HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY – VNU HCMC

OFFICE FOR INTERNATIONAL STUDY PROGRAMS

FACULTY OF CHEMICAL ENGINEERING

SYNTHESIS OF SILVER NANOPARTICLES

BY GREEN CHEMISTRY METHOD USING
THE SOLUTION OF SQUID LEAF
EXTRACT (Eclipta prostrata L.) AS A
REDUCING AND STABILIZING AGENT

Lecturer: Tran Hai Ung

Subject: Introduction to Engineering
Class: CC01
Group: 8
Group members:
Đoàn Huỳnh Ngọc Hân 2252197
Lê Phan Hiếu Hạnh 2252192
Đặng Hoàng Minh Nhật 2252568
Nguyễn Văn Thuận 2252788
Đỗ Phương Uyên 2252912

1
CHAPTER 1: INTRODUCTION
1.1. Research question
1.1.1. The need for research
Over the past several decades, the field of nanoscience and technology has
developed very rapidly, producing materials with sizes between 0.1 and 100 nm.
Nano silver has always attracted the attention of scientists because of its unique
properties as well as its range of applications. At the nanoscale, silver increases
the antibacterial activity by up to 50,000 times the ionic size [1]. Since most
bacteria are not resistant to silver antibiotics, the silver nanoparticles are not
ineffective. In addition, silver nanoparticles can generate active oxygen from air
or water, thereby destroying the cell membrane of bacteria. Nano is widely used
in life and is incorporated into polymers such as polyvinyl alcohol (PVA),
polyethylene (PE),...
Although nanosilver is widely applied in human daily life and synthesized
from many different methods, one of them can be toxic after synthesis. To raise
the spirit of environmental protection and human health, the synthesis of nano
silver from green materials such as squid grass leaves can reduce harmful
chemicals and solvents.
The project "Synthesis of silver nanoparticles by green chemistry
method using the solution of squid leaf extract (Eclipta prostrata L.) as a
reducing and stabilizing agent" is a new research direction to create a nano-
silver solution that is friendly to the environment. Environmental, does not contain
harmful chemicals.
1.1.2. Based on scientific and practical
Nanotechnology is a science that has been studied for many years and is
growing rapidly. As a science that is applied to many fields such as chemistry,
biomedicine, electronics,... Due to its high antibacterial activity and harmlessness
to humans and the environment, nano silver synthesis technology is widely used.
promoted in recent years.
The topic was conducted based on the results of research on synthesizing
silver nanoparticles and testing their antibacterial activity by the research works.
1.2. Hypothesis to be tested and research questions
1.2.1. Hypotheses to be tested
- Can squid leaf extract reduce Ag+ ions to Ag metal?
- The resulting AgNPs are nano-sized.
- AgNPs particles have antibacterial activity.
1.2.2. Research question
- Can squid leaf extract reduce Ag+ ions to Ag metal?
- Are Ag particles nano-sized?
- Are AgNPs granules antibacterial?
- Can the compounds in the squid grass extract be used as agents in the
silver nano-separation process?
1.3. Review of documents related to the research topic
For decades, nanosilver has always attracted great attention from scientists
around the world due to its unique properties and wide applicability. Specifically,
silver nanoparticles have been studied and used as catalysts [3], biological probes
[4], and imagers [5]. In particular, thanks to its effective antibacterial activity [6],
silver nanoparticles are believed to be a potential solution in the problem of
biological infections.
In the world, the production of silver nanoparticles has been published a lot
with different fabrication methