ANTIBACTERIAL EVALUATION OF AFANG LEAF

EXTRACT AND ITS SYNTHESIZED SILVER

NANOPARTICLES

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 CHAPTER ONE

INTRODUCTION

1.0 BACKGROUND OF THE STUDY

Nanoscience has been the subject of substantial research in recent years. It has

been explored by researchers in various fields of science and technology (Kholoud

et al. 2010). The novel properties of NPs have been exploited in a wide range of

potential applications such as in medicine, cosmetics, renewable energies,

environmental remediation, biomedical devices (Quang Huy, 2013), electronics,

optics, organic catalysis, vector control, sensor, etc., have drawn extensive

attention to this field of study (Mousavand et al. 2007). Among the metals, silver

nanoparticles have shown potential applications in various fields such as the

environment, bio-medicine, catalysis, optics and electronics (Rao et al., 2000).

Silver nanoparticles are mostly smaller than 100 nm and consist about 20–15,000

silver atoms. In its nanoscale form, silver exhibits unique physicochemical and

biological activities. This has made them useful as sensor, vector control,

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antimicrobial, anticancer, and antiplasmodial agents, catalysts, among others

(Elemike et al. 2014; Vinod et al. 2014; Kathiravan et al. 2014; Saraschandra and

Sivakumar 2014; Namita and Soam 2014).

Concerted effort has been made to synthesize diverse range of silver nanop articles

varying in size, geometry, and morphology because of their potential applications,

particularly in electronics (P. V. Kamat, 2002), electrochemical sensing (L. M. Liz-

Marzán, 2006), catalysis (F. Zhang, Y. Pi et al., 2007), and antimicrobial properties

(T. Sakai et al., 2006). The size, geometry, dispersion and stability often determine

the suitability of the nanoparticles for certain applications. Synthesis may involve

physical means such as ultraviolet light, microwaves, photo-reduction, or chemical

reduction using hydrazine, ascorbic acid, sodium borohydride, glucose, and

organic stabilizers or biological means using plant extract, microorganism or plant

sap. Several physical and chemical methods have been used to synthesize and

stabilize silver nanoparticles (Senapati et al., 2005, Klaus-Joerger et al., 2001).

The most popular chemical approaches, including chemical reduction using a

variety of organic and inorganic reducing agents, electrochemical techniques,

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physicochemical reduction, and radiolysis are widely used for the synthesis of

nanoparticles.

Although these means are fast and easy, they are either expensive or toxic

particularly the chemical method and may lead to non eco-friendly byproducts thus

the need for environmental, nontoxic synthetic protocols for nanoparticles

synthesis. In the global efforts to reduce generated hazardous waste, “green”

chemistry and chemical processes are progressively integrating with modern

development in science and industry (Sharma et al., 2009) leading to the

developing interest in biological approaches which are free from the use of toxic

chemicals as by products. Biological methods can be used to synthesize

nanoparticles without the use of any harsh, toxic and expensive chemical

substances. The bioreduction of metal ions by combinations of biomolecules found

in the extracts of certain organisms (e.g., enzymes/proteins, yeast, fungi, bacteria

and plants) is environmentally benign, yet chemically complex (Ankamwar et al.,

2005). It has been elucidated that biomolecules with carbonyl, hydroxyl, and amine

functional groups have the potential for metal ion reduction and capping of the

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newly formed particles during their growth processes (Harekrishna et al., 2009,

He et al., 2007). Biomolecules in plants and spices extract are essential oils

(terpenes, eugenols, e.t.c.), polyphenols, carbohydrates, e.t.c. and can reduce and

stabilize Ag+ to Ag0. It provides advancement over chemical and physical methods

as it is cost effective and environment friendly.

1.1 LITERATURE REVIEW

Disease-causing microbes are becoming resistant to drug therapy and therefore

poses great public health problem. Many researchers are now engaged in

developing new effective antimicrobial reagents with the emergence and increase

of microbial organisms resistant to multiple antibiotics, which will increase the

cost of health care. Colloidal silver has been known for a long time to possess

antimicrobial properties and also to be non-toxic and environmentally friendly. It

has been used for years in the medical field for antimicrobial applications such as

burn treatment (Parikh et al. 2005; Ulkur et al 2005), elimination of

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microorganisms on textile fabrics (Jeong et al. 2005; Lee et al. 2007; Yuranova et

al. 2003), disinfection in water treatment (Russell and Hugo 1994; Chou et al.

2005), prevention of bacteria colonization on catheters (Samuel and

Guggenbichler 2004; Alt et al. 2004; Rupp et al. 2004), etc. It has also been found

to prevent HIV from binding to host cells (Sun et al. 2005). The mechanism of the

bacterial effect of AgNP as proposed is due to the attachment of AgNPs to the

surface of the cell membrane, thus disrupting permeability and respiration

functions of the cell (Kevitec et al. 2008). It is also proposed that AgNPs not only

interact with the surface of a membrane but can also penetrate inside the bacteria

(Morones et al. 2005), but the effects of silver nanoparticles (AgNP) on

microorganisms have not been developed fully. Researchers believe that the

potential of colloidal silver is just beginning to be discovered (Dorjnamjin et al.,

2008).

1.2 Nanotechnology
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Nanoparticles are viewed as the fundamental building blocks of nanotechnology

(Mansoori et al., 2005). They are the starting points for preparing many

nanostructured materials and devices and their synthesis is an important

component of the rapidly growing research efforts in nanoscience and

nanoengineering (Mansoori et al., 2007).

In nanotechnology, a nanoparticle is defined as a small object that behaves as a

whole unit in terms of its transport and properties. Nanoparticles can equally be

called ultrafine particles since their sizes range from 1 to 100 nm. Fine particles

ranges from 100 to 2,500 nm, while coarse particles are sized between 2,500 and

10,000 nm (Williams, 2008). A nanometer is one billionth of a meter (10 -9 m),

roughly the width of three or four atoms, smaller than the wavelength of visible

light and a hundred-thousand the width of human hair.

