Gauteng, KwaZulu-Natal, Limpopo, Mpumalanga and North
West. Z. capense is distributed widely in the eastern and northern parts of South Africa
(van Wyk et al., 2009). Distribution also extends sporadically into the southern regions
(Figure A1.2).

8
 Figure A2.1 Leaflets of Z. capense (S.F.van Figure A2.2 Geographical distribution of Z.
Vuuren). capense in South Africa (SANBI).

Small Knob wood is a forest and woodland tree that grows singly, among other species of
trees. It can also be found in rocky areas and along rivers. It always needs enough
moisture to thrive, and grows larger where there is not too much wind. It is found in a
large backward curved C-shape, from the limestone ridges south east of Johannesburg,
round eastward to the Drakensberg, all along the coast southwards, to Mossel-Bay
(Coates Palgrave, 2002).

2.3.3 Zanthoxylum capense Traditional Medicinal uses

Z. capense has a reputation of being widely used as a medicine by the African and
European people. The bark is taken as a tonic in persons suffering from sores (Bryant,
1970; Hutchings et al., 1996) as well as a snakebite treatment. The whole plant is burned
on a hot plate and the fumes inhaled for dizziness (Oluwole et al., 2002). An infusion of
the leaf lends its uses for gastric (Hutchings et al., 1996) and intestinal disorders as well as
intestinal parasites (Watt and Breyer-Brandwijk, 1962; Hutchings et al., 1996). The fruit
has been used for flatulent colic, palsy, as a stomach remedy and for fever (Watt and
Breyer-Brandwijk, 1962; Forbes, 1986; Hutchings et al., 1996; van Wyk et al., 2009).

9
Table 2.2: Ethnomedicinal use of Zanthoxylum capense

Plant Part Popular Uses References

Used

Leaves Fever, stomach ache, Amabeoku and Kinyua,

flatulent colic, toothache 2010
and epilepsy

Root/Bark TB and other respiratory Luo et al, 2011

diseases

Leaves Gastric and intestinal Bryant, 1966; Hutchings

disorders, anthelmintic, et al, 1996.
cough, bronchitis, pleurisy

Leaf, Fruits Syphilis and Buwa and Van Staden

Gastrointestinal disorders 2006; McGaw and Eloff
2010.

A decoction of the bark is given to cattle for gall sickness. It is also used in combination
with other medicinal plants to treat tuberculosis, paralysis, epilepsy, infertility and
impotency, pleurisy, as an anthelminthic, a purgative, enema, parasiticide as well as for
blood purifying (Watt and Breyer-Brandwijk, 1962; Watt, 1967; Hutchings et al., 1996; van
Wyk et al., 2009).

The root bark as well as a paste of the inner stem bark is similarly used (Smith, 1895; Watt
and Breyer-Brandwijk, 1962; Pujol, 1990; Hutchings et al., 1996; van Wyk et al., 2009). Z.
capense is widely used to disinfect anthrax-infected meat as well as in the treatment of
anthrax (Watt and Breyer-Brandwijk, 1962).

A decoction of the root is used as a mouthwash (Watt and Breyer-Brandwijk, 1962; Pujol,
1990; Hutchings et al., 1996; Steyn et al., 1998; van Wyk et al., 2009) for aphthae in
children and a lotion for acne. Powdered root taken by mouth has been used for pimples

10
and blood poisoning. Powdered bark is used to treat paralysis and relieve toothache by
loosening the tooth (Watt and Breyer-Brandwijk, 1962; Hutchings et al., 1996). Other uses
of the root of Z capense include the treatment of bronchitis and for violent chronic
coughing when used in combination with other plants.

2.3.4 Chemical constituents of Zanthoxylum capense

Phytochemical study carried by Luo et al 2012 and 2013), lead to the isolation of sixteen
chemically diverse compounds whose structures are represented in Figure (LUO et al,
2012; LUO et al, 2013). The isolated compounds included the benzophenantridine-type
alkaloids decarine (1), norchelerythrine (2), dihydrochelerythrine (3), 6-
acetonyldihydrochelerythrine (4), tridecanonchelerythrine (5), 6- acetonyldihydronitidine
(6), zanthocapensine (7) and a quinazoline- and a quinoline-type alkaloid rutaecarpine (8)
and skimmianine (9). Moreover, three lignans named (–)-sesamin (10), (–)-episesamin
(11), and (–)-savinin (12), two neolignans, zanthocapensol (13) and zanthocapensate (14),
an amide N-isobutyl (2E, 4E)-2, 4-tetradecadienamide (15) and the pentacyclic triterpene
lupeol (16) were also isolated (Luo et al, 2012; Luo et al, 2013).

Figure 2.3: Chemical structures of the alkaloids 1 − 9 isolated from Zanthoxylum

capense (Luo et al, 2012; Luo et al, 2013).

11
Figure 2.4: Chemical structures of the lignans 10 − 12 and neolignans 13 − 14 isolated
from Zanthoxylum capense (Luo et al, 2012; Luo et al, 2013).

Figure 2.5: Chemical structures of N-isobutyl-(2E, 4E)-2, 4-tetradecadienamide (15) and

lupeol (16) isolated from Zanthoxylum capense (Luo et al, 2012; Luo et al,
2013).

The benzophenanthridine class of alkaloids is a specific group within the isoquinoline

alkaloids, which occur only in higher plants, mainly in the Papaveraceae and Rutaceae
families (Zenk 1994; Ishikawa 2001). In previous studies, this type of compounds has
shown interesting biological activities, namely antitumor, antibiotic and anti-tuberculosis
(Ishikawa 2001, Parhi et al 2012).

12
2.4 Nanotechnology

Nanotechnology is the application of science to control matter at a molecular level and is

an interdisciplinary field which involves physics, chemistry, biology and engineering. A
nanometer refers to one billionth of a meter (Sahoo et al., 2007). The physicist, Richard P.
Feynman explained the concept of nanotechnology in 1959 through his talk “There’s
plenty of room at the bottom”. It only became known as ‘nanotechnology’ in 1974 when
Professor Norio Taniguchi devised the term. Although it seems like novel science, it has
been used for aesthetic and curative purposes for centuries (Singh et al., 2010).

Nanomaterials offer greater permeability and sensitivity allowing more unique

interactions than larger particles. The merging of nanotechnology and biotechnology
seems logical considering that nanostructures are capable of interacting with molecular
and DNA components which are at the nanometer scale (Singh et al., 2010).
“Bionanotechnology is a subset of nanotechnology where the biological world provides
the inspiration and/or the end goal” (Goodsell, 2004). Exclusive interactions with similarly
sized biological materials and the ability to traverse these materials allow for the
discovery of new and intriguing biological applications.

South Africa has shown interest in nanotechnology with regard to the impact it will have
on the mineral markets. In 2005, the National Nanotechnology Strategy (NNS) was
launched and funding of around 600 million USD was allocated to research and
development. India is currently leading in the trilateral nanotechnology development
programme with Brazil and South Africa. There is a concern that natural resources might
become superfluous due to the development of cheaper, stronger and more functionally
rich materials. The focus areas of the National Nanotechnology Strategy (NNS) are:
establishing characterization centres throughout the country enabling researchers with
access to advanced machines; creation of Research and Innovation networks to enable
collaborations between different institutes; initiatives to increase human capital
resources and flagship projects highlighting the benefits of nanotechnology in the
chemistry, water, energy, health and mineral sectors (Wetter, 2010).

13
2.5 Nanoparticles

A nanoparticle (NP) (10-9m) refers to a small object that in terms of its properties,
functions as an entire unit. The physical and chemical properties have a molecular and an
atomic origin and thus behave differently in comparison to their bulk compounds. There
are two groups of NPs, organic and inorganic: the organic group is comprised of carbon
NPs and fullerenes whilst the inorganic group consists of noble metal (Ag and Au) and
semiconductor (titanium dioxide and zinc oxide) NPs. Inorganic NPs provide better
material properties with wide availability, good biocompatibility and functional
adaptability (Singh et al., 2010). Inorganic NPs show important novel chemical, physical
and biological characteristics as well as good functionality owing to their nano-scale size.
An example to prove this is the colour difference between metallic gold which is yellow
whereas Au NPs that have a wine red hue (Daniel and Astruc, 2004; Bhattacharya and
Mukherjee, 2008).

Properties of NPs are derived from the high quantity of surface area and the high surface
area to volume ratio which allows them to have a significant surface energy. As their
diameter decreases, the available surface area of the particle itself increases and thus has
an influence on potential applications (Christian et al., 2008; Singh et al., 2010). NPs also
offer additional advantages when they are chemically modified to form a suspension,
emulsion or aerosol. In addition, some chemicals can also modify the function and
indirectly stabilise against aggregation or coagulation by changing the surface layer of the
particle (Singh et al., 2010). The full potential of NPs depends on stability,
biocompatibility and directed specificity to the target sites with regard to systemic
administration (Mody et al., 2010).

Due to their unique and tuneable properties, they are able to find applications in
photothermal therapy (Jain et al., 2007), catalysis (Xiao and Xia, 2010), optoelectronics
(Mohanpuria et al., 2008), water purification (Jain and Pradeep, 2005), quantum dot
lasers for detection (Ledenstov et al., 1996), Surface Enhanced Raman Spectroscopy
(SERS) detection and biosensing (Stuart et al., 2005).

14
2.6 Classification of nanoparticles

Nanoparticles fall into two categories: organic and inorganic nanoparticles. Organic
nanoparticles may include carbon nanoparticles (fullerenes) in other hand, inorganic
nanoparticles may include magnetic nanoparticles, noble metal nanoparticles (like gold
and silver) and semiconductor nanoparticles (like titanium dioxide and zinc oxide). There
is a growing interest in inorganic nanoparticles as they provide superior material
properties with functional versatility. They have been examined as potential tools for
medical imaging as well as for treating diseases due to their size features and advantages
over available chemical imaging drugs agents and drugs. Gold nanoparticles have been
used extensively in imaging, as drug carriers and in thermotherapy of biological targets
(Cheon & Horace, 2009). Inorganic nanoparticles (metallic and semiconductor
nanoparticles) exhibit intrinsic optical properties which may enhance the transparency of
polymer- particle composites. For these reasons, inorganic nanoparticles have found
special interest in studies devoted to optical properties in composites. Size dependent
colour of gold nanoparticles has been used to color glass for centuries (Caseri, 2009).

