THE ANTIBACTERIAL ACTIVITY OF SILVER NANOPARTICLES

FROM THE BIOSYNTHESIS OF WATER HYACINTH (Eichhornia crassipes)

An Undergraduate Research

Presented to the

School of Pharmacy

Centro Escolar University

In Partial Fulfillment

Of the Requirements of the Degree

Bachelor of Science in Pharmacy

By

Shalyn Anne Carbonel

Aliezah Jean Fidelino

 Febbie Gay Saavedra

Clarence Lourdes Sipe

Jannelle Tan

CHAPTER 1

The Problem and Its Setting

Introduction

Good things come in small packages. For years scientists have been proving this phrase

to be true, emphasizing that the things with the most value or quality are small. This is seen

through the extensive research on nanotechnology, especially in the pharmaceutical industry with

the use of nanoparticles in the treatment of infections, inflammation, allergies, and even cancer.

According to the UNESCO Science Report: Towards 2030, nanotechnology has become a

research priority for many countries due to its many potential applications.

The use of nanoparticles, defined by Britannica, as materials with dimensions within the

nano range of less than 100 nm. The biosynthesis of such nanoparticles has found use in

improving the pharmaceutical industry with uses in treating many conditions. According to

Shobha, Vinutha, and Anada, research of the biosynthesis of metals used in medicine from plants

has shown a significant difference when compared to chemically synthesized nanoparticles that

are non-eco-friendly, toxic, and have low productivity.

The activity of silver as an antibacterial agent has been proven and used in different

preparations for years. Silver, a precious metal that was first described of having antimicrobials
back in 400 B.C. by Hippocrates, has been used to fight infections for thousands of years. A

team led by James Collins of Boston University has concluded that silver ions attack bacterial

cells in two main ways: it makes the cell membrane more permeable, and it interferes with the

cell’s metabolism, leading to the overproduction of reactive, and often toxic, oxygen compounds.

These two mechanisms could potentially be used to make today’s antibiotics more effective

against resistant bacteria.

One of todays global health problems is Antimicrobial resistance, the ability of a

microorganism to stop an antimicrobial from working against it, resulting in standard treatments

to become ineffective, for infections persist and spread (WHO). In the Philippines, where

inappropriate antibiotic use is pretty common, there is an increasing amount of resistant

microorganisms. From the 2015 ARSP, there are reported continued high rates of

methicillin/oxacillin resistance among staphylococci, increasing resistance among the bacterial

organisms Pseudomonas aeruginosa and Acinetobacter baumannii, high resistance rate of

Haemophilus influenzae to ampicillin. Many studies have shown that the combination of

antibiotics and silver nanoparticles can both enhance the antibacterial effect and help prevent the

increasing resistant strains of microorganisms.

Water lily (Eichhornia crassipes), also known as water hyacinth, have been menaces for

our rivers and seas. They can cover entire surfaces of rivers, causing the depletion of oxygen and

many fish to be killed. Its excessive growth has also caused reduction in light penetration and

can kill other plants living in the same bodies of water. The water lily has been home to

mosquitos that, according to the Philippine Council for Health Research and Development, carry

diseases such as dengue, malaria, and encephalitis. Other than to the health of human society,

water lily causes hindrances to water transport, clog irrigation, hydropower, and water supply
systems, and even cause flooding. In recent studies of the Department of Environmental and

Natural Resources, water lilies have been considered as pests and water pollutants.

The researchers have noted that although, water lilies may be harmful to the environment,

they can be harvested and used to create silver nanoparticle products that can bring both health

and economic benefits to society.

Background of the Study

Since ancient times, silver has been recognized as an effective antimicrobial agent against

bacteria, fungi, and viruses. According to Nurit et. al, silver and its compounds have been used to

treat burns, wounds, a variety of infections, disinfect medical devices, and even in water

purification. Although not much is known about the mechanism of action of silver in the

elimination of bacteria, it has been tested that the affectivity is size dependent (Poulose et. al),

with smaller sized nanoparticles being more effective in treating infections.

With the emergence of antibiotic-resistant strains silver is starting to gain more ground in

research and development. With silver, it seems that bacteria are less prone to develop resistance

compared to conventional antibiotics i.e. penicillin. Some questions do arise in the use of silver

to treat infections, including: the definition and determination of silver minimal inhibitory

concentration (MIC) and breaking point, the ease of emergence of resistant strains, whether

silver really kills biofilm or just planktonic cells, and the side effects of Ag on humans.

Several bactericidal mechanisms have been hypothesized with the use of silver

nanoparticles. In E. coli, as a representative of Gram-negative bacteria, Ag nanoparticles were

shown to cause “pits” in the cell wall by increasing the membrane permeability and inactivating

the respiratory chain. Investigation by Choi et. al showed that the Ag ion, which has an affinity
for sulfur and nitrogen, can inhibit and disrupt protein structure by binding to thiol and amino

groups. Silver nanoparticles were shown also to have synergistic antibacterial effects both on

Gram-positive and Gram-negative bacteria when provided in combination with antibiotic

(Khurana et. al). Even with all the questions that arise from the use of silver nanoparticles in

antibiotic treatment, out of the metal nanoparticles, silver is still the most promising.

Setting of the Study

The research will be done at Centro Escolar University in Manila, Philippines. Centro

Escolar University (CEU) is a private higher education institution that was founded on June 3,

1907 by Librada Avelino and Carmen de Luna. Originally, the school was a private all-girls

institution named Centro Escolar de Senoritas before becoming a university in 1933. CEU

upholds the philosophy of Ciencia y Virtud, science and virtue. It continues to improve curricula

and facilities and maintains a highly professional and dynamic teaching force and university

staff. For over a century, Centro Escolar University has been providing to society many

professionals in a number of different fields.

The first college of CEU opened in 1921, the College of Pharmacy. Today the College of

Pharmacy is now known as the School of Pharmacy, lead by Dean Cecilia D. Santiago. The

School of Pharmacy has been accredited with a Level IV status by the Philippine Association of

Colleges and Universities Commission on Accreditation (PACUCOA) as certified by the

Federation of Accrediting Agencies of the Philippines (FAAP). The facilities are equipped with

the latest and state-of-the-art laboratory equipment and library resources.

CEU is located in the San Miguel district, in a primarily middle-class residential area of

Manila. Malacañang Palace, the official residence of the President of the Republic of the
Philippines is located in this area. Just outside the gates of Malacañang Palace is where Mendiola

street, the home of various colleges and universities, is located. Mendiola street is the location of

San Beda College, College of the Holy Spirit, La Consolacion College, and Centro Escolar

University.

Figure 3.3

Map of Mendiola

The water lilies are collected from the Pasig River, near the MMDA Flood Control Unit

in Manila. The Pasig River connects Laguna de Bay to Manila bay and stretches for 25

kilometers. The water flows towards manila bay during the wet season when the water lilies were

collected.

Conceptual Framework of the Research

Nowadays the growing demand of medication for the prevention, treatment and

management of bacterial infections caused by bacteria has become the problem biggest problem

of the world due to growing resistance against antibacterial agents.

The researchers’ goal and target is to discover an eco-friendlier and biological approach

towards the development of antibacterial agents with the use of metal (silver) nanoparticles.
 The researchers focus on the effectivity of the biosynthesized Silver nanoparticles from

the leaves of Water hyacinth (Eichhornia crassipes) Family Nymphaceae for the management

and treatment of infections caused by bacteria.

This study is composed of preparation and filtration of the plant leaf broth extract from

the boiled water hyacinth leaves (Eichhornia crassipes), synthesis and characterization of the

biosynthesized silver nanoparticles, and subjecting to physical and instrumental tests, and

determination of its efficacy as an antibacterial agent against various bacteria.

General Objectives:

The study aims to determine the antibacterial activity of silver nanoparticles from the

biosynthesized Eichhornia crassipes.

Specific Objectives:

1. Biosynthesize silver nanoparticles from the leaves of water hyacinth (Eichhornia

crassipes)

2. To differentiate the antibacterial activity of the biosynthesized silver nanoparticles

against Bacillus subtilis, Pseudomonas fluorescens, Klebsiela pneumonia, Escherichia

coli, Staphylococcus aureus.

3. To compare the efficacy of the biosynthesized silver nanoparticles to the drug

tetracycline as an antibacterial.

Hypothesis

The study tries to test the null hypothesis as follows:

 There are no significant differences on the antibacterial activity of the biosynthesized

silver nanoparticle from Water hyacinth leaves against the commercially available antibacterial

(Tetracycline).

Significance of the Study

The results of the research could be of importance to the following:

The Environmental Community. The researchers will be utilizing the use of

water hyacinth from the Pasig River. Water hyacinths, considered to be pollutants and

home of disease carriers (such as dengue and malaria by mosquitos) when reduced in

number would help lessen clogging of sewages and provide no room for growth and

reproduction of parasites thus prevents floods and spread of diseases.

Bacterial Infection Carriers. The study aims to show the antibacterial activity of

Silver Nanoparticles to five common strains of bacteria namely Bacillus subtilis,

Pseudomonas fluorescens, Klebsiela pneumonia, Escherichia Coli and Staphylococcus

Aureus. Silver Nanoparticles is also said to be more efficacious in combating bacterial

actions due to the smaller size of the particles and larger surface area which leads to the

increase of membrane permeability and cell destruction (Biosynthesis of metallic

nanoparticles using plant derivatives. Palaniselvam Kuppusamy et. Al). This study would

be beneficial to those who are carriers of bacterial infections since it provides an

alternative, or possibly a more effective way of managing bacterial infections aside from

the commercially and synthetically available medication in the market.

Pharmaceutical Companies. From this research, pharmaceutical companies can

come up with a different approach of formulating an antibacterial from a natural source:

 biosynthesis. It is also a simple one-step, cost effective, environment friendly and

relatively reproducible and often results in more stable materials.

Scope, Limitation and Delimitation

This study is focus on the determination of the antibacterial activity of the biosynthesized

Silver Nanoparticles from Water hyacinth (Nymphaea nouchali Burm.) Fam. Nymphacaea to

inhibit the growth of some broad spectrum antimicrobial agents by following the steps to the

conducted:

1. Collection and preparation of the plant sample.

2. Preparation of Broth by boiling of the leaves

3. Synthesis of silver nanoparticles from water hyacinth through the addition of freshly prepared

silver nitrate (AgNO3) to the plant leaf broth extract.

4.. Characterization of the biosynthesized silver nanoparticles through the different

spectrophotometer instruments (UV Visible Absorption and FTIR Spectroscopy, X-ray

Diffraction Analysis, Scanning Electron Microscope, Energy Dispersive Spectrum, Dynamic

Light Scattering Method and Transmission Electron Microscopy).

6. Testing the antibacterial activity of the biosynthesized silver nanoparticles by agar diffusion

method.

Definition of Terms

The following unfamiliar terms are defined according to their use in the study for a

clearer understanding of their meaning:

Antibacteria agent. Agents that destroys, interfere or suppress the growth and reproduction of

bacteria.

Antimicrobial Agents. These are substances or the chemicals that are used to either inhibit or

slow down the microbial growth.

Antibiotic. A medicine (such as penicillin or its derivatives) that inhibits the growth of or

destroys microorganisms

Bactericidal. A substance that kills bacteria

Bacteriostatic. A biological or chemical agent that stops bacteria from reproducing, while not

necessarily killing them otherwise.

Nanoparticles. A microscopic particle with at least one dimension less than 100 nm.

Nanotechnology. Science, engineering, and technology conducted at the nanoscale, which is

about 1 to 100 nanometers

Biosynthesis. The process involved in the preparation of biological molecules by the use of

reagents or catalysts derived from natural substances occurring in a living organism.

Zeolites. These are solids with a relatively open, three-dimensional crystal structure built from

the elements aluminum, oxygen, and silicon, with alkali or alkaline-Earth metals (such as

sodium, potassium, and magnesium) plus water molecules trapped in the gaps between them.

CHAPTER 2

Review of Related Literature and Studies

 This chapter includes the botanical description, chemical composition and medicinal uses

of the plant. This summary of relevant facts serves as the foundational knowledge about the

study on the determination of the antimicrobial activity of the biosynthesized Silver

Nanoparticles from Water hyacinth (Nymphaea nouchali Burm.) Fam. Nymphacaea to inhibit

the growth of some broad spectrum antimicrobial agents.