Nanoparticles can be made of materials of diverse chemical nature, the most

common being metals, metal oxides, silicates, non-oxide ceramics, polymers,

organics, carbon and biomolecules. Nanoparticles exist in several different

morphologies such as spheres, cylinders, platelets, tubes, flowers, cubes etc. They

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possess unique physiochemical, optical and biological properties which can be

manipulated to suit a desired application. Nanoparticles are of great interest due to

their externally small size, and large surface to volume ratio. They exihibit utterly

novel characteristics compared to the large particles of the bulk material and have

been included in fields of science as diverse as surface science, organic chemistry

molecular biology, semi conductor physics, microfabrication, material science,

inorganic chemistry and so on.

The concepts that seeded nanotechnology were first discussed in 1959 by

renowned physicist Richard Feynman in his talk “There's Plenty of Room at the

Bottom”, in which he described the possibility of synthesis via direct

manipulation of atoms. In 1974, “Norio Taniguchi now used the word

nanotechnology to describe precision manufacturing materials at the nanometer

level which refers to the synthesis, manipulation, and control of matter at nano

dimensions that will make most products lighter, stronger, cleaner, less

expensive and more precise.

1.3 Physiochemical Properties of Nanoparticles

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 Nanoparticles also often possess unexpected optical properties as they are

small enough confine their electrons and produce quantum effects e.g.

gold nanoparticles appear deep red in dark solutions.

 A unique property among nanoparticles is quantum confinement in

semiconductor particles, surface plasmon resonance in some metal

particles and super paramagnetism in magnetic materials. For example,

ferroelectric materials smaller than 10 nm can switch their magnetization

direction using room temperature thermal energy. Thus this property is

not always desired in nanoparticles thus making them unsuitable for

memory storage.

 Suspensions of nanoparticles are possible since the interaction of the

particle surface with the solvent is strong enough to overcome density

differences, which otherwise usually result in a material either sinking or

floating in a liquid.

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  The high surface area to volume ratio of nanoparticles provides a

tremendous driving force for diffusion, especially at elevated

temperatures. Sintering can take place at lower temperatures, over shorter

time scales than for larger particles.

1.4 Methods of Nanoparticles Synthesis

Currently, many methods have been reported for the synthesis of nanoparticles

which include chemical, physical, biological and photo-induced approach.

1.4.1 Chemical Approach:

The chemical approach is the most used method since it for provides an easy way

to synthesize nanoparticles in solution. This consists of the chemical reduction of a

metal salt in solution followed by the crystallization of zero-valence metal

particles. The particle synthesis is usually conducted in the presence of a

stabilizing agent that prevents excessive molecular growth and/or aggregation of

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the metal nanoparticles. Hence when nanoparticles are produced by chemical

synthesis, three main components are needed: a salt (e.g. AgNO3), a reducing agent

(e.g. ethylene glycol) and a stabilizer agent (e.g. PVP) to control the growth of the

nanoparticles and prevent them from aggregating.

In one study, Oliveira and coworkers (2005) prepared dodecanethiol-capped silver

NPs, according to Brust procedure (Brust et al., 2002) based on a phase transfer of

an Au3+ complex from aqueous to organic phase in a two-phase liquid-liquid

system, which was followed by a reduction with sodium borohydride in the

presence of dodecanethiol as stabilizing agent, binding onto the NPs surfaces,

avoiding their aggregation and making them soluble in certain solvents. They

reported that small changes in synthetic factors lead to dramatic modifications in

nanoparticle structure, average size, size distribution width, stability and self-

assembly patterns.

1.4.2 Physical Approach:

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In physical processes, nanoparticles are synthesized by evaporation-condensation,

exploding wire technique, chemical vapour deposition, microwave irradiation,

pulsed laser ablation, supercritical fluids, sonochemical reduction, and gamma

radiation with evaporation-condensation and laser ablation being the most important

physical approaches. The absence of solvent contamination in the prepared thin films and

the uniformity of NPs distribution are the advantages of physical synthesis methods in

comparison with chemical processes.

Siegel and colleagues (2012) demonstrated the synthesis of AgNPs by direct metal

sputtering into the liquid medium. The method, combining physical deposition of

metal into propane-1, 2, 3-triol (glycerol), provides an interesting alternative to

time-consuming, wet-based chemical synthesis techniques. Silver NPs possess

round shape with average diameter of about 3.5 nm with standard deviation 2.4

nm. It was observed that the NPs size distribution and uniform particle dispersion

remains unchanged for diluted aqueous solutions up to glycerol-to-water ratio 1 :

20.

1.4.3 Biological Approach:

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As stated earlier in the chemical method of synthesis, three main components are

needed: a salt (e.g. AgNO3), a reducing agent (e.g. ethylene glycol) and a stabilizer

agent (e.g. PVP) to control the growth of the nanoparticles and prevent them from

aggregating. In biological synthesis of nanoparticles, the reducing agent and the

stabilizer are replaced by molecules produced by living organisms. These reducing

and/or stabilizing compounds can be utilized from bacteria, fungi, yeasts, algae or

plants.

The development of efficient green chemistry methods for synthesis of

nanoparticles has become a major focus of researchers. In the global effort to

reduce generated waste and toxic materials, “green” chemistry and chemical

processes are progressively integrating with modern developments in science and

industry. They have investigated in order to find an eco-friendly technique for

production of well-characterized nanoparticles. Various approaches using plant

extracts have been used for the synthesis of nanoparticles. These approaches have

many advantages over chemical, physical, and microbial synthesis because there is

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no need of the elaborate process of culturing and maintaining the cell, using

hazardous chemicals, high-energy and wasteful purifications.