2.6.1 Silver Nanoparticles

Ag ions have been used for the treatment of burns and wounds for centuries. In the year
1700, silver nitrate was used in its solid form to treat venereal diseases, abscesses and
fistulae from salivary glands. In the 19th century, AgNO3 was used in varying
concentrations to treat fresh burn wounds. In 1881, eye inflammations of new-borns
(opthalmia neonatorum) were cured using AgNO3 eye drops. The use of silver slowly
declined when penicillin came into use. Recently however, clinicians have gone back to
the use of silver in wound dressings (Klasen, 2000; Lansdown, 2002).

Ag is inert in its metallic form but gets ionized when it comes into contact with the
moisture of the wound. The ionized silver is very reactive which brings about binding to
bacterial proteins which results in structural change of the cell and this eventually leads
to death. These ions are also responsible for inhibiting bacterial replication by denaturing
bacterial DNA and RNA. The amount of silver and the rate at which it gets released
determines its antimicrobial ability (Lansdown, 2002; Rai et al., 2009).

15
Ag NPs are clusters of silver atoms that are metallically bonded together and are well
known for their antibacterial activity. In aqueous solutions, they release Ag ions which
accounts for this activity (Chaloupka et al., 2010). Ag NPs are now being seen as an
affordable and valuable method for enhancing water filtration in developing countries
(Jain and Pradeep, 2005). In animal models there is also evidence of potent anti-
inflammatory activity and better wound healing with a scar-less endpoint (Tian et al.,
2007). Ag NPs biosynthesized from fungi have also been incorporated into textile fabrics
yielding a positive antibacterial effect (Duran et al., 2007). The preservation of food by
inhibition of microorganism growth has also been performed by integrating Ag NPs into
plastics, food storage bags, chopping boards and refrigerator surfaces (Chaudhry et al.,
2008). Vegetable oil was used to make an environmentally safe metal NP embedded paint
which showed extremely promising antibacterial activity against Gram-positive and Gram-
negative bacteria. This could be the future of coating several surfaces to eliminate
bacterial problems (Kumar et al., 2008). Also, a promising future prospect for HIV
therapeutics could lie in Ag NPs, as studies have shown the prevention of key interactions
of CD4+ cells with envelope proteins of HIV-1 by Ag NPs in in vitro assays (Lara et al.,
2010).

Antibacterial activity against biofilm formation by Pseudomonas aeruginosa and

Staphylococcus epidermidis has been established. This is very important as pathogenic
bacteria are often seen to form biofilms and the exopolysaccharide substance that is
produced is able to reduce the susceptibility of the microorganism to drug administration.
Antibiotics kill the microorganisms only, whereas the Ag NPs are able to inhibit the
microorganisms as well as inhibit the ability to synthesize the exopolysaccharide
(Kalishwaralal et al., 2010). Another important aspect of Ag NPs is that they have been
found to be non-toxic to humans in small concentrations (Rai et al., 2009). Exposures to
concentrations lower than 3 parts per million did not produce a significant immune
response (Klippstein et al., 2010). The European Protection Agency is in the process of
determining environmental limits for Ag NPs since they are becoming more popular
commercially. It is not seen as being harmful to the environment by some since the
nature of the NPs become unstable rapidly and forms the harmless clumps of silver metal
(Maass, 2008; Levard et al., 2012).
16
2.6.2 Gold Nanoparticles

Au NPs are considered to be the most stable NPs and have applications in size-related
optical and electronic properties in the field of biology. This colloidal gold as it is known
has been used to stain glass and ceramic from around the 4th or 5th century B.C in China
and Egypt (Daniel and Astruc, 2004). Au NPs were also used in the middle ages to
diagnose syphilis, cure heart problems and dysentery. Jeremias Benjamin Richters in 1818
gave a possible reason as to why there is a difference in the colour of gold solutions in
comparison to their bulk form which states “pink or purple solutions contain gold in the
finest degree of subdivision, whereas yellow solutions are found when the fine particles
have aggregated”. In 1857, Faraday was responsible for producing gold colloidal solutions
and observing their optical effects (Ostwald, 1909; Daniel and Astruc, 2004). Although
bulk Au is known to be inert, Au NPs have very reactive cores that are especially useful in
catalytic activities. Their combination of low inherent toxicity, high surface area and
tuneable stability provide them with unique qualities that should enable new delivery
strategies (Ghosh et al., 2008).

Au NPs are now being considered as perfect anti-angiogenic compounds since they are
easy to synthesize and characterize, biocompatible and able to bind easily to thiols which
would in turn inhibit or denature the function of angiogenesis inducer proteins. Various
diseases such as cancer, arthritis, etc. depend on angiogenesis thus Au NPs could lead to
new therapeutic measures for these diseases (Bhattacharya and Mukherjee, 2008).
Selective killing of target bacteria by irradiating Au NP-attached bacterial surface with a
laser has also been accomplished (Zharov et al., 2006). Strategies are emerging that could
allow Au NPs to become the next generation of diagnostic tools, as these NPs show great
sensitivity and specificity that can easily replace conventional molecular methods such as
PCR and PCR-based approaches. Future trends of Au NPs in diagnostics include their
functionalization, allowing modification of the Au NP biomolecule interface and
modulation of the properties of the biomolecule (Daniel and Astruc, 2004). There is not
much research with regard to the toxicity of Au NPs to human cells and this need to be
taken into consideration when applying these particles for medical uses.

17
2.7 Methods for Nanoparticle Synthesis

Generally, nanoparticles are prepared by a variety of chemical and physical methods

which are quite expensive and potentially hazardous to the environment which involve
use of toxic and perilous chemicals that are responsible for various biological risks. The
development of biologically-inspired experimental processes for the syntheses of
nanoparticles is evolving into an important branch of nanotechnology. Generally, there
are two approaches which are involved in the syntheses of silver nanoparticles, either
from ‘‘top to bottom’’ approach or a ‘‘bottom to up’’ approach (Fig 2.6). In bottom to top
approach, nanoparticles can be synthesized using chemical and biological methods by
self-assemble of atoms to new nuclei which grow into a particle of nanoscale as shown in
Fig. 2.7a while in top to bottom approach, suitable bulk material break down into fine
particles by size reduction with various lithographic techniques e.g. grinding, milling,
sputtering and thermal/laser ablation (Fig 2.6 and 2.7b).

Figure 2.6: Different approaches of synthesis of metal nanoparticles.

18
Figure 2.7: Protocols employed for synthesis of nanoparticles (a) bottom to top
approach and (b) top to bottom approach.

In bottom to top approach, chemical reduction is the most common scheme for syntheses
of metalic nanoparticles (Elghanian et al., 1997, Hurst et al., 2006). Different organic and
inorganic reducing agents, such as sodium borohydride (NaBH4), sodium citrate,
ascorbate, elemental hydrogen, Tollen’s reagent, N, N- dimethyl formamide (DMF) and
poly (ethylene glycol) block copolymers are used for reduction of metallic ions in aqueous
or non-aqueous solutions (Tran et al., 2013, Iravaniet al., 2014). Capping agents are also
used for size stabilization of the nanoparticles. One of the biggest advantages of this
method is that a large quantity of nanoparticles can be synthesized in a short span of
time. During this type of syntheses; chemicals used are toxic and led to non-ecofriendly
by-products. This may be the reason which leads to the biosyntheses of nanoparticles via
green route that does not employ toxic chemicals and hence proving to become a
growing wanton want to develop environment friendly processes.

In case of top to bottom approach; nanoparticles are generally synthesized by

evaporation–condensation using a tube furnace at atmospheric pressure. According to
Revaprasadu and Mlondo (2006), nanoparticles synthesized by chemical routes will be the
preferred method of choice in future applications, due to the control of the size, shape,
and ease of management of these nanoparticles.

19
Table 2.3: Summary of advantages and disadvantages associated with each of the
synthesis methods.
Advantages
Physical synthesis method
Co-precipitation Simultaneous incidence of
nucleation, growth, and/ or
agglomeration processes
Laser ablation Enables colloidal NPs
solutions to be formed in a
variety of solutions
Vapour-phase Can produce spherical
particles
Inert Gas Condensation Ability to control the
morphology of single state
NPs
Chemical Synthesis method
Turkevich Particle size can be
controlled within a range of
10 to 100 nm with limited
polydispersity
Solvothermal Choice among various
solvents thereby increasing
versatility of synthesis
Sol-gel Size and stability control has
been achieved by some
materials
Biosynthesis Method
Microorganisms
Easy, low-cost,
Plant extracts environmentally-friendly
Algal
biomass/cyanobacteria
Disadvantages
Products are scarcely
soluble and produced
under high supersaturation
Produces NPs in small
quantities and requires a
high energy per unit
Requires supersaturated
vapour and optimal
condensation kinetics with
high temperatures
Ultra-high vacuum
chambers are needed with
different gases and high
pressures
Uses organic solvents that
are harmful to the
environment and requires
purification
High pressures and above
boiling point temperatures
required.
Complexity of process and
the synthesized
precipitates are generally
amorphous
Incubation periods to grow
microorganisms
Bio-sustainability
Growth periods and
conditions required
20
2.7.1 Physical Synthesis

Physical synthesis follows the top-down method which involves NPs being directly
generated from bulk materials by way of generation of atoms through various distribution
techniques. These distribution or removal techniques can be mechanical, chemical,
electrochemical, etc. based on the bulk substrate and the desired outcome. Milling is one
of the most widely used techniques in the manufacturing industry. There is a certain
amount of control when it comes to the dimensions of the final product in that the travel
path of the mill is precisely controlled by computer aided numerical systems (Singh et al.,
2010).

The most important physical approaches in nanoparticle synthesis include evaporation-

condensation and laser ablation. Various metal nanoparticles such as silver, gold, lead
sulphide, cadmium sulphide, and fullerene have previously been synthesized using the
evaporation-condensation method. The absence of solvent contamination in the
prepared thin films and the uniformity of nanoparticles distribution are the advantages of
physical approaches in comparison with chemical processes. (Kruis and Rellinghaus, 2000,
Magnusson et al., 1999). It was demonstrated that silver nanoparticles could be
synthesized via a small ceramic heater with a local heating source (Jung et al., 2006). The
evaporated vapour can cool at a suitable rapid rate, because the temperature gradient in
the vicinity of the heater surface is very steep in comparison with that of a tube furnace.
This makes the formation of small nanoparticles in high concentration possible. This
physical method can be useful as a nanoparticle generator for long-term experiments for
inhalation toxicity studies, and as a calibration device for nanoparticle measurement
equipment (Jung et al., 2006).