Literature and Studies

Water hyacinth (scientific name:Eichorniacrassipes (Mart.) Solms-Laub. Is a medicinal

plant coming from the family of Pontederiaceae. It’s common Filipino terms include: Water

hyacinth, Floating Water hyacinth, Water-Orchid and JacintheD’eau.Water hyacinth is a free-

floating perennial plant that can grow to a height of 3 feet. The dark leave blades are circular to

elliptical in shape attached to a petiole. The water hyacinth has large, blue to violet in color with

a six sepals, pale lilac and can grow up to 4 centimeters long (Godofredo U. Stuart Jr., M.D.

2017).The said species originated in the Amazon Basin of Brazil and it has been introduced to

some tropical and subtropical regions around the world. (Ebersole 2008).It varies in size from cm

to over a meter in height. The plant system consists of individual shoots each with up to ten

expanded leaves arranged spirally (Fernandez, 2004).

Water hyacinth was introduced into many parts of the world, including the Philippines, as

an ornamental garden pond plant due to its beauty. Most Filipinos know it as “water lily”. Water

hyacinth is considered the most productive plant on earth as it yields more than 200 tons of dry

matter per hectare per year under normal conditions (Tacio, 2009).The plant is for more
productive than the crops that have been carefully cultivated by man under near ideal conditions

of fertilization, irrigation and pest control (Bunton, 2009).

In the Philippines, it is used as an antifungal, antimicrobial, antibacterial,

hepatoprotective, anti-inflammatory and aids against cancer. In India, its flowers are used for

stomach ache and the roots are used as a treatment for pneumonia. In Bangladesh, roots and

flowers are used in the treatment of hepatic disorders and abdominal swelling (Godofredo U.

Stuart Jr., M.D. 2017).

In Bangladesh, the Mennonite Central committee has been experiencing with paper

production from water hyacinth for some years. They have already established two projects that

make paper from water hyacinth stems. The water hyacinth fiber alone when blended with waste

paper or jute the result is reportedly good. Similar small scale cottage industries, paper making

projects have been successful in a number of countries including the Philippines, Indonesia and

India. (Michaelangelica, 2009).

In the Philippines, water hyacinth is used to make baskets and making domestic use. In

India, water hyacinth is also used to produce similar goods for the tourist industry. Charcoal

briquetting is an idea which has been proposed in Kenya to deal with the rapidly expanding

carpets of water hyacinth which are evident on many parts of Lake Victoria. The proposal is to

develop a suitable technology for the briquetting of charcoal dust from the pyrolysis of water

hyacinth. (Michaelangelica, 2009).

Water hyacinth can also be used to aid the process of water purification either for

drinking water. In sewage systems, the root structures of water hyacinth provide a suitable
environment for aerobic bacteria to function. Aerobic bacteria feed on nutrients and produce

inorganic compounds which in turn provide food for the plants (Fernandez, 2004).

Flower extract of the said plant even showed an antimicrobial activity against B. cereus,

S. aureus and P. aeruginosa, the methanolic extracts from the Water hyacinth were evaluated

against various pathogenic microorganism such as S. aureus, Pseudomonas aeruginosa, B.

subtilis, E. coli and Candida albicans.(Godofredo U. Stuart Jr., M.D. 2017).

Many human illnesses are caused by the widespread infections caused by various

bacteria. Bacteria are considered to be a tiny single-celled organisms/eukaryote. In various

studies being conducted it was said that some strains of bacteria specifically the “good bacteria”

are beneficial on our health especially it works by keeping our gut healthy (probiotics) and are

considered to be harmless to people but some are responsible for a range of infections like

cellulitis, boil, impetigo, gonorrhea, syphilis, urinary tract infection (UTI) and meningitis. These

disease-causing varieties are called pathogenic bacteria. (State of Victoria 2018). In order to cure

infectious diseases, researchers have discovered some antibacterial agents, which are considered

to be the most promising chemotherapeutic agents. Gradually, due to various issues arising

during the use of antibacterial agents, such as the resistance phenomenon, an enormous increase

in the number and types of the newly added antibacterial agents has been observed. Because of

this growing antimicrobial resistance of some pathogenic microorganism, new drugs are now

frequently entering into the market along with the existing drugs.(Hamid Ullah and Saqib Ali,

2017)
 Antimicrobial agents are the substances that are used to either inhibit or slow down the

microbial growth. Antimicrobial chemotherapy is the study that helps to understand the

mechanisms that undergo within the body to cure a disease. These antimicrobial agents that are

used for treatment of infections and diseases are called as antibiotics. Examples of such

antibiotics include: Penicillin, cephalosporin, polymyxin, bacitracin. These are some of the

antibiotics used to treat bacterial infections. (OMICS International, 2018)

Antibacterial agents are substances that interferes, kill or prevent the growth of bacteria.