The first successfully reported synthesis of nanoparticles assisted by living plants

appeared in 2002 when it was shown that gold nanoparticles, ranging in size from

2-20 nm, could form inside alfalfa seedlings. Subsequently it was shown that

alfalfa could form silver nanoparticles when exposed to a silver rich medium.

Other works on plants and plant parts that have been used for the synthesis of

silver nanoparticles are Thevetia peruviana latex (Rupiasih et al. 2013), Wrightia

tinctoria (Bharani et al. 2011), Solanum xanthocarpum (Muhammad et al. 2012),

Opuntia ficus (Silva-de-Hoyos et al. 2012), Sphaeranthus amaranthoides

(Swarnalatha et al. 2012), Punica granatum (Naheed et al. 2012) Citrullus

colocynthis (Satyavani et al. 2011), Eucalyptus chapmaniana (Ghassan et al.

2013), Acacia auriculiformis (Nalawade et al. 2014), Ficus benghalensis,

Azadirachta indica (Debasis et al. 2015), e.t.c.

The biomolecules present in these plants are responsible for the formation and

stabilization of silver nanoparticles (Iravani et al. 2014). Nanoparticles produced

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by plants are more stable and the rate of synthesis is faster than in the case of

microorganisms. Moreover, this method is simple, cost effective, energy-saving

and reproducible. The nanoparticles are more various in shape and size in

comparison with those produced by other organisms. The advantages of using

plant and plant-derived materials for biosynthesis of metal nanoparticles have

interested researchers to investigate mechanisms of metal ions uptake and

bioreduction by plants, and to understand the possible mechanism of metal

nanoparticle formation in plants.

1.4.4 Photo-induced Approach:

The photo-induced synthetic strategies can be categorized into two distinct

approaches, that is the photo-physical (top down) and photochemical (bottom up)

ones. The former could prepare the NPs via the subdivision of bulk metals and the

latter generates the NPs from ionic precursors. The NPs are formed by the direct

photo-reduction of a metal source or reduction of metal ions using photo-

chemically generated intermediates, such as excited molecules and radicals which

are known as photosensitization in the synthesis of NPs.

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Huang and coworkers (2008) reported the synthesis of silver NPs in an alkaline

aqueous solution of AgNO3/carboxymethylated chitosan (CMCTS) using UV light

irradiation. CMCTS, a watersoluble and biocompatible chitosan derivative, served

simultaneously as a reducing agent for silver cation and a stabilizing agent for the

silver NPs. The diameter range of produced silver NPs was 2–8 nm, and they can

be dispersed stably in the alkaline CMCTS solution for more than 6 months

The main advantages of the photochemical synthesis are;

 It is a clean process, with high spatial resolution, and convenience of use.

 It has great versatility; the photochemical synthesis enables one to fabricate

the NPs in various mediums including emulsion, surfactant micelles,

polymer films, glasses, cells, etc.

1.5 Applications of Nanoparticles

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There is wide applicability of nanoparticles due to their interesting optical,

conductive, physio-chemical, electronic, antimicrobial properties.

1.5.1 Medical and Pharmaceutical Applications

Nanoparticles can be made to control and sustain release of the drug during the

transportation and as well as the location of the release since the distribution and

subsequent clearance of the drug from the body can be altered. An increase in

the therapeutic efficacy and reduction in the side effects can also be achieved.

Targeted drugs may be developed.

The surface change of protein filled nanoparticles has been shown to affect the

ability of the nanoparticles to stimulate immune responses. Researchers are

thinking that these nanoparticles may be used in inhalable vaccines. Researchers

are developing ways to use carbon nanoparticles called nanodiamonds in medical

applications. For example, nanodiamonds with protein molecules attached can be

used to increase bone growth around dental or joint implants.

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Other medical and pharmaceutical applications include; tissue engineering, bio

detection of pathogens, tumour destruction via heating (hyperthermia), drug and

gene delivery, separation and purification of biological molecules and cells.

1.5.2 Biosensing

A biosensor is an analytical device used for the detection of an analyte, which

combines a biological component with a physicochemical detector (Florinel-

Gabriel, 2012). The sensitive biological element can be tissues,

microorganisms, organelles, cell receptors, enzymes, antibodies, nucleic acids,

e.t.c. or a biologically derived material or biomimetic component that interacts,

binds or recognizes the analyte under study. Nanomaterials are exquisitely

sensitive chemical and biological sensors. Their large surface area to volume

ratio can achieve rapid and low cost reactions, using a variety of designs

(Gerald, 2009).

Biosensing can have the following applications;

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  Environmental applications e.g. the detection of pesticides and river

water contaminants such as heavy metal ions.

 Determining the presence of pathogen and food toxins in food analysis.

 Determining levels of toxic substances before and after bioremediation.

 Determination of drug residues in food, such as antibiotics and growth

promoters.

 Drug discovery and evaluation of biological activity of new compounds.

1.5.3 Optical Applications

The optical properties of noble metals nanoparticles have been of great interest

because of many applications in optical devices (optical limiters, solar cells,

medicals imaging, surface enhanced spectroscopy, surface plasmonic devices)

and bio-applications (Haglund et al. 1993).

1.5.4 Optoelectronics

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Optoelectronics is the study and application of electronic devices that source,

detect and control light. The light includes invisible forms of radiation such as

gamma rays, X-rays, ultraviolet and infrared, in addition to visible light.

Optoelectronic devices are electrical-to-optical or optical-to-electrical

transducers, or instruments that use such devices in their operation. It can

function as an emitter of optical radiation, such as a light-emitting diode (LED),

or as a photovoltaic (PV) device that can be used to convert optical radiation

into electrical current, such as a photovoltaic solar cell.

In optoelectronics;

 Nanoparticles can be applied in the production of optocouplers, a

component that transfers electrical signals between two isolated circuits

by using light. They prevent high voltages from affecting the system

receiving the signal.