2.7.2 Chemical Synthesis

The bottom-up strategy is the basis of chemical synthesis methods. The foundation of this
type of synthesis of NPs is that a salt of the metal is dissolved in a solvent and reduced to
the zero valence state. Molecular components are the starting materials, followed by
chemical reactions, nucleation and growth to form complex clusters (Brust et al., 1994;
Ju- Nam and Lead, 2008). Metal atoms that are produced by reduction in solutions are
primarily insoluble, which leads to clusters called ‘embryos’ through slow aggregation.
21
The moderately sized embryos will either dissociate or grow to become stable. Once the
embryos have reached a certain size, they separate from the solution as solid particles
and are referred to as nuclei. The nuclei grow to nanosize particles and thereafter
diffusion of metal atoms onto the particles occurs and/ or the nanosize particles
aggregate to form the final NPs. The density and the shape of the NPs depend on the
dominant mechanism. NPs produced via aggregation will predominantly have a spherical
shape and a lower density (Goia and Matijevic, 1998). If the surface is not protected by a
capping agent, interactions between particles will generally occur so as to reduce their
high surface energy which causes the aggregation. Capping agents can be organic or
biological molecules as well as polymers. They are responsible for charge or steric
stabilization mechanisms which prevent further agglomeration making them integral
components of NPs (Feldheim and Foss, 2002; Ju-Nam and Lead, 2008).

The conventional method for synthesizing Au NPs is by the Turkevich method. This
method consists of the reduction of HAuCl4 salt in a boiling solution of sodium citrate. The
average particle size can be controlled to be in the range of 10 to 100 nm with limited
polydispersity. Citrate molecules act as both reducing and capping/stabilizing agents and
functionalization of the particles are possible from this step. The chemical reduction of Ag
and Au NPs can be expressed in equations (1) and (2) respectively:

AgNO3 + NaBH4→Ag + ½H2 + ½B2H6 + NaNO3 (1) (Van Dong et al., 2012)

2HAuCl4 + 3C6H8O7 (citric acid) → 2Au + 3C5H6O5 (3-ketoglutaric acid) + 8HCl + 3CO2 (2)

(Tabrizi et al., 2009).

2.7.3 Biosynthesis

Biosynthesis like chemical synthesis methods uses the bottom-up strategy whereby the
main reaction is the reduction of the metal salts to form NPs. Biosynthetic methods
employing either biological microorganisms or plant extracts have emerged as a simple or
viable alternative to chemical and physical synthesis methods. Green synthesis provides
various advancements as it is cost effective, environmentally friendly, easily scaled up for
large scale synthesis and there is no need to use high pressure, energy, temperature and
toxic chemicals (Veerasamy et al., 2011). In biosynthesis, it is taken that the natural
22
materials extract acts as reducing agents for the generation of metal NPs (Dubey et al.,
2010). NPs are synthesized by bacteria, fungi, algae and plants. Since the noble metal NPs
are in contact with humans in their applications, there is a growing need to develop eco-
friendly processes to synthesize NPs to prevent the exposure of toxic chemicals (Song and
Kim, 2009). However, most of the methods are still in the developmental stage and
various problems are often experienced with the stability of NPs, control of crystal growth
and aggregation of particles.

Plant Biosynthesis - Plants as autotrophs have an advantage over other systems in terms
of their morphological characterization, molecular distribution, primary and secondary
metabolite systems which allows them adaptability in a range of conditions (Jha et al.,
2009). They have many advantages:

 they are found along a wide range of ecological boundaries making them easily
available;

 they are safe to handle;

 they are stocked with valuable metabolites; and

 they allow any chemical protocol to be seen as ‘green’.

Plants metabolites are seen as treasures which have not been used to its full capacity
especially when relating it with metallic NP synthesis (Kalishwaralal et al., 2010). Using
plants for NP synthesis can be advantageous over other biological processes because:

 They are easily available;

 It eliminates the need for expensive tissue culture facilities and expertise; and

 It can be suitably scaled up for large scale synthesis of nanoparticles under non-
aseptic conditions (Jha et al., 2009).

Intracellular synthesis of Ag or Au NPs was found in living alfalfa plants. This was possible
via the uptake of Au and Ag ions respectively, from solid media (Gardea- Torresday et al.,
2002; Gardea-Torresday et al., 2003; Sharma et al., 2007). Recently, the extracellular
synthesis of NPs using leaf extracts has become a popular area of research due to its

23
rapidity and rather than using whole plants, is more economical due to the easier
downstream processing. Table 2 provides a summary of many of the studies undertaken
with aqueous leaf extracts to synthesize Ag and Au NPs.

Table 2.4: Leaf extracts used to synthesize Ag and Au NPs and the resultant shapes and
size ranges.

LEAF EXTRACT NPS SYNTHESIZED AND SHAPES REFERENCE

Azadirachta indica Ag NPs – Spherical and polydispersed (Shankar et al., 2004)

(Neem)
Au NPs – spherical, triangular,
hexagonal

Ag / Au Bimetallic NPs

Tamarindus indica Au NPs – Triangular (Ankamwar et al., 2005)

(Tamarind)

Aloe vera Ag NPs – Spherical (Chandran et al., 2006)

Au NPs – Triangular

Cinnamomum Ag NPs – Spherical (Huang et al., 2007)

camphora
Au NPs – Spherical, triangular

Coriandrum sativum Au NPs – Spherical, triangular, truncated (Narayanan and

(Coriander) Sakthivel, 2008)

Magnolia kobus and Au NPs – Spherical, triangular, (Song et al., 2009)

Diopyros kaki hexagonal, pentagonal

Rosa rugose Ag NPs- Spherical (Dubey et al., 2010)

Au NPs- Spherical, triangular, hexagonal

Moringa oleifera Ag NPs – Spherical (Prasad and Elumalai,

2011)

24
Coleus amboinicus Ag NPs – Spherical and triangular (Narayanan and
Sakthivel, 2011)

Mentha piperita Ag NPs – Spherical (MubarakAli et al., 2011)

Ficus benghalensis Ag NPs – Spherical (Saxena et al., 2012)

Prosopis juliflora Ag NPs – Triangular, pentagonal, (Raja et al., 2012)

hexagonal)

Iresine herbstil Ag NPs – Spherical (Dipankar and Murugan,

2012)

Amaranthus spinosus Au NPs – Spherical and triangular (Das et al., 2012)

Pepper leaves Ag NPs – Spherical (Mallikarjuna et al.,

2012)

Aloe barbadensis Ag NPs – Spherical, quasi-spherical (Elizondo et al., 2012)

Au NPs – Quasi-spherical, triangular, rod

Artimisia nilagirica Ag NPs – Square Spherical, Triangular (Vijayakumar et al.,

2013)
Hexagonal

2.8 Size and Shape of Nanoparticles

The intrinsic properties of a metal NP are mainly determined by its size, shape,
composition, crystallinity and structure (Sun and Xia, 2002). Chemical reactivity is highly
dependent on surface morphology. The number of edges and kink sites as well as the
surface-area-to-volume ratio can dictate unique surface chemistries (Yang et al., 2008).

Studies show that the interaction between the reducing or stabilizing agents and metallic
ions strongly affect the size and shape of the particles. The size and shape of NPs could be
controlled by the addition of various polymer molecules and organic salts. The reason for
the various particle morphologies was the diversity of the interactions between the polar

25
groups of the stabilizing agents and the metal. The very strong interaction between the
organic polar groups and Ag for example results in very thin and large plates as the
particle shape. The addition of alkali salts to the reaction solution yielded particles, which
were morphologically not uniform and show a very broad size range distribution
(Widoniak et al., 2005).

It was found that Ag NPs possess a more uniform spherical shape and Au NPs are able to
form anisotropic shapes such as triangular and polygonal plates (Dubey et al., 2010). The
main difference of shape control between Ag and Au NPs are related to the comparative
advantage of reductive and protective biomolecules (Armendariz et al., 2004). It was
found that the NP size and spherical structures could be controlled by changing the
reaction temperature and leaf broth concentration with smaller sizes being formed at
higher temperatures and leaf broth concentrations (Song et al., 2009).

The main interest in anisotropic particles is due to their sharp edges, which lead to high
local electric field gradients under illumination, making them attractive candidates for a
number of applications such as optical biosensing and SERS (Grzelczak et al., 2008). Au
and Ag nanotriangles may have potential applications in the treatment of cancer
hyperthermia and optical coatings (Chandran et al., 2006).

Once materials are prepared in the form of very small particles their physical and
chemical properties change significantly. Nano-dimension, percentage of surface
molecule compare to bulk molecule is high and this enhances the activity of the particle in
the nano dimension and the normal properties of the particle like heat treatment, mass
transfer, catalytic activity, etc. are all increased. But as compared to non-metal
nanoparticles, metal nanoparticles have more industrial applications. Many new
developments are offered by nanoparticles in the field of biosensors, biomedicine and bio
nanotechnology-specifically in the areas:

 Drug delivery

 As medical diagnostic tools,

 As a cancer treatment agent (Gold nanoparticles).

26
Nanoparticles and nanostructure are become developing in human medical application,
including imaging or the delivery of therapeutic drugs to cell, tissues and organs. Many
drug loaded nanoparticles interact with organs and tissues and are taken up by cells.
Several studies have shown that the tissue, cell and even cell organelle distribution of
drugs may be controlled and improved by their entrapment in colloidal nanomaterials,
like micellar structures which act as nanocontainers (Alexiou et al., 2000; Savic et al.,
2003). Magnetic nanoparticles have wide range of applications, such as the
immobolization of proteins and enzymes, bioseparation, immunoassays, drug delivery,
and biosensors (Chen et al., 2002). Nanoparticles of ferromagnetic materials may have
important applications; their reduced sizes can support only single magnetic domains.
Due to their potential technological applications as recording media in the synthesis of
arrays of 4 nm diameter FePt nanoparticles with an extremely narrow size distribution
has promoted significant research efforts (Varlan et al., 1996).