Antibacterials are not only used as a treatment against bacterial infections but nowadays it has

been used to disinfect surfaces and eliminate potentially harmful bacteria.( OMICS International,

2018)

The advent of nanotechnology is considered to be the biggest engineering innovation

since the Industrial Revolution. (Aston 2005). Over the past few decades, inorganic nanoparticles

like semiconductor quantum dots, whose structures exhibit a significantly, improved physical,

chemical, and biological properties and functionality due to their nanoscale size. Nanophasic and

nanostructured materials are attracting a great deal of attention because of their potential for

achieving specific processes and selectivity, especially in biological and pharmaceutical

applications. (Seoul National University, 2007)

Back then during ancient time silver has been employed as the most widely used

inorganic antibacterial agent to fight infections and control spoilage. The silver, silver ion, and

silver compounds antibacterial activity have been extensively scrutinized. Silvers’

epidemiological history has recognized its nontoxicity in normal use. The processes that

contributed to its bacterial effect are perhaps the catalytic oxidation by metallic silver and
reaction with dissolved monovalent silver ion. As a result, silver ions have been drastically used

as an antibacterial constituent in dental resin composites in synthetic zeolites and in coatings of

medical devices. (Tamil Nadu Agricultural University, 2010)

The size of metallic nanoparticles plays an important role in its bactericidal effect

because it safeguards the large surface area of particles with the bacterial cells. Therefore, a large

contact surface area is probable to heighten the degree of bacterial eradication. The nanoscaled

material synthesis and eradication in terms of physicochemical properties is of boundless interest

in the creation of bactericidal materials. The concentration of nanoparticles is directly

proportional on its extent of inhibition as well as on the initial bacterial population. With time the

growth rate of bacteria increases at all concentration and its growth was somewhat affected at 10

µg ml–1 of AgNO3 but greater decline was perceived under same concentration of silver

nanoparticles. (Tamil Nadu Agricultural University, 2010)

Upon Ag+ management, the silver ions mechanism of inhibitory action takes place. DNA

loses its replication ability and expression of ribosomal subunit proteins, as well as other cellular

proteins and enzymes essential to ATP production, becomes inactivated (Yamanaka et al., 2005).

There are reports conducted regarding to the bactericidal activity of silver nanoparticles,

it was made known that the interface between silver nanoparticles and elements of the bacterial

membrane caused structural fluctuations and damage to membranes, finally resulting to cell

death. (Pal et al., 2007)

According to Associate Professor O’Connor of University of Melbourne research team,

the benefit of nanoparticle is that it attacks bacteria in multiple ways.

Resistance for the bacteria is difficult to develop because of its different forms of attacks. By

disrupting the membrane around the bacteria, nanoparticles do its job. Cell membrane plays an

important role in bacteria so that it stays functioning. Once it got holes in it or to leak, as a result

it will not di its function and will be disrupted and eventually it will die.

O’Connor and her team has done tests where they’ve incorporated the nanoparticles as a coating

on the surface of a medical implant, or as part of a tissue-engineering scaffold. The antimicrobial

components are then gradually released into their surrounding environment, and prevent

infections forming.
 CHAPTER 3

Methods and Procedure

This chapter deals with the methodology and procedure used to derive Silver

Nanoparticles from the leaves of Water hyacinth (Eichhornia crassipes) from the family of

Pontederiaceae to determine its antibacterial activity on Bacillus subtilis, Pseudomonas

fluorescens, Klebsiela pneumonia, Escherichia Coli and Staphylococcus Aureus

Method of Research Used

The study will utilize an experimental method of research where systematic procedure,

controlled observation and measurements to test hypotheses were done. The researchers will

synthesize Silver Nanoparticles from the leaves of water lilies to be used as antibacterial on

Bacillus subtilis, Pseudomonas fluorescens, Klebsiela pneumonia, E. Coli and S. Aureus.

3.1 Research Procedures

Eichhornia crassipes leaves will be collected from Pasig river. The leaves will be

thoroughly washed under tap water to remove the adhered dust particles present on the surface

and then rinsed with distilled water for three times. The cleaned leaves will be completely dried

at room temperature on a blotting paper. The dried leaves will be chopped into small pieces and

stored in an air-tight container at room temperature for further use.