 Nanoparticles are also applied in optical fibers which are used most to

transmit light between the two ends of the fiber and find wide usage in

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 fiber-optic communications. They are also used for illumination, and are

wrapped in bundles so that they may be used to carry images, thus

allowing viewing in confined spaces e.g. fiberscope.

1.5.5 Energy and Electronic Applications

Quantum Dots;

A quantum dot (QD) is a nanocrystal made of semiconductor materials that is

small enough to exhibit quantum mechanical properties. Specifically, its excitons

are confined in all three spatial dimensions. The electronic properties of these

materials are intermediate between those of bulk semiconductors and of discrete

molecules (Brus, 2007, Norris, 1995, Murray et al., 2000).

Quantum dots are applied in;

In textile technology, various kinds of organic dyes are used but more flexibility is

being required of these dyes, and the traditional dyes are often unable to meet the

expectations (Walling et al., 2009). To this end, Quantum dots have quickly filled

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in the role, found to be superior to traditional organic dyes on several counts. One

of the most immediately obvious being brightness (owing to the high extinction co-

efficient combined with a comparable quantum yield to fluorescent dyes

(Michalet et al., 2005) as well as their stability (allowing much less

photobleaching).

Also in biology, the usage of quantum dots for highly sensitive cellular imaging

has seen major advances over the past decade (Spie., 2014). Another application

that takes advantage of the extraordinary photostability of quantum dot probes is

the real-time tracking of molecules and cells over extended periods of time (Dahan

et al., 2003).

In light emitting devices, because Quantum dots naturally produce monochromatic

light; they can be more efficient than light sources which must be color filtered.

They are used to improve existing light-emitting diode (LED) design.

1.5.6 Antibacterial Applications

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Silver nanoparticles serves as an inorganic antibacterial powder and play a critical

role in the suppression and killing of pathogenic microorganisms such as S. aureus,

E. coli, etc. This innovative anti-infective products has broad spectrum, non-

resistance, durable, has a non-oxidized appearance and is unaffected by pH effects.

Ag-Nps are incorporated in apparel, foot wears, paints, wound dressings,

appliances, cosmetics and plastics for their antibacterial properties. The colloidal

silver is capable of disinfecting water through sterilization.

1.5.7 Other Applications of Nanoparticles

Generally, nanoparticles are used or being evaluated for use, in many fields. The

list below introduces several of the other uses under development. They include;

Applications in Manufacturing and Materials: Titanium dioxide and zinc oxide

nanoparticles are commonly used in sunscreen, cosmetics and some food products

while silver nanoparticles are used in food packaging, clothing, disinfectants and

household appliances. Nano silver and carbon nanotubes are used for stain-

resistant textiles; and cerium oxide as a fuel catalyst. Zinc oxides nanoparticles can

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be dispersed in industrial coating to prevent wood, plastic and textile from

exposure to UV rays.

Applications in Water Purification: Nanotechnology is also being applied to or

developed for application to a variety of industrial purification processes.

Purification and environmental cleanup applications include the desalination of

water, water filtration, wastewater treatment, groundwater treatment, and other

nanoremediation.

Applications in the Environment: Researchers are using photocatalytic copper

tungsten oxides nanoparticles to break down oil into biodegradable compounds.

The nanoparticles are in a grid that provides high surface area for the reaction. It is

activated by sunlight and can work in water, making them useful for cleaning up

oil spills.

1.6 Silver Metal

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Silver is a chemical element with symbol Ag (Latin name; argentum). It has its

electronic configuration as [Kr] 4d10 5s1 (no. of electron per shell; 2, 8, 18, 18, 1)

and has an atomic number 47. It is very ductile, malleable metal (slightly less so

than gold), with a brilliant white metallic luster that can take a high degree of

polish. The electrical conductivity of silver is the highest of all metals, even higher

than copper. Pure silver has the highest thermal conductivity of any metal.

The most common oxidation state of silver is +1 (e.g. silver nitrate, AgNO 3), other

oxidation states include; +2 compounds (e.g. silver (II) fluoride, AgF 2), +3 (e.g.

potassium tetrafluoroargentate (III), KAgF4) and even +4 compounds (e.g.

potassium hexafluoroargentate (IV), K2AgF6) (Riedel et al., 2009).

Silver is found in a native form as an alloy with gold (electrum), and in ores

containing sulphur, arsenic, antimony or chlorine. Some ores include; argentite

(Ag2S), chlorargyrite (AgCl) and pyrargyrite (Ag 3SbS3). The metal is primarily

produced as a byproduct of electrolytic copper refining, gold, nickel, and zinc

refining. Naturally occurring silver is composed of two stable isotopes, 107

Ag and

109
Ag, with 107 Ag being slightly more abundant (51.8% natural abundance).
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1.6.1 Some Uses of Silver

Silver is used to make solder and brazing alloys, and as a thin layer on bearing

surfaces can provide a significant increase in resistance and reduce wear under

heavy load, particularly against steel. It is used in photography, in the form of

silver nitrate and silver halides, for the development of coloured films. Some

electrical and electronic products use silver for its superior conductivity, even

when tarnished. Small devices, such as hearing aids and watches, commonly use

silver oxide batteries due to their long life and high energy-to-weight ratio. Silver,

in the form of electrum (a gold–silver alloy), was coined to produce money. Silver

coins and bullion are also used as an investment to guard against inflation and

devaluation.

Silver salts have been used since the middle ages to produce a yellow or orange

color to stain glass. Using a process called sputtering, silver, along with other

optically transparent layers, is applied to glass, creating low emissivity coatings

used in high-performance insulated glazing. Silver can be alloyed with mercury at

room temperature to make amalgams that are widely used for dental fillings. Silver
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and silver alloys are used in the construction of high-quality musical wind

instruments of many types. Flutes, in particular, are commonly constructed of

silver alloy or silver plated both for appearance and for the frictional surface

properties of silver. Brass instruments, such as trumpets and baritones, are also

commonly plated in silver.

