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CHAPTER 1,2,3 AgNP
CHAPTER 1,2,3 AgNP
CHAPTER 1,2,3 AgNP
An Undergraduate Research
Presented to the
School of Pharmacy
In Partial Fulfillment
By
Jannelle Tan
CHAPTER 1
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
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
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
microorganisms. From the 2015 ARSP, there are reported continued high rates 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
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
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 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
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
(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.
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
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
Federation of Accrediting Agencies of the Philippines (FAAP). The facilities are equipped with
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.
Nowadays the growing demand of medication for the prevention, treatment and
management of bacterial infections caused by bacteria has become the problem biggest problem
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
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
General Objectives:
The study aims to determine the antibacterial activity of silver nanoparticles from the
Specific Objectives:
crassipes)
tetracycline as an antibacterial.
Hypothesis
silver nanoparticle from Water hyacinth leaves against the commercially available antibacterial
(Tetracycline).
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
Bacterial Infection Carriers. The study aims to show the antibacterial activity of
actions due to the smaller size of the particles and larger surface area which leads to the
nanoparticles using plant derivatives. Palaniselvam Kuppusamy et. Al). This study would
alternative, or possibly a more effective way of managing bacterial infections aside from
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:
3. Synthesis of silver nanoparticles from water hyacinth through the addition of freshly prepared
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
bacteria.
Antimicrobial Agents. These are substances or the chemicals that are used to either inhibit or
Antibiotic. A medicine (such as penicillin or its derivatives) that inhibits the growth of or
destroys microorganisms
Bacteriostatic. A biological or chemical agent that stops bacteria from reproducing, while not
Nanoparticles. A microscopic particle with at least one dimension less than 100 nm.
Biosynthesis. The process involved in the preparation of biological molecules by the use of
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
of the plant. This summary of relevant facts serves as the foundational knowledge about the
Nanoparticles from Water hyacinth (Nymphaea nouchali Burm.) Fam. Nymphacaea to inhibit
plant coming from the family of Pontederiaceae. It’s common Filipino terms include: Water
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
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
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.
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
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
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
Many human illnesses are caused by the widespread infections caused by 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
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)
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
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
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
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
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
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
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
components are then gradually released into their surrounding environment, and prevent
infections forming.
CHAPTER 3
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
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
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
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
Filtration
Characterization
Antibacterial Activity
3.2 Characterization
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.
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
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
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
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
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.