2.9 Antibacterial Activity of Nanoparticles

Antibacterial activity via nanoparticles is one of the most important applications in

nanotechnology. Antibacterial agents are able to locally destroy bacteria without causing
toxicity to the surrounding tissue generally (von Nussbaum et al., 2006). However, with
the broad usage of antibiotics, bacterial resistance is on the rise particularly due to
genetic mutations. These antibiotic resistant strains are responsible for the production of
new, more fatal diseases. The drug resistant strains that cause these diseases bring about
the need for high dosage treatment methods which produce adverse side effects and
increased cost (Hajipour et al., 2012). Certain classes of NPs have antimicrobial activity
and the ability to be a carrier for antibiotic delivery in vitro and in animal models. The
mechanism of antibacterial activity of the NPs depends on the bacterial species as well as
the intrinsic properties, surface modification and composition of the nanoparticles
(Hajipour et al., 2012). In nanoparticles, the energy of the system increases due to the
increase in ratio of surface to bulk atoms which reduces the system stability and in turn
enhances the antimicrobial properties in comparison with the bulk form. A distinct
advantage of nanoparticles lies in the avoidance of non-specific interaction between the
drug and unaffected tissue (Sun and Xia, 2002).
27
Silver (Ag) nanoparticles have proved to be a successful broad spectrum antibacterial
agent in many medical streams. Silver nanoparticles are highly toxic to multidrug resistant
bacteria, which in turn indicate the potential for use in biomedical applications (Jain et al.,
2009). The mechanism of the antimicrobial effect of silver lies in it being released into
solution and higher ratios of nanoparticles to bacteria resulting in quicker bactericidal
activity. The findings were related to previous studies and there are a number of methods
by which Silver nanoparticles inactivate Escherichia coli namely: direct effect of damaging
the function of proteins and lipids in the cell wall and cytoplasmic membrane which
affects: transport; respiration metabolism; indirect production of reactive oxygen species
and possible contact with the DNA once the cell wall and cytoplasmic membrane have
been damaged (Figure 1. Some reports show that Staphylococcus aureus bacteria display
higher resistance to the Ag NPs than E. coli which is probably due to the thicker
peptidoglycan component in the cell wall thus requiring a longer treatment time
(Chamakura et al., 2011).

Figure 2.8: Mechanisms of toxicity of Ag NPs on bacterial cell (Hajipour et al., 2012)

28
Studies conducted thus far on antibiotic properties of functionalized Au NPs have shown
that they display effective antibacterial activity on certain species, such as E. coli and
Pseudomonas aeruginosa. However, these results are variable and the NPs do not
function with the same level of success on other bacterial species (Pissuwan et al., 2010).

Microbes are unlikely to develop resistance against silver, as they do against conventional
and narrow-target antibiotics, because the metal attacks a broad range of targets in the
organisms which means that they would have to develop a host of mutations
simultaneously to protect themselves (Pal et al., 2007). Antimicrobial NPs offer many
advantages and benefits that conventional agents cannot provide. Table 2.8 provides a
summary of the advantages and disadvantages that antimicrobial NPs have over free
antimicrobial agents.

Table 2.8: A comparison of the advantages and disadvantages of antimicrobial NPs Vs

free antimicrobial agents, (Huh and Kwon, 2011).

29
 CHAPTER THREE
EXPERIMENTAL METHODOLOGY

3.1 Reagents and chemicals

All chemicals and reagents, unless otherwise indicated, were obtained from suppliers and
used without further purification. All the chemicals used in this study were purchased from
Sigma-Aldrich, namely Gold salt, Silver salt, methanol, sodium dichloromethane, ethanol;
methanol extracts (extraction volume 200 ml/ 10 g ground dried plant materials).

3.2 Collection and Preparation of Plant Materials

The plant samples (leaves and bark) were collected from the indigenous plant nursery
located at the Walter Sisulu national botanical gardens, Roodepoort, Gauteng, South Africa.
The plant materials were washed with soap and tap water until no foreign material
remained. The samples were shade-dried for 5 days and ground into fine powder using an
electrical blender. The powdered samples were stored in an air tight container and
protected from sunlight for further use.

3.2.1 Preparation of Leaf Extracts

Aqueous extractions of leaves bark were carried out according to the method outlined by
Dubey et al. (2010) with minor modifications. For the aqueous extract, 20 g of thoroughly
dried leaves and bark were boiled in 250 ml autoclaved distilled water for 20 minutes. This
was filtered with Whatman No. 1 filter paper and the filtrate was sterilized with a syringe
filter through a 0.22 μm pore filter (Millipore). These extracts were kept in the refrigerator
at 4°C and were used within ± 5 days.

3.2.2 Preparation of 0.1 M NaAuCl4 solution

A 0.1 M NaAuCl4 solution was prepared by dissolving 0.2 g of NaAuCl4 salt in approximately
1 ml deionized water. This solution was further diluted and made up to 4 ml with deionized
water and the solution was stored in brown bottle.

30
3.3 Phytochemical Screening of Plant Extracts

These were carried out according to methods outlined by Harborne (1973); Trease and
Evans (1989) and Sofowara (1993). Healthy leaves and bark of Z. capense were dried in an
oven and ground to a fine powder using an electric blender. Phytochemical screening for the
presence or absence of terpenoids, steroids, alkaloids, flavonoids, tannins, reducing sugars
and phlobatannins were carried out on powdered specimens of the plants. The aqueous
extract was prepared by boiling 2 g of the powdered sample in 20 ml distilled water.

3.3.1 Terpenoids (Salkowski’s Test)

For the presence of terpenoids, 5 ml of each extract was mixed with 2 ml of chloroform.
Concentrated H2S04 (3 ml) was carefully added to form a layer. A reddish brown colouration
of the interface was formed showing a positive result for the presence of terpenoids.

3.3.2 Steroids Test

For the presence of steroids, 0.5 g powdered plant samples were boiled in 5 ml ethanol for
five minutes. This was treated with 2 ml acetic anhydride. 2 ml H2SO4 was carefully added,
drop-wise, to this and observed for a colour change from violet to blue/green indicating the
presence of steroids.

3.3.3 Alkaloids (Dragendroff’s Test)

For the presence of alkaloids, 2 ml of aqueous extracts were treated with a few drops of
Dragendorff’s reagent (Potassium Bismuth Iodide Solution) (Merck). Formation of a red
precipitate indicated the presence of alkaloids.

3.3.4 Flavonoids Test

For the presence of flavonoids, 1 g of the powdered plant samples was heated with 10 ml
ethyl acetate in a steam bath for three minutes. The mixtures were filtered and 4ml of the
filtrate of each was shaken with 1 ml diluted (1:10) ammonia solution. A positive result was
indicated by a yellow colouration.

31
3.3.5 Tannins

For the presence of tannins, 0.5 g of the powdered plant samples were each boiled in 20 ml
distilled water and a few drops of 0.1 % ferric chloride was added to each. These were
observed for a brownish green or black colouration to indicate a positive result.

3.3.6 Phlobatannins

For the presence of phlobatannins, 2 ml of aqueous extract of each of the plant samples was
boiled with 2 ml 1% Hydrochloric acid. The presence of phlobatannins was indicated by the
presence of a red precipitate at the base of the test tubes whereas a negative result was
indicated by the absence of a precipitate.

3.3.7 Reducing Sugars

For the present of the reducing sugars, 2 ml of Benedict’s solution was added to 10 ml of
water extracts of each sample, samples were boiled in a boiling water bath. The presence of
reducing sugars was indicated by yellow, orange and red colouration which also indicated
increasing relative concentrations of sugars present.

3.4 Biosynthesis of Silver and Gold Nanoparticles from Leaf and Bark Extracts
of Zanthoxylum capense

3.4.1 Synthesis of Silver Nanoparticles

1 M AgNO3 (Sigma) was dissolved in sterile distilled water. This was used to prepare 0.5 mM
AgNO3 solution. 100 µl of aqueous solution of 0.5 mM, AgNO3 were reduced using 2.5 ml of
the aqueous leaf and bark of Zanthoxylum capense extracts at room temperature
(approximately 25°C). The colourless solution was mixed with a stirrer bar and visually
observed for a yellow-brown colour change indicative of the formation of Ag-NPs (Dubey et
al., 2010). The solutions that produced a positive colour change were then stored at 4°C in
dark glass bottles.

3.4.2 Synthesis of Gold Nanoparticles

1 M HAuCl4 (Sigma) was prepared in sterile distilled water. This was used to prepare a 0.5
mM HAuCl4 solution. 100µl of aqueous solution of 0.5mM, HAUCL4 were reduced using 2.5
ml of the aqueous leaf and bark of Zanthoxylum capense extracts at room temperature
32
(approximately 25°C). The pale yellow solution was mixed with a stirrer bar and visually
observed for a maroon-purple colour change indicative of the formation of Au NPs (Dubey
et al., 2010). The solutions that produced a positive colour change were then stored at 4°C
in dark glass bottles.

3.5 Characterization of the synthesized silver and gold Nanoparticles

3.5.1 UV-visible Spectra Analysis

A scan in the wavelength range of 200 nm to 800 nm was carried out to confirm the
synthesis and stability of the NPs. Samples were poured into quartz cuvettes, placed into
the SHIMADZU UV-2540 spectrophotometer and absorbance measurements in relative light
units (AU) were taken. Cary WinUV software was used to analyse results. A cuvette of
distilled water was used as the blank. Ag NPs and Au NPs were expected to show a sharp
peak in the ranges 395 nm - 425 nm and 520 nm - 600 nm respectively according to
literature (Link and El-Sayed, 2000; Ju-Nam and Lead, 2008). The range with regard to this
study was refined to a smaller range ie. 400 nm - 425 nm for Ag NPs and 520 nm - 550 nm
for Au NPs so as to provide uniformity amongst the particles produced with respect to their
size and shape.

3.5.2 Transmission Electron Microscopy (TEM) Analysis

The JEOL JEM-1010 Transmission Electron Microscope at the University of Johannesburg

(Kingsway Campus) was used. The NPs samples were prepared by centrifugation of the NPs
solutions in an Eppendorf Centrifuge 5810 at 13000 rpm for 45 minutes. The supernatant
was siphoned off and the resulting suspension was re-suspended in distilled water. Each
specimen used for analysis was prepared by immersing a formva-coated copper grid into the
respective solution, removing the grid and allowing it to dry for a few minutes before
analysis. The size and morphology of the NPs were noted.

3.5.3 Fourier-Transformation Infrared (FTIR) Spectroscopic Analysis

The FTIR measurement was used to identify if the same biomolecules were responsible for
the synthesis and capping of NPs for the different extracts and between the Ag and Au NPs.
NPs samples were centrifuged in the Eppendorf Centrifuge 5810 at 13000 rpm for 45
minutes. The resulting suspension was re-suspended in distilled water and repeated thrice
33
to remove any free biological material that may interfere with the results (Jain et al., 2009).
The Perkin Elmer, Spectrum 100 FTIR spectrophotometer, was used to analyse the samples.
A drop of distilled water was used as a blank and this background was removed from the
sample scan. A drop of sample was then placed on the machine and a scan measuring
transmittance percentage over the range of wavenumbers from 500 cm-1 to 4000 cm-1 was
taken. E-FTIR software was used to analyse the results and ascertain peak values which was
then interpreted.

3.5.3 Dynamic light scattering (DLS) (Zetasizer)

The Ag-NPs and Au-NPs were further characterized using the DLS analyser (Zetasizer NANO
series equipped with 50 mW, 532 nm laser, model ZEN3600; Malvern Instruments, Malvern,
UK). The zeta potential was done to measure the charge on the surface of capped gold and
silver nanoparticle which provided information about the stability of the nanoparticle
dispersion. The size distributions and zeta potentials of the silver nanoparticles and gold
nanoparticles with plant extract were measured using Malvern Nano ZS Nano series
(Malvern Instruments, United Kingdom) following the dynamic light scattering (DLS)
technique (Nguyena et al., n.d.). The measurements were done with a 173° detector and
temperature set at 25°C. The samples were characterised for both size and surface charge
suspended in deionised water and sonicated for 5 minutes. The zeta potential was recorded
in millivolts (mV), while particle size was recorded in nanometres (nm).

3.6 Antibacterial Activity

The antibacterial activity of methanolic plant extracts and aqueous Ag-NPs and Au-NPs were
carried out on selected bacteria by agar disk diffusion assay method and the minimum
inhibitory concentration (MIC) method (Eloff, 1998). MIC is defined as the lowest
concentration of an antimicrobial agent that kills all the organisms in a given sample and is
carried out generally by dilution methods. In this test, microorganisms are tested on their
ability to produce visible growth in microplate wells containing broth and dilutions of the
potential antibacterial compounds.

34
3.6.1 Disk-Diffusion Antibacterial Assay

The fourteen bacteria used as test organisms were as follows: Bacillus cereus (ATCC10876),
B. subtilis (ATCC19659), Enterococcus faecalis (ATCC13047), Mycobacterium smegmatis
(MC2155), Staphylococcus epidermidis (ATCC14990), Staphylococcus aureus (ATCC 25923),
Escherischia coli (ATCC25922), Enterobacter cloacae(ATCC13047), Klebsiella pneumoniae
(ATCC13882), K. oxytoca (ATCC8724), Proteus mirabilis (ATCC7002), P. vulgaris(ATCC6380)
and Pseudomonas aeruginosa(ATCC27853). These strains are clinical pathogens obtained
from Davies Diagnostics, and are differentiated by their reference codes following the
species name designating their site of isolation from the host.

Stock cultures were prepared on Nutrient Agar plates (Biolab), sealed and stored at -70°C.
When required the cultures were grown in Mueller Hinton Broth (MHB) (Biolab) for 24h at
37°C. The concentration of bacterial cells was adjusted to visually match that of a 0.5
MacFarland Standard which corresponds to 108 CFU/ml (Matasyoh et al., 2007). 100 μl of
each bacterial sample was swabbed onto Mueller Hinton Agar (Biolab) plates. All the NP
solutions were centrifuged at 13 000 rpm for 60 minutes, the supernatant was siphoned off
and the NP pellet was weighed and re-suspended in DMSO (an important polar aprotic
solvent that dissolves both polar and nonpolar compounds and is miscible in a wide range of
organic solvents as well as water) to form 30 μg/ml solutions (Novak, 2002). 15 μl of all
solutions was pipetted onto 5.5 mm sterile filter paper (Whatman No. 5 disks and air dried
in a biological safety cabinet.

The treated discs were placed on the surface of the inoculated bacterial plates and
incubated at 37°C for 16 hours. Control disks with DMSO served as the negative control,
whilst AgNO3 (1 μg/ml) and HAuCl4 (1 μg/ml) were the positive controls and 1mM
Ciprofloxacin was used as the reference drug control. All tests were carried out in triplicate.
Streptomycin is a fluoroquinolone synthetic antimicrobial agent that is active against Gram-
positive and Gram-negative bacteria and thus regarded as a broad spectrum antibiotic. It
has been used to treat urinary tract infections, sexually transmitted diseases as well as
respiratory and gastrointestinal infections (Rosemary et al., 2006).

35
3.6.2 Minimum inhibitory concentration assay (MIC)

Microbiological assays are performed in order to determine the potency or concentration

of a test substance e.g. essential oil or extract to be able to determine its effects on the
growth of a micro-organism (Hewitt and Vincent, 2003). A fundamental principle to these
assays is that they depend on the comparison of the effect of a standard reference
substance e.g. an antibiotic such as ciprofloxacin, and a sample whose potency is to be
determined on a “biological system” e.g. a culture of micro-organisms. The comparison is
between the reference standard and the sample of unknown potency i.e. differences in
antimicrobial activity can be seen between a known antimicrobial agent and a sample that
has never been tested before (Hewitt and Vincent, 2003).

One of the simplest and widely preferred techniques used to analyse the effectiveness of an
antimicrobial‟s action is to determine the minimum inhibitory concentration (MIC) (Eloff,
1998; Hewitt and Vincent, 2003). The minimum inhibitory concentration is the lowest
concentration of an antimicrobial that completely inhibits the growth of an organism
(Bannister et al., 2000). It is based on the principle of contact of a test organism to a series
of dilutions of test antimicrobial/substance (van Vuuren, 2007).

3.6.2.1 Preparation of resazurin solution

Resazurin dye (0.02 g) was dissolved in 100 mL sterile water. Vortex mixer was used to
homogenize the solution. This solution was then referred as Resazurin dye solution.
Resazurin is an oxidation-reduction indicator used for the evaluation of cell growth,
particularly in various cytotoxicity assays (McNicholl et al., 2006). It is purple non-
fluorescent and non-toxic dye becomes pink and fluorescent when reduced to resorufin by
oxidation reduction within viable cells. Resorufin is further reduced to hydroresorufin
(uncoloured). Resazurin reduction test has been used from decades to demonstrate
bacterial and yeast contamination of milk (Bigalke. 1984).

36
3.6.2.2 Antibacterial screening

In vitro antimicrobial activity was determined using the modified broth micro dilution
method incorporating resazurin as an indicator of cell growth in 96-well micro titre plates
(Eloff, 1998). Sterile 96-well plates were marked appropriately with the plant extracts and
organisms to be tested respectively. Under aseptic conditions (laminar flow unit), 200 μl of
sterile water was added to outer wells of a 96 well micro-titre plate to minimize the
evaporation of culture medium of the test wells during incubation.

Rows A, B, C, E, F and G of a micro-well plate were filled with 100 μl sterile Mueller Hinton
Broth (MHB) using a micropipette. In Row A, column 1 to 5, NPs aqueous solution was
added to make a concentration of 128 μg/ml. From that well, 100 μl was transferred into
column 2 and was taken up and released three times to ensure adequate mixing. This was
done until column 6 which was a concentration of 0.125 μg/ml. The final 100 μl from this
well was discarded. Serial dilutions were then done in a vertical orientation, from A to H,
using a multi-channel pipette. The negative control was the solvent DMSO; one well filled
with MHB served as a sterility control and a well filled with MHB plus the test organism was
the growth control.

Bacterial culture (100 μl) was added to each well except the sterility controls. The
microplates were sealed and incubated overnight at 37°C for 12 hours. Following this, 10 μl
of 0.02% indicator Resazurin sodium salt was added to each well and incubated for 30
minutes. Inhibition of growth was indicated by purple/ blue suspensions whereas growth
was seen as red/pink/yellow coloured suspensions. The MIC was determined by the lowest
concentration at which there is no bacterial growth. Tests were performed in replicates of
five.

37
Well Description Selected test material Selected test material
and Well (100µl) + bacterial (100µl) + bacterial
Number suspension (100 µl) + suspension (100 µl) +
resazurin indicator solution resazurin indicator solution
(10µl) (10µl)
1 2 3 4 5 6 7 8 9 10 11 12
128 A - o o o o o      -
Serial Dilution in Tests Wells ( in

64 B - o o o o o      -

32 C - o o o o o      -

16 D - o o o o o      -

8 E - o o o o o      -

4 F - o o o o o      -

2 G - o o o o o      -
µg/ml)

1 H - o o o o o      -

Figure 3.1: Layout of 96-well test plate showing wells for testing activity of selected plant
extracts on individual test bacteria i.e. 2 types of bacteria per plate ( • and o
)(green and blue), d.H2O control wells (- )(orange).

38
 CHAPTER FOUR

RESULTS AND DISCUSSION

4.1 Phytochemical screening of plant extracts

Major phytochemical classes were tested to establish a connection between the compounds
present in the leaves and bark of the plant. Phytochemicals act as both reducing agents and
capping or stabilising agents. It is known that phenolic compounds are responsible for the
reduction in biosynthesis (Huang et al., 2007). The separation techniques used are based on
the polarity of the phytochemicals. The results of the phytochemical screening found in the
leaf and bark extracts of the plants are summarized in Table 4.1.

Table 4.1: Preliminary qualitative phytochemical analysis of crude extracts of Zanthoxylum

Capense
ZANTHOXYLUM CAPENSE
PHYTOCHEMICAL TEST LEAVES BARK
Terpenoids √ √
Tannins √ √
Alkaloids √ √
Flavonoids √ √
Steroids √ √
Phlobatannins √ √
Reducing sugars √ √

(√ = present)

Phytochemical analysis conducted on the plant extracts revealed the presence of

constituents which are known to exhibit medicinal as well as physiological activities
(Safowra, 1993). Analysis of the plant extracts revealed the presence of phytochemicals
such as alkaloids, tannins, flavonoids, saponins, phlabotannins, steroids and terpenoids.

39
Z. Capense aqueous extracts formed a reddish brown colour interface between the layers
which showed a positive result for steroids; formed a reddish colour owing to presence of
phlobatannins and produced a yellow colour change to indicate the presence of flavonoids.
Phlobatannins have been reported for its wound healing properties, these are anti-
inflammatory and analgesic (Ayinde et al., 2007) and antioxidant (Okwu and Omogbai,
2007), and epidemiologic studies recommend that coronary heart disease is opposed by
dietary flavonoids. Steroids are responsible for cholesterol-reducing properties, and also
help in regulating the immune response (Shah et al., 2009).

The leaf and the bark extracts of Z. Capense produced a brownish-black colouration
indicating the presence of tannins, and reddish brown colour for terpenoids. Tannins have
amazing stringent properties. They are known to hasten the healing of wounds and inflamed
mucous membranes. Terpenoids have been found to be useful in the prevention and
therapy of several diseases, including cancer, and are also reported to have medicinal
properties such as anti-carcinogenic, antimalarial, antiulcer, antimicrobial and diuretic
activities (Aharoni et al., 2005),

The leaf and bark extracts of Z. Capense formed a red precipitate giving a positive result for
the presence of alkaloids and tested positive for the present of reducing sugars. Plants
having alkaloids are used in medicines for reducing headache and fever. These are
attributed for antibacterial and analgesic properties (Pietta, 2000).

It was deduced by Shankar et al., (2004) that reducing sugars (aldoses) and terpenoids and
polyphenolic compounds play a key role in the reduction of Ag and Au ions and the
formation of corresponding NPs while ketones/ aldehydes bind to emerging spherical NPs to
form large nanotriangles and hexagons. Euphorbia prostate has been reported to possess
phytochemicals such as anthraquinones, flavonoids, phenols, phlobatannins,
polysaccaharides, saponins, tannins and terpenoids which was able to produce Ag NPs that
were toxic against parasites (Kamgang et al., 2007; Zahir and Rahuman, 2012). The results
produced in this study show that the major component responsible for the reduction of the
metal salts was tannins, reducing sugars, phlabotannins, saponins and flavonoids as these
was present in the plant.

40
4.2 Biosynthesis of Silver and Gold Nanoparticles from Aqueous Extracts

The green method of synthesizing silver and gold nanoparticles from silver and gold salts is
classed as the “bottom-up” approach as atoms assemble together, mounting into a
nanoscale particle (Kavitha et al. 2013). Plant extracts have phytochemicals and
biomolecules that are known to play the role of reducing agents in the synthesis of metallic
nanoparticles, by donating electrons (Kasthuri et al. 2009). The review by Kavitha et al
(2013) states that phytochemicals such as alkaloids, terpenoids, saponins, hydroxyflavones,
isoflavones, tannins, quinines, polyols and polyphenols; and biomolecules such as oxalic
acid, sucrose, fructose, salanin, thiamine, azadirone, curcacycline A and B as well as other
proteins, enzymes and carbohydrates have been reported to act as reducing agents in
mediating the synthesis of nanoparticles.

The phytochemicals and biomolecules extracted from the plant Zanthoxylum capense
aqueous extracts, believed to be acting as reducing agents are the saponins, tannins,
alkaloids, flavanoids, steroids, carbohydrates and proteins.

Scheme 4.1: Possible mechanisms for the green synthesis of silver and gold nanoparticles
using plants extracts.

41
The possible mechanism for the green synthesis for both silver and gold nanoparticles in this
research is depicted in Scheme 4.1 above. Babu et al. (2013) suggested the possible
mechanism of the reduction of gold salt in Au3+ state to gold nanoparticles in the Au0 state.
Their report stated that the phytochemicals and biomolecules having hydroxyl and carbonyl
groups first bind with the gold salt ions Au3+ to form gold complexes, which are later
reduced to gold seed particles and agglomerated to “clusters” (Babu et al. 2013). These
clusters act as nucleation centers, catalyzing the reduction of other Au3+ ions in solution.
These clusters accumulate to form gold nanoparticles.

Table 4.2 summarizes the results of the colour changes. The positive dark brown colour
change can be seen in Figure 4.1a and the positive reddish purple colour change when
mixed with AgNO3 and HAuCl4 (Fig 4.1b) respectively. The colour change was almost
instantaneous with gold nanoparticles whereas silver nanoparticles required stirring for few
hours. It was noticed that the Ag-NPs solution that formed from Z. capense turned from
yellowish-orange to dark brown after two hours.

Table 4.2: Results of colour change with 2 ml aqueous leaf and bark extracts when added
to (0.5 mM) AgNO3 and HAuCl4 solutions at 25°C

NPs/Plants Z. Capense (Leaves) Z. Capense ( Bark)

Silver (Ag) give the colour change give the colour change

Gold (Au) give the colour change give the colour change

It can also be stated that while some reduction reactions with the plant extract are
spontaneous, others require extra energy in the form of temperature, as shown by Ag-NPs
formation by Z. capense extract in comparison to Au NPs. It was found that as temperature
increases, so does the absorbance values. However, this does not necessarily mean that a
high absorbance produces a monodisperse solution of nanoparticles as is seen from UV-vis
scans which produced broad peaks (Philip et al., 2011).

42
 A B

Figure 4.1: Positive colour change results for (a) shows the colour change before and after
the reduction of Ag+ into Ag-NPs by Z. capense aqueous leaf extract reaction
with 0.5 mM solution of AgNO3 and (b) Z. capense aqueous leaf extract with
0.5 mM HAuCl4 (Au-NPs)

Visual observation for both gold and silver, the colour change from light yellow to dark
brown for sliver and from light yellow to purple for gold for aqueous plant extracts. The
colour change indicates the formation of nanoparticle.

Silver and gold salts are seen to react differently with the plant and at different parameters
possibly due to their different reduction requirements and the variation in each plants
phytochemistry. This leads to the conclusion that shape and size of NPs can be controlled by
varying the concentration of plant extract and reaction temperature.

4.3 Characterization Results and Analysis

4.3.1 UV-Vis spectra analysis

Qualitative analysis for the formation of silver and gold nanoparticles can easily be followed
using spectrophotometer. UV-Vis spectroscopy is a crucial and efficient technique in
determining the formation and stability of, not only gold, but other metal nanoparticles
(Sujitha and Kannan. 2013; Basavegowda et al. 2014). Colloid silver and gold nanoparticles
solutions show a very intense colour absent in the bulk materials as well as for individual
43
atoms. The intense colour is because of the “collective oscillation, or resonances, of free
conduction electrons” (Sujitha and Kannan 2013). These resonances are known as surface
plasmon resonances and their oscillation frequencies are usually located in the visible region
of the spectrum.

Colour of silver and gold nanoparticles are attributed to SPR arising due to the collective
oscillation of free conduction electrons induced by an interacting electromagnetic field
(Grzelczak et al., 2008). According to Mie’s theory, only a single SPR band is expected in the
absorption spectra of spherical NPs, whereas anisotropic particles could give rise to two or
more SPR bands depending on the shape of the particles. The analysis of SPR absorption
band can also provide valuable information on the size, structure and aggregation
properties of nanoparticles which makes UV-vis spectrophotometry a vital characterization
technique for NPs.

The excitation of surface plasmon vibrations of silver and gold nanoparticles exhibits
yellowish-brown and red purple colour, respectively which makes it easy to follow the
formation of gold and silver nanoparticles in the aqueous solution (Mirkin et al., 1996). In
the case of gold nanoparticles, the colour of gold solution was pale yellow and after the
addition of boiled aqueous extracts of Zanthoxylum capense it transformed into red purple
colour within 30 minutes. The reduction continued for 5 hours at 25°C. But, in the case of
silver the colourless solution of silver nitrate after the addition of boiled leaf extract of
Zanthoxylum capense took at least 2 hours to change its colour from colourless to light
yellow and further yellow to yellowish–brown but the complete reduction took
comparatively more time, i.e., 48 hours at 25°C as compared to that for the complete
reduction of gold nanoparticles. The most possible reason could be difference in their redox
potential.

44
Figure 4.2: UV-visible absorption spectra of representative Silver nanoparticles
synthesized using leaf (L) and Bark (B) extracts of Zanthoxylum capense
(0.5Mm of AgNO3).

45
The UV-Vis absorption spectrum of the Ag-NPs is shown in Figure 4.2. Absorption spectra of
Ag nanoparticles formed by reacting with leaf and back extracts of Zanthoxylum capense has
absorbance maxima at 443 nm and 419 nm respectively. It is well known that colloidal silver
nanoparticles exhibit absorption at the wavelength from 390 to 420 nm due to Mie
scattering (Kleemann, 1993) 20]. Hence, the band at 420-430 nm can be attributed to the
property of Mie scattering. This may not include the protecting agent, because the Mie
scattering responds only to the silver metal (Aoki et al., 2003). A remarkable broadening of
peak at around 350 nm to 480 nm indicates that the particles are polydispersed. It was
observed that the peak was blue shifted in the absorption spectrum from 350nm to 480 nm
with increasing reaction time.

From different literatures it was found that the silver nanoparticles show SPR peak at
around 420nm. So we confirmed that Zanthoxylum capense leaf and bark extracts has
potential to reduce Ag ions into Ag nanoparticles. The intensity of absorption peak increases
with increasing time period. This characteristic colour variation is due to the excitation of
the SPR in the metal nanoparticles the insets to Figure of graph represent the plots of
absorbance at λmax (i.e., at 420 nm) versus time of reaction

Figure 4.3 shows the UV-Vis spectra of the synthesized gold nanoparticle capped with
aqueous bark extract of Zanthoxylum capense. During the synthesis of the gold
nanoparticles, the colour change observed was from yellow to ruby red as seen on figure
4.1(b). This red colour observed is because gold atoms agglomerate to the nanoscale
forming gold nanoparticles, and it was observed that gold nanoparticle surface plasmon
resonance (SPR) band appeared at 548 and 567 nm. SPR is directly proportional to the
density of the nanoparticle in solution (Ahmad et al. 2013; Basavegowda et al. 2014).

These results are in agreement with literature; the purple colour of spherical gold
nanoparticles is observed when the surface plasmon resonance peaks from 560 – 595 nm
(Daniel and Astruc 2004). Furthermore, the nature of the surface plasmon resonance peak
of the synthesized gold nanoparticles indicates that the gold nanoparticles synthesized
dispersion has a narrow range. Such a conclusion from UV-Vis spectroscopy has been made
elsewhere in literature. Pandey et al. (2012) commented on mono and poly dispersion
indications from UV-Vis spectroscopy analysis of their synthesized gold nanoparticle.
46
 548 nm

Figure 4.3: UV-visible absorption spectra of representative gold nanoparticles synthesized

using the using leaf (L) and Bark (B) extracts of Zanthoxylum capense (0.5Mm of HAuCL4)

47
4.2.2 FT-IR spectra analysis

FT-IR spectrum analysis was carried out to obtain data of the functional groups in metallic
interactions. Understanding the functional groups capping agents on the gold and silver
nanoparticle surface is crucial as it helps identifying the potential biomolecules that are
stabilizing the nanoparticles.

a)

b)
Figure 4.4: FTIR absorption spectrum obtained from (A) silver nanoparticles biologically
synthesized by reduction of Ag+ ions and (B) gold nanoparticles biologically synthesized by
reduction of AuCl4- ions using bark broth of Zanthoxylum capense.

48
FTIR gives the information about functional groups present in the synthesised nanoparticles
for understanding their transformation from simple inorganic AgNO3 to elemental silver by
the action of the different phytochemicals which would act simultaneously as reducing,
stabilizing and capping agent. FTIR spectrum illustrates the bio fabrication of silver and gold
nanoparticles mediated by the plant extracts.