1.7 Recent Works on Nanoparticles

Though much work have been done on silver nanoparticles but greater works are

underway as this field of study has proved to enhance the applicability of

nanoelectronics, photonics, biomarker, bio-diagnostic, biosensors and related

materials used in polymers, textiles, fuel cell layers, composites and solar energy

materials. High surface areas can be achieved using solutions and using thin film

by sputtering targets and evaporation technology using pellets, rod and foil.

Caixia and coworkers 2009, investigated the fabrication of novel Pd-Cu bimetallic

nanocomposites with hierarchically hollow structures through a simple galvanic

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replacement reaction using dealloyed nanoporous copper (NPC) as both template

and reducing agent. The reaction process was monitored by UV-Vis absorbance

spectra and X-ray diffraction (XRD), which clearly demonstrated a structure

evolution from NPC precursor to a Pd-rich PdCu alloy structure upon the

completion of the reaction. Structural characterization by use of SEM and TEM

revealed that the replacement reaction between NPC and [PdCl4]2− solution results

in a nanotubular mesoporous structure with a nanoporous shell, which comprised

interconnected alloy nanoparticles with size around 3 nm.

Omid Akhavan and Elham Ghaderi 2009, investigated the effect of an electric field

on the antibacterial activity silver nanorods against E. coli bacteria. It was found

that the grown silver nanorods show strong and fast antibacterial activity. Applying

an electric field in the direction of the nanorods (without any electrical connection

between the nanorods and the capacitor plates producing the electric field)

promoted their antibacterial activity.

In another experiment, Elemike et al 2014, investigated silver nanoparticles for

antibacterial activities using pineapple leaf extract. The synthesized pineapple leaf

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nanoparticles were reddish brown in aqueous solution and susceptible to the

growth of S. aureus, S. pnuemoniae and E. coli which showed great antibacterial

properties thereby placing the agents in broad spectrum plane.

1.8 Gnetum africanum (Afang Leaf)

Gnetum africanum is a vine gymnosperm species found natively throughout

tropical Africa. It has numerous common names most notably, ukase or afang in

Nigeria. It is also referred to as a form of ‘wild spinach’ in English.

It is traditionally a wild vine and is considered to be a wild vegetable. It is a

perennial plant that grows approximately 10m long, with thick papery-like leaves

growing in groups of three.

Gnetum africanum is found mainly in the humid tropical forest regions of Central

African Republic, Cameroon, Gabon, Democratic Republic of the Congo and

Angola. In Nigeria, it is mostly found in the southern part of the country (Calabar

and Akwa Ibom) and used in preparing special delicacies.

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Primarily, the leaves are used as vegetable for soups and stews, commonly afang

soup. The leaves may further be used as a remedy for nausea, sore throats, or as a

dressing for warts. The stem of the plant may also be eaten for medicinal purposes,

including the reduction of pain during childbirth. It is a good source of protein and

is strong in essential and non-essential amino acids. The plant is also used

medicinally as anti-inflammatory, anticarcinogenic and antioxidant.

1.9 Aim of Work

The aim of this research is to synthesize silver nanoparticles from Gnetum

africanum leaf extract and evaluate its antibacterial function.

1.9.1 Objective of the Research

The objectives of the research are:

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 To synthesize silver nanoparticles using Gnetum africanum aqueous leaf

extract.

 To characterize the synthesized nanoparticles.

 To study the antibacterial properties of the nanoparticle and the ordinary

aqeous leaf extract.

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 CHAPTER TWO

MATERIALS AND METHODS

2.1 Materials

Reagents (silver nitrate, ethanol), Gnetum africanum plant, Bacteria strains (S.

aureus, E. coli and S. typhi), Culture media (Nutrient agar, Salmonella Shigella

Agar, Eosin Methylene Blue Agar, Sorbitol Agar), Petri dishes.

2.2 Instruments

CamSpec M501 single beam UV-Vis spectrophotometer, Autoclave, Oven,

Centrifuge.

2.3 Preparation of the leaf extract

The whole plants were collected from Ikot-Ekpene, Cross River State, Nigeria.

Fresh leaves of Gnetum africanum plant was washed to remove dust particles and

dirt, sun dried, chopped into smaller pieces and blended into powder. 2g of the

blended leaf was measured and dispersed into a 250 ml conical flask along with

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200 ml of distilled water. The mixture was then boiled for 60 minutes, allowed to

cool and filtered off.

2.4 Synthesis of the silver nanoparticles

For the synthesis of silver nanoparticles, 45 ml of the extract and 450 ml of 1mM

silver nitrate solution was poured into a conical flask and heated to 70 °C using a

magnetic stirrer heater. The mixture was then boiled until there was a colour

change from a light yellow to dark brown while being sampled at intervals for UV-

Visible determination. The dark brown solution was then centrifuged, collected in

a ceramic crucible and dried in an oven at a temperature of 50 °C. The resulting

particles were then used for the antibacterial analysis.

2.5 UV-Vis spectrophotometer analysis

The reduction of monovalent Ag+ ions to Ag0 was monitored by measuring the

UV-Vis spectrum of sample aliquots of silver nanoparticles (AgNPs) solution of

the reaction medium at 15minutes interval after diluting small aliquot of the

sample in to distilled water. UV-Vis spectral analysis was done using CamSpec

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M501 single beam UV/Vis spectrophotometer at the range of 200-700 nm and

absorption peaks were observed between 400-430 nm regions due to the excitation

of surface plasmon vibrations in the AgNPs solution.

2.6 Preparation of Culture Media

2.6.1 Sorbitol-MacConkey pt. I & II Agar (exclusive for E. coli)

This agar was prepared by weighing 2 g of part I Sorbitol-MacConkey agar and 0.5

g of part II Sorbitol-MacConkey agar in 50 ml of distilled water in a conical flask,

after which it was autoclaved for 15minutes at 121 0C. After autoclaving, it was

allowed to cool before pouring into a test tube and the test organism was

introduced into it and was incubated at 37 0C for 24 hours.

2.6.2 Salmonella Shigella Agar (exclusive for S. typhi)

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This agar was prepared by weighing 3.15 g of the salt agar into 50 ml of distilled

water in a conical flask, after which it was autoclaved for 15 minutes at 121 0C.

After autoclaving, it was allowed to cool before pouring into a test tube and the test

organism was introduced into it and was incubated at 37 0C for 24 hours.

2.6.3 Eosin Methylene Blue (EMB) Agar (exclusive for S. aureus)

This agar was prepared by weighing 2.27 g in 50 ml of distilled water in a conical

flask. It was autoclaved for 15 minutes at 121 0C and allowed to cool. This was

poured into a test tube and the test organism introduced into it which was later

incubated at 37 0C for 24 hours.

2.7 Sensitivity Test

2.7.1 Determination of Sensitivity of Isolate with Extract

Nutrient agar plates were prepared and sterilized, and were allowed to cool and

dispensed into three sterile petri-dishes. 0.1 ml of the test organism in a broth

culture were swabbed on the surface of the prepared solid media using sterile swab

stick after which 0.3 g of the extract was measured and mixed in 1ml of distilled

35
water. Perforated disk was added on it for about 15 minutes. Then three of the disk

were placed far apart on the dishes and incubated at 37 0C for 24 hours. After

incubating, the diameter of the zones of inhibition around each disk was measured

in millimeters and recorded.

Similar procedure was used for the antibiotic sensitivity disc and the nanoparticle.

2.8 Determination of MIC

A serial dilution was done using 4 test tubes labeled A, B, C, D containing 9ml of

distilled water in each test tube, 1ml of aliquot of extract prepared earlier described

in 2.6.1 was added to test tube A and mixed thoroughly. With the help of a syringe,

1ml was collected from test tube A into test tube B and thoroughly mixed. This

process was carried out for test tubes C and D respectively. 1.05 g of Mueller

Hinton agar and 0.75 g of Agar Agar was dissolved in 50 ml of distilled water,

heated for 15 minutes, allowed to cool and poured into three sets of four plates

labeled A, B, C, D (i.e. each set for a different bacterial isolate), after which the

different bacterial isolates were poured in it. 0.1 ml was collected from the test

36
tubes A, B, C, D each and was dispensed each set of plates A, B, C, D respectively

and were incubated at 37 °C for 24 hours. Similar procedure was done for the

nanoparticle solution accordingly. The minimum inhibitory concentration of the

extracts was obtained by observing the lowest concentration of extracts with no

growth.

37
 CHAPTER THREE

RESULTS AND DISCUSSION

3.1 UV-Vis spectrophotometer analysis

The bioreduction of the Ag+1 to Ag0 proceeds gradually after the addition of

Gnetum africanum aqueous leaf extract Fig. 3a to silver nitrate solution. The

bioreduced Ag0 gave reddish-brown coloured solution as shown in Fig. 3b which

shows the formation of silver nanoparticles (Elemike et al. 2014). This indicates

the green route to the reduction of silver ions and stabilization of silver

nanoparticles produced. The optical properties of metallic nanoparticles arise from

a complex electrodynamic effect that is strongly influenced by the surrounding

dielectric medium. Light impinging on metallic particles causes optical excitations

of their electrons. The principal type of optical excitation that occurs is the

collective oscillation of electrons in the valence band of the metal (Peter et al.

2010).

38
Under the temperature of investigation 70 °C, the colour of the silver nanoparticles

changed from light yellow to light brown within 45 minutes and finally to dark

brown after 60 minutes. Within this range of time, the surface plasmon bands

moved from a higher wavelength to lower region indicating blue shift. This shows

that the particles are disintegrating into smaller particles a situation that may

possibly be termed “digestive ripening”. Generally, the surface plasmon resonance

(SPR) bands are influenced by the size, shape, morphology, composition, and

dielectric environment of the synthesized nanoparticles (Kelly et al. 2003;

Stepanov 1997).

The peak intensity of the surface plasmon absorption for AgNPs occurred in the

optical region of the UV–Visible spectrum between 420-422 nm. It was observed

that the absorption intensity increased with increase in the reaction time. The

maximum absorbance was recorded at a wavelength of 421 nm at the 120 th minute

at a heating temperature of 70 °C. The formation of silver nanoparticles at 70 °C

underscores the ability of G. africanum aqueous leaf extract to reduce silver ion

and stabilize it without employing chemical reduction route.

39
Figure 3a: G. africanum leaf extract mixed with Figure 3b: colour change showing the

1 mM of AgNO3 in the ratio 1 : 10 formation of AgNPs after 120 minutes

3.2 KINETIC STUDY

The absorbance spectra and growth pattern of the reaction synthesis was monitored

and analysed using CamSpec M501 single beam spectrophotometer shown in fig.

3c.

40
 4
3.5
3 0min
2.5 5mins
15mins
Absorbance

2 30mins
1.5 45mins
60mins
1
75mins
0.5 90mins
0 105mins
100 200 300 400 500 600 700 800 120mins
-0.5

Wavelength (nm)

Figure 3c: UV-Visible absorption spectra of AgNPs synthesized by reacting G. africanum leaf

extract with AgNO3 in ratio 1 : 10

The kinetic study carried out on the synthesized silver nanoparticles was aimed at

determining the exact order of reaction of the nanoparticle synthesis followed. To

plot the graph, the highest intensity was used in extrapolating the other intensities

at different time intervals (from 0 – 120 minutes i.e. 423nm). The first and second

order plots were drawn and their R2 values were compared to suggest the order of

reaction.