The plant leaf broth extract will be prepared by taking 20 grams of thoroughly washed

and finely cut leaves in 200 ml sterile distilled water in a beaker. The mixture will be boiled for

30min and will be cooled at room temperature and filtered through Whatman filter paper no.1
until no insoluble material appeared in the broth. The filtered broth extract will be kept at 4 C for

further studies.

Clear leaf extract will be obtained to be used for the synthesis of silver nanoparticles. 10

ml of leaf broth will be added to 90 ml of a 1mM solution of AgNO3 for reduction of silver ions

and the reduction reaction temperature will be optimized at 90C heated in a water bath for 10

min. A color change from yellow to brown designates the formation of colloidal AgNPs. To be

followed by the preparation of different concentration of AgNPs: 10, 20, 30 and 40 mcg/mL

Collection of Plant Samples

Segregation of plant parts

Washing and Drying of Leaves

Cutting of leaves into small pieces

Preparation of Broth: Boiling of Leaves

Filtration

Synthesis of Silver Nanoparticles

Characterization

Antibacterial Activity
3.2 Characterization

UV Visible Absorption and FTIR Spectroscopy

The synthesized silver nanoparticles will be initially characterized by UV visible

absorption spectroscopy. The samples will be taken in a 1cm quartz cuvette and measured in a

JASCO V 650 spectrophotometer containing double beam in identical compartments each for

reference and test solution from 200 nm to 900 nm. The presence of functional groups in C.

viscosa fruit extract synthesized AgNPs were identified by FTIR analysis using KBr pellet

technique at the range of 4000-400 cm1 or at a resolution of 4 cm1 in the diffuse reflectance

mode.

X-ray diffraction Analysis

X-ray diffraction analysis will be performed to examine the crystallographic structure of

the purified AgNPs. In the procedure for preparing samples for XRD, a thin film of sample will

be applied onto a glass slide by dropping 100 lL of the sample and drying for 30 min. The XRD

pattern will be recorded using Rigaku Miniflex 600 X-ray diffractometer with operating voltage

of 40 kV at a 15 mA current strength. The samples will be subjected to Cu Ka radiation with

nickel monochromator in the 2h range of 20–80.

Scanning electron microscope (SEM) and energy dispersive spectrum (EDS)

The same sample preparation procedure will be used for scanning electron microscope

(SEM) and energy dispersive spectrum (EDS) measurements. The morphology of AgNPs will be

examined using FE-SEM, and the presence of silver will be confirmed by EDAX.
 Dynamic light scattering method

The average size and stability of the C. viscosa mediated synthesized silver nanoparticles

will be determined by dynamic light scattering (DLS) technique and the zeta potential. For this

analysis, the samples will be filtered (0.2 mm) and the clear nanoparticles solution will be used

and the measurement will be taken at the respective range. Dynamic light scattering method will

be employed for the zeta potential and particle size analysis of the colloidal SNPs using Malvern

Zetasizer nanosizer (size range 0.1–10,000 nm).

Transmission Electron Microscopy

The AgNPs will be analyzed through TEM determining their size and shape.

Transmission Electron Microscopy image will be taken using JOEL JEM SX 100. The sample

will be placed on a copper grid and left to dry for 60 min under vacuum. The sample will then be

subjected to transmission electron microscopy studies.

3.3 Antibacterial Activity

Silver nanoparticles will be tested for their antibacterial activity by the agar diffusion

method on Muller-Hinton agar plates. The bacterial strains Bacillus subtilis, Pseudomonas

fluorescens, Klebsiela pneumonia, Escherichia Coli and Staphylococcus Aureus will be utilized

for this antibacterial analysis. Wells will be made by standard cork borer at equal distance and

will be filled with silver nanoparticles.

The incubated bacterial culture (107 /mL) will be swabbed uniformly using a sterile

cotton swab. Various concentration of (10-40 mg/mL) silver nanoparticles will be poured into

each wells. After which, agar plates will be incubated at 37 C for 24 h and then the zone of
inhibition (mm) will be measured. Standard antibiotic tetracycline will be used as a positive

control. Three replicates will be used for each treatment and ANOVA will be done by using MS

Excel software.