Figure 4.4 shows the FTIR spectrum of the Zanthoxylum capense synthesized gold and silver
nanoparticle colloid solution when capped with the extract.

For the synthesized silver nanoparticles (Fig 4.4 a), the peak at 1634 cm-1 can be assigned to
amide 1 bond of proteins responsible for stabilization of the nanoparticles by acting as a
capping ligand through -C-O-C-, -C-O-, and –C=C- functional group (Naheed et al., 2011). The
IR bands at 1162 cm-1 and 1360 cm-1 can be assigned to stretching and bending modes of C-
O and O-H of alcohols. The absorption bands around 2013 cm-1 and 3176 cm-1 are assigned
to C-H bending for aromatic compounds as well as O-H stretch for carboxylic acids.

The IR bands in Gold nanoparticles at 3743 cm-1 and 3065 cm-1 can be assigned to to
stretching modes of O-H of alcohols groups. Absorption band around 1158 cm-1, 1303 cm-1
and 1629 cm-1 are assigned to C-O stretch for alcohols and C=C for alkenes. IR bands around
981 cm-1 for Au nanoparticles and 985 cm-1 for Ag nanoparticles are assigned to C-O-C
vibrations for proteins and other heterocycles in the plant extract (Li et al., 2003).

The presence of various groups like –C=O, -C=C, -C-O etc. can be attributed to heterocyclic
water soluble components present in the extracts of Zanthoxylum capense. The
bioreduction reaction, in the present study, was done in the aqueous medium. Therefore, it
was inferred that some of the bio-organics of the aqueous leaves extract such as proteins,
flavonoids, phenols and polysaccharides bind to Ag atoms and are responsible for the
synthesis and stabilization of Ag-NPs. Such interactions between metal nanoparticles and
plant constituents have been reported in the literature (Phillips. 2011).

The FTIR results presented direct evidence for the formation of a chemical bond between
silver (Ag) and gold (Au) nanoparticles and the nitrogen of the amide group present in the
amino acids. It has been reported that either through free amino groups or cysteine in
amplitude of peaks and a small shift was observed in both cases, which may be attributed to
the difference in capping species and nature of co-ordination with metal surface for silver
49
residues, the protein is able to bind to the NPs that lead to the stabilization of NPs by
surface bound protein (Gole et al., 2001; MubarakAli et al., 2011).

Nanoparticles synthesized using plant extracts are surrounded by a thin layer of some
capping organic material which allows the stability of NPs in solution. The nature of
stabilizing agents plays an important role in size distribution and the shape of nanoparticles
which greatly determines the NPs functional properties (Widoniak et al., 2005). Chemical
materials and polymers have been used to stabilize NP solutions but these stabilizers are
only thermally removable at extremely high temperatures of more than 200°C and this can
be seen as a possible interference to the NPs themselves. Some chemical stabilizers like
ethanol are only able to keep a nanoparticle solution stable for 48 hours before complete
precipitation. Many physical and chemical synthetic methods have been used to synthesize
NPs but there are still problems experienced with stability, aggregation, control of
nanocrystal growth, shape and size. Green chemistry is showing promising results in order
to combat these problems (Korbekandi et al., 2009).

50
4.2.4 Transmission Electron Microscopy (TEM) analysis

Primarily the TEM analysis was done to see if the altered method used in this work was
reproducible in the synthesis of silver and gold nanoparticles. Samples were analysed to
investigate the reproducibility of the method. In each sample, images were taken and their
corresponding core size distribution histograms were used to compare with other samples.
Secondly, TEM analysis was to observe the shape of the gold and silver nanoparticles, and
when looking at Figures 4.5 and 4.6 the shape of the silver and gold nanoparticles
synthesized are nearly spherical in nature and are poly dispersed. For the measurements of
the silver and gold nanoparticles diameters, Image J software was used.

a) b)

c)

Figure 4.5: TEM images of Ag NPs (0.5mM) taken at (a) 100nm (b) 50 nm and (c) size-
distribution histogram of the Ag NPs.

51
Tem analysis for silver and gold nanoparticles was observed. From TEM images it can be
seen that silver nanoparticles were polydispersed and ranged in size from 2-30 nm. At
higher magnification the nanoparticles are more clearly observed. They were spherical in
shape and few nanoparticles were agglomerated. Dense nanoparticles can be seen at lower
magnification (figure 4.5 (a)) spherical nanoparticles are clearly observed and are seemed to
be separated by interparticles distance. Images clearly indicate presence of darker material
covered by a light material capping (Rajesh et al., 2009). And the capping can be assigned to
polyphenols, flavonoids, sterols, proteins or reducing sugars like glucose and fructose
present in Zanthoxylum capense.

Figures 4.6 (a-c) showed the TEM analysis of nearly spherical Au-NPs. The particle sizes are
distributed in the range of 5-40 nm, with an average particle size of 18 nm. (Figure 4.6 c)).
This large variation in particle size was due to the presence of a few irregular shaped
particles. The nanoparticles appear to be considerably smaller than the average particle size
observed with the DLS analyzer, presumably arising from the dry state of the TEM
measurements. Also, the large size of particles observed by DLS is due to the fact that the
measured size also includes the bio-organic compounds enveloping the core of the Au-NPs.

It is evident that the gold nanoparticles are surrounded by a faint thin layer of other
materials. The synthesized Au-NPs were capped by the plant constituents (polysaccharides,
protein, polyphenolic and flavonoidal compounds) that prevented their aggregation.
Inherent capping offers the additional advantage of the stability in the green chemical
synthesis (Morones et al., 2005).

52
 a) b)

c)

Figure 4.6: TEM images of Au NPs (0.5mM) taken at (a) 100nm (b) 50 nm and (c) size-
distribution histogram of the Au NPs.

53
 4.2.5 Particle size and potential charge analysis

4.2.5.1 Nano particle size analysis

The dynamic light scattering (DLS) analysis is used to measure the shell thickness of a
capping or stabilizing agent enveloping the metallic nanoparticles along with the actual size
of the metallic core. Figure 4.7 and 4.8 shows the particle size distribution of the
biosynthesized Ag-NPs using DLS measurements. After analysing data, it was found that Ag
nanoparticle size were in the range of 50-800 nm for the leaf Ag-NPs extracts and 37.84-
91.28 nm for the bark Ag-NPs. The average particle size of the Ag-NPs was found to be
around 78 nm and 50 nm respectively.

Figure 4.7: Size distribution graph of the hydrodynamic size of the silver nanoparticles
treated with aqueous Leaf extract of Zanthoxylum capense

54
Figure 4.8: Size distribution graph of the hydrodynamic size of the silver nanoparticles
treated with aqueous bark extract of Zanthoxylum capense.

Figure 4.9 and 4.10 shows the particle size of the gold nanoparticles samples. After
analysing data, it was found that Au nanoparticles size was in the range of 105-1000 nm and
43.82-615.1 nm respectively. Similarly, the analysis shows the graphical representation of
average particle size distribution of Au nanoparticles. The average particle size of the Au-
NPs was found to be around 164 nm and 68 nm respectively. The hydrodynamic radius (size
of the particle including the capping agent of Au NPs was found to be 719.6 and 432.5nm for
gold nanoparticles.

The polydispersity index (PDI) was 0.384 and 0.530 respectively; Polydispersity index
represents the ratio of particles of different sizes to total number of particles. Higher the
polydispersity index less is the monodispersed particles. Synthesis of monodispersed
nanoparticles by biological method has been a challenging task in nanotechnology. The large
size particles observed by DLS is due to the bio-organic compounds enveloping the core of
the GNPs (Prathna et al. 2011). Poly disparity index (PDI) is a measurement for distribution
of metal nanoparticles with from 0.000 to 0.5. PDI greater than 0.5 values indicate the
aggregation of particles. Form the table 4.3 it was clear that all the nanoparticles
synthesized from Z. Capense aqueous extracts does not aggregate except for bark
synthesized gold nanoparticles that have a PDI of 0.530.

55
Table 4.3: Synthesized silver and gold nanoparticles from aqueous extracts of the plant Z.
capense, size, PDI, and zeta potential.

ZANTHOXYLUM CAPENSE
SI NO NPs/ Extracts DLS (Size) PDI Zeta Potential
1 Silver (Leaves) 78.82 0.449 -32.6

Silver (Bark) 50.75 0.468 -30.6

2
Gold (Leaves) 164.2 0.384 -27.1
3
Gold (Bark) 68.06 0.530 -31.9
4

Figure 4.9: Size distribution graph of the hydrodynamic size of the gold nanoparticles
treated with aqueous Leaf extract of Zanthoxylum capense

56
Figure 4.10: Size distribution graph of the hydrodynamic size of the gold nanoparticles
treated with aqueous bark extract of Zanthoxylum capense.

57
4.2.5.2 Zeta potential analysis

Zeta potential analysis was carried out to investigate the stability of the silver and gold
nanoparticles synthesized. Zeta potential (ZP) values reveal information regarding the
surface charge and stability of biosynthesized nanoparticles. Figure 4.11 and 4.12 shows the
corresponding average Zeta Potential value (−32.6 and -30.6 mV for silver nanoparticles)
and (-27.1 and -31.9 mV for gold nanoparticles) suggesting higher stability of colloidal Ag-
NPs and Au-NPs.

Zeta Potential Distribution

200000

150000
Total Counts

100000

50000

0
-100 0 100 200
Apparent Zeta Potential (mV)

A
Silver NP B (0.5mM)

Figure 4.11: Zeta potential distribution graph illustrating the zeta potential of Z. capense
aqueous leaf and bark silver nanoparticles (0.5mM)

58
The net charge of -30.8 and mV reported in Figure 4.11 for the synthesized silver
nanoparticles indicate that the nanoparticles apply electric repulsive forces on each other.
This is typical of green methods (using plant extracts) for the synthesis of metallic
nanoparticles. The zeta potential values for green synthesis are generally lower than those
of other synthesis processes and as a result generally have stability of a few weeks,
compared to the few months for the other processes (Kavitha et al. 2013).

Zeta Potential Distribution

200

200000

150000
Total Counts

100000

50000

0
-100 0 100 200
Apparent Zeta Potential (mV)
B
Gold NP B (0.5mM)

Figure 4.12: Zeta potential distribution graphs illustrating the zeta potential of Z. capense
aqueous leaf and bark gold nanoparticles (0.5mM)

59
Nanoparticles are very small in size for which they are energetically very unstable.
Therefore, the particles undergo agglomeration/aggregation to stabilize themselves. So
there were some potential charges on the surface of the nanoparticles which makes them
stable. These charge potential we got from this analysis. Zeta potential (Surface potential)
has direct relation with the stability of a form/structure as mentioned below (Table: 4.4).