For a first order reaction, the equation is Ln A0 – Ln A = K1T. The graph is

obtained by plotting Ln A against T where slope = -K. For a second order reaction,

41
the equation is 1/A – 1/A0 = K2T. The graph is obtained by plotting 1/A against T

where slope = K.

Intensity – Time Table for Silver Nanoparticle at 421nm

Time, t Absorbance 1/A Ln (1/A) √t At/Ae

,A

0 0.097704 10.23 2.23 0 0.081

5 0.189101 5.29 1.67 2.24 0.156

15 0.225438 4.44 1.49 3.87 0.186

30 0.811657 1.23 0.21 5.48 0.671

45 0.801969 1.25 0.22 6.71 0.663

60 1.069602 0.94 -0.06 7.75 0.882

75 0.603877 1.66 0.51 8.66 0.499

90 1.172351 0.85 -0.16 9.49 0.969

105 1.392401 0.72 -0.31 10.25 1.151

120 1.209955 0.83 -0.19 10.95 1

42
 1st Order Plot
2.5
2
1.5
f(x) = − 0.0180437663738635 x + 1.55038526737556
1 R² = 0.719005946808513
0.5
0
0 20 40 60 80 100 120 140
-0.5

Fig. 3d: A plot of Ln A against time

2nd Order Plot

12
10
8
6
f(x) = − 0.0540409924487594 x + 5.68923408845739
4 R² = 0.552822126907166
2
0
0 20 40 60 80 100 120 140

Fig. 3e: A plot of 1/A against time

Since the R2 value of the 1st order plot is greater than the 2nd order plot, therefore

the reaction is 1st order at 80°C. From the values for 1st order k = 0.719 and 2nd

order = 0.5528, this supports the 2nd order reaction since its value is lower showing

that it is the slowest step and the rate determining step. The reaction followed 1st

43
order but since the value fell between 0.5528 ≤ R2 ≤ 0.719, it can equally be said

that it followed the 2nd order reaction.

Intraparticle diffusion

For the intraparticle diffusion the equation is At = K√t + c. The graph is obtained

by plotting At against √t where K is the slope.

intraparticle diffusion
1.6
1.4
1.2 f(x) = 0.117160347209329 x − 0.00882317074901107
1 R² = 0.827654794110832
0.8
0.6
0.4
0.2
0
0 2 4 6 8 10 12

Fig. 3f: a plot of At against √t

Since the graph did not pass through zero point, diffusion control is not the sole

controlling mechanism hence it can be said to be a pseudo first order.

3.3 Antibacterial Evaluation

44
The silver nanoparticles were tested for their antimicrobial property against gram-

positive (S. aureus) and gram-negative (E.coli and S. typhi) bacteria after

incubation. The results of the antibacterial studies of silver nanoparticles against

drug resistant microorganism revealed that the silver nanoparticles showed

bactericidal activity against the organisms tested. The plant extract equally showed

some antibacterial activity.

All three test organisms (S.aureus, E.coli and S.typhi) were susceptible to the

influence of the silver nanoparticles and the plant extract. The bactericidal activity

of the silver nanoparticles was highest against S. aureus and E. coli with an

average zone of inhibition of 21 mm.

Table 3.1: Zone of Inhibition for Antibiotic Sensitivity Disc

Organisms Zone of Inhibition(mm)

CRX NIT GEN AUG CPR OFL CXM CAZ

45
S. typhi 0.0 20.0 20.0 14.0 28.0 16.0 0.0 0.0

8.0 26.0 18.0 12.0 30.0 20.0 0.0 0.0

5.0 26.0 18.0 16.0 30.0 20.0 0.0 0.0

Mean values 4.0 24.0 19.0 14.0 29.0 19.0 0.0 0.0

(mm)

E. coli 10.0 30.0 15.0 12.0 25.0 16.0 0.0 0.0

8.0 30.0 17.0 12.0 20.0 16.0 0.0 0.0

8.0 28.0 16.0 13.0 20.0 18.0 0.0 0.0

Mean values 9.0 29.0 16.0 12.0 22.0 17.0 0.0 0.0

(mm)

S. aureus 0.0 28.0 24.0 5.0 27.0 16.0 0.0 0.0

0.0 28.0 24.0 8.0 28.0 20.0 0.0 0.0

46
 0.0 30.0 22.0 6.0 30.0 16.0 0.0 0.0

Mean values 0.0 29.0 23.0 6.0 28.0 17.0 0.0 0.0

(mm)

KEY: AUG=Augumetin, CPR=Ciprofloaxacin, NIT=Nitrofurantion,

GEN=Gentamicin, CRX=Cefuroxime, CAZ=Cetlazidime, OFL=Ofloxacin,

CXM=Cefixime

An antibiotic sensitivity disc was used as reference for the evaluation of the

antimicrobial activity between the nanoparticle and the plant extract. This was

obtained from Ablik Biologicals (IVD B.No: 100NZ53N). It comprises the

following antibiotics;

Code Antibiotic Concentration

CAZ Ceftazidime 0.03mg

CRX Cefuroxime 0.03mg

GEN Gentamicin 0.01mg

47
CXM Cefixime 0.005mg

OFL Ofloxacin 0.005mg

AUG Augmentin 0.03mg

NIT Nitroflurantin 0.3mg

CPR Ciproflaxcin 0.005mg

Table 3.2: Zone of Inhibition for Extract

E. coli S. aureus S. typhi

Extract 15.0 14.0 20.0

(300mg/ml)

20.0 12.0 20.0

20.0 11.0 18.0

Mean value (mm) 18.0 9.0 19.0

48
Table 3.3: Zone of Inhibition for Nanoparticle

E. coli S. aureus S. typhi

Nanoparticle 20.0 22.0 20.0

(30mg/ml)

22.0 20.0 20.0

22.0 20.0 20.0

Mean value (mm) 21.0 21.0 20.0

Different concentrations of the AgNPs (30, 15, 7.5, 3.73 mg/ml) and the leaf

extract (300, 150, 75, 37.5 mg/ml) were prepared to assess the minimum inhibitory

concentration towards bacteria concentration of 106 CFU/ml. From the results

obtained as shown in table 3.4 and 3.5, it was observed that even at low

concentration of 7.5 mg/ml, the growth of E. coli and S. aureus was still inhibited

but in the case of S. typhi, there was growth at this concentration therefore the

minimum inhibitory concentration for S. typhi was 15 mg/ml.

49
In the case of the leaf extract, growth was observed at 75 mg/ml thereby placing

the substrate at a minimum inhibitory concentration of 150 mg/ml. The silver

nanoparticle has exhibited greater antibacterial activity than the ordinary leaf

extract. This could be as a result of the capping and stabilizing effect of the organic

compounds from the extract towards the silver metal thereby resisting the attack of

bacterial organisms.

The antibacterial property of silver has been known for thousands of years and

silver nanoparticles have equally proved to be more effective by binding to

bacterial surfaces, altering the membrane function and inhibiting replication.

Table 3.4: MIC Result for Nanoparticle

CONCENTRATION (mg/ml)

Nanoparticle 30 15 7.5 3.75 MBC

S. typhi - - + + ≥15

E. coli - - - + ≥7.5

S. aureus - - - + ≥7.5

50
KEY: - = inhibition of test organism, + = resistance of test organism

Table 3.5: MIC Result for Extract

CONCENTRATION (mg/ml)

Extract 300 150 75 37.5 MBC

S. typhi - - + + ≥150

E. coli - - + + ≥150

S. aureus - - + + ≥150

KEY: - = inhibition of test organism, + = resistance of test organism

51
Ceftazadime (0.03 mg) and cefixime (0.005 mg) showed no zone of inhibition for

any of the three bacteria samples compared to the extract (300 mg) and the

nanoparticle (30 mg). Cefuroxime (0.03 mg) had a very low zone of inhibition

against the gram negative bacteria (i.e. 4.0 mm for S. typhi and 9.0 mm for E. coli)

while it had no zone of inhibition for the gram positive bacteria (S. aureus).

Augmentin (0.03 mg) had a zone of inhibition of 14.0 mm for S. typhi, 12.0 mm

for E. coli but a very low zone of inhibition for S. aureus, 6.0 mm. Gentamicin

(0.01 mg) and ofloxacin (0.005 mg) both showed good bactericidal activity against

all three bacteria species. Gentamicin had a mean diameter of inhibition of 19.0

mm for S. typhi, 16.0 mm for E. coli and 23.0 mm for S. aureus while ofloaxicin

had 19.0 mm for S. typhi, 17.0 mm for E. coli and 17.0 mm for S. aureus.

Nitrofurantin (0.3 mg) and ciprofloxacin (0.005 mg) both displayed an excellent

bactericidal activity against all three bacteria strains. Nitrofurantin had a mean

diameter of inhibition of 24.0 mm for S. typhi, 29.0 mm for E. coli and 29.0 mm

for S. aureus while ciprofloxacin had 29.0 mm for S. typhi, 22.0 mm for E. coli and

28.0 mm for S. aureus.

52
From the above results, both the nanoparticle and extract had a better bactericidal

effect on the bacteria than ceftazadime and cefixime which showed no inhibition.

The action of cefuroxime against the bacteria samples was more bacteriostatic (i.e.

it prevents growth) than bactericidal compared to the extract and nanoparticle,

although augmentin had a greater effect even though both were of the same

concentration (0.03 mg). Gentamicin and ofloxacin both showed good bactericidal

activity against all three bacteria samples and even inhibited S. aureus better than

the plant extract at a lower concentration of 0.005 mg. Nitrofurantin (0.3 mg) and

ciprofloxacin (0.005 mg) both displayed an excellent bactericidal activity against

all three bacteria strains greater than both the nanoparticle and plant extract even at

lower concentrations.

The main component of the cell walls of gram-positive bacteria is a rigid network

composed of three macro-molecular concentric shells making it resistant to

mechanical rupture, while gram-negative bacteria have a network that is only one

molecule thick, together with up to 25% mass of lipoprotein and

lipopolysaccharide (Ma et al. 2003; Olajire et al., 2010). Due to the small size of

53
the silver nanoparticle, this enabled the penetration into the cell wall of the E.coli

thereby affecting the cell membrane and finally death of the cell.

Our research equally showed that nitrofurantin and ciprofloxacin are better

antibiotics for the treatment of some drug resistant microorganism compared to

other standard drugs used. Gentamicin and ofloxacin are also good antibiotics. The

concentration of the other antibiotics would have to be increased before they can

exhibit excellent antibacterial functions. The plant extract and nanoparticles are

bacteriostatic at low concentration and bactericidal at high concentration although

the plant extract have a low bactericidal effect compared to the nanoparticle.

CHAPTER FOUR

54
 CONCLUSION AND RECOMMENDATION

In summary, silver nanoparticles are green synthesized using leaves extract of

Gnetum africanum. Green synthesis method is a simple, non toxic, rapid method.

In the present study, it has been demonstrated that the extract of Gnetum africanum

plants are capable of producing silver nano particles. These environmentally

benign silver nanoparticles were further confirmed by using UV-Vis spectroscopy.

It is confirmed that silver nanoparticles are capable of rendering high antibacterial

efficacy and hence has a great potential in the preparation of antibacterial drugs.

As metal reduction is achieved by potential medicinal plant extracts,

biocompactibility is also ensured. Success of such a rapid time scale for synthesis

of metallic nanoparticles is an alternative to chemical synthesis protocols and

provides low cost bioreductant with enhanced stability.

The broad spectrum antibacterial activity displayed by this new AgNPs is

potentially important and we recommend that further research should evaluate the

55
cytotoxicity of the nanoparticles as their applications in the formulation of novel

antibacterial therapeutic drugs seem promising.

56
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