Table: 4.4: A table showing the stability of the nanoparticles according to the potential
charge

Zeta potential [mV] Stability behaviour of the colloid

from 0 to ±5 Rapid coagulation or flocculation
from ±10 to ±30 Incipient instability
from ±30 to ±40 Moderate stability
from ±40 to ±60 Good stability
more than ±61 Excellent stability

60
4.3 Antibacterial activity of Zanthoxylum capense leave and bark extracts,
(Nanoparticles)

4.3.1 Disk Diffusion Antibacterial Assay

Table 4.5 provides a comprehensive summary of the average results of the disk diffusion
zones of inhibition. The negative control DMSO gave a negative result with no inhibition
against all the bacteria. Streptomycine showed antibacterial activity with eight species i.e. E.
aurogenes, K. oxytoca, M. smegmatis, P. vulgaris, P. aeruginosa, P mirabilis, B. cereus and S.
epidermidis and had no activity on the rest of the bacteria. The Au-NPs solution exhibited
activity against six bacteria. The highest zone of inhibition against M. smegmatis was by Ag-
NPs (7.5 mm) and B. subtilis (6.8 mm) as well as S. epidermidis (6 mm) in comparison to Au-
NPs and the aqueous extracts which showed smaller zones of inhibition. For majority of the
bacteria, the zones of inhibition vary between the samples tested. HAuCl4 results are not
included since it did not work.

Table 4.5: Antibacterial activity from the aqueous leaf and bark extracts of Z. capense and
aqueous nanoparticles.

Zone of inhibition (mm)

Zanthoxylum Capense Controls
Bacteria Leaves Bark AuNPs AgNPs AgNO3 Positive (-)
control control
1: Bacillus cereus (ATCC10876) nil nil 0 5 16 2.6 0
2: B. subtilis (ATCC19659) 0 nil 4.2 6.8 11 nil 0
3: Enterococcus faecalis 3.2 3.7 nil 2.3 9 nil 0
(ATCC13047
4: Mycobacterium smegmatis 2 3.3 3.4 7.5 8 2.1 0
(MC2155)
5: Staphylococcus epidermidis 3.3 3 4.3 nil 8 1 0
(ATCC14990),
6: Staphylococcus aureus (ATCC nil nil 1.65 6 11 nil 0
25923),
7: Escherischia coli (ATCC25922), 1.7 1.60 nil nil 20 nil 0
8: E. aurogenes nil nil 2 3.2 5 4.6 0
9: Klebsiella pneumoniae 2.9 0.6 nil nil 11 nil 0
(ATCC13882)
10: K. oxytoca (ATCC8724), 4.1 2 nil nil 5 5.1 0
11: Proteus mirabilis (ATCC7002 2.5 2.1 nil 0.8 0 0
12: P. vulgaris(ATCC6380) 2.6 4 5.8 4 9 4.2 0
13: Pseudomonas nil nil nil nil 7 3.4 0
aeruginosa(ATCC27853)
14: Proteus mirabilis (ATCC7002) 2.5 2.1 nil 3 4 1.1 0
61
The Z. capense plants extracts solution exhibited activity against nine out of fourteen
bacteria. The best activity for the aqueous plant extracts (4.1 mm) was seen against K.
oxycota. The lowest zone of inhibition found was for K. pneumonia (0.6 mm). Z. capense
aqueous Ag-NPs had its highest activity against M. smegmatis with a zone of inhibition of
7.5 mm, B. subtilis with a zone of 6.8 mm as well as S. aureus and Bacillus cereus with the
zone of 6 mm and 5 mm respectively. Its lowest activity was against Proteus mirabilis with a
zone of 0.8 mm. however, Ag -NPs showed moderate activity for any other bacteria as well.

Aqueous Au-NPs showed moderate antibacterial activity with its highest zone of inhibition
of 5.8 mm against P. vulgaris. The aqueous Au- NPs produced their lowest activity with
zones of 1.65 mm against S. aureus (as was seen with plant extracts). It was also noticed
that these Au- NPs had moderate activity against all strains S. epidermidis as well as B.
subtilis.
The antibacterial activity of Ag and Au NPs were investigated by Kim et al. (2007), while the
Ag NPs with an average diameter of 13.5 nm showed positive results against E. coli and S.
aureus, the Au NPs of approximately 30 nm displayed no activity. The best activity on
average in this study was shown by Z. capense Au NPs which were in the size range of 5 nm
to 45 nm and therefore is agreeable with the above investigation.

The antimicrobial activity in terms of inhibition zones significantly varied with test microbes
and types of NPs. This differential antimicrobial activity observed in this study can be
attributed to the differential size range of Au-NPs in the same sample. There is a
discrepancy when it comes to finding a correlation between the antibacterial activity of NPs
and specific bacteria by different researchers because experiments produce NPs that are
poorly defined and characterized and the usage of different strains of bacteria. There is no
standard to use and thus a lot of work needs to be done if a reference system is to be
envisioned.

There are contrasting reports when it comes to the antibacterial activity of Au-NPs. Some
studies show positive results for Au-NP bactericidal activity and believe that it is initially
mediated by strong electrostatic attractions to the negatively charged bilayer of the cell
membrane due to cationic Au-NPs being found to be moderately toxic while anionic
particles were unreactive. Another study showed that Au-NPs did not have any bactericidal
62
activity on their own (Burygin et al., 2009). Although the Au-NPs synthesized by the plant
leaf extracts in this study were biologically inert, there is the possibility of them being
engineered to possess antibacterial functionality. Au NPs that were chemically synthesized
had bactericidal activity against E. coli and did not produce any ROS-related processes which
could be the cause of low toxicity of Au NPs to mammalian cells (Brust et al., 1994).

4.3.2 Minimum Inhibitory Concentration Assay

The results of the MIC assay for the leaf extracts and aqueous Au-NPs of plant Z. capense
against each of the bacteria can be seen in Table 4.6. DMSO which served as the negative
control and the sterility control wells produced pink solutions when the indicator was
added.
The MIC results of the Z. capense Au-NPs (Table 4.6) showed poor antimicrobial activity
against K. pneumoniae, P. mirabilis and E. coli with mean MIC values at 16, 64 and 16 mg/ml
respectively. Some antimicrobial activity was seen against P. valgaris (4.0 mg/ml).
Noteworthy antimicrobial activity was seen against S. aeruginosa, M. smegmatis and B.
cereus whereby mean MIC values of 2.0, 1.0 and 2.0 mg/ml were obtained. Thus Z.capense
Au-NPs was the most active in inhibiting the growth of M. smegmatis and can be considered
a moderate antimicrobial inhibitor. Ag NPs results were not included since it was not
conclusive

63
Table 4.6: Minimum Inhibitory Concentration results of Methanol leaves extract and Au-
NPs synthesized from aqueous leaf extracts of Z. capense.

MIC (mg/ml)
Bacteria
Zanthoxylum capense
Aqueous leaves Aqueous Au-NPs
extracts
Bacillus cereus ˃32 2
(ATCC10876)
Enterococcus faecalis 32 -
(ATCC13047
Mycobacterium 32 2
smegmatis (MC2155)
Staphylococcus 32 -
epidermidis (ATCC14990),
Escherischia coli 32 16
(ATCC25922),
Klebsiella pneumoniae 32 16
(ATCC13882)
Proteus mirabilis ˃32 ˃64
(ATCC7002
P. vulgaris(ATCC6380) ˂1 4

Pseudomonas ˂1 1
aeruginosa(ATCC27853)

64
 CHAPTER FIVE

CONCLUSSION AND RECOMENDATIONS

Nanotechnology is a quite recent and promising field due to the novel and useful
applications of the nanomaterials (NM)) which form the basis for many technological fields
and scientific areas. This field has opened the door for the production of uncountable new
materials due to the discovery of fascinating and unique properties of the matter in the
nanoscale. The influence of the nanotechnology on modern society is both explained and
justified by the scale of the variety of scientific and technological applications using nano-
based ideas. Although engineered nanoparticles are designed to fulfil a special purpose and
we may have an idea about their original properties during their production, the fate and
properties of the NPs.

The present work reports a simple, novel and successful synthesis of silver and gold
nanoparticles using Zanthoxylum capense aqueous leaf and bark extracts as a novel
reducing and stabilizing agent of silver and gold salts. UV-Vis spectral analysis confirmed the
surface plasmon resonance of biosynthesized Ag-NPs and Au-NPs. The DLS and TEM images
studies had shown that the synthesized Ag-NPs and Au-NPs have a size below 30 nm and
50nm respectively. Zeta potential value for biosynthesized Ag-NPs was (-32.6 and -30.6 mV),
Au-NPs was (−27.1 and -31.9 mV) indicating the stability of the nanoparticles. From FT-IR
spectra, it was found that biomolecules responsible for capping and stabilization of Ag-NPs
and Au-NPs were heterocyclic water soluble compounds present in the Zanthoxylum
capense aqueous extracts.

Moreover, Antibacterial potential of both Ag and Au nanoparticles as a function of

nanoparticles concentration was tested against different bacteria, gram positive as well as
gram negative. The test was performed by both Disc diffusion assay and minimum inhibitory
concentration (MIC) method. From the study, nanoparticles were observed to have
moderate antimicrobial potential. When the result was compared with the effect by
antibiotics like Streptomycin, nanoparticles were found to be more potent than antibiotics.

65
Green synthesis of NPs is a necessary technology that requires more research. The results of
this study show that Z capense is a plant that can produce useable NPs. Monodispersity and
stability are problems with biosynthetic procedures that need to be resolved. Au NPs have
proven to have antibacterial activity. Although, Ag NPs formed from the plants does not
have good antibacterial properties, these particles are still useful for other applications.

From the study presented in this thesis, it can be recommended that some investigations be
carried out on these Au and Ag- NPs to optimize the antimicrobial activity of other agents.
The development of new antibacterial agents through nanoformulation is required to be
explored.

Compare the effect of the plant used on the different metal nanoparticles, varying the
synthesis parameters such as Ph., temperature as well as volume. Different solvents should
be used to extract the plant materials before synthesis.

A more comprehensive toxicity assessment to ensure the safety of the gold nanoparticles,
both in the environment and in living systems should be done; also analysis on the fate of
the gold and silver nanoparticles in living systems and in the environment are all key factors
in determining the safety of the nanoparticles synthesized for biomedical applications.

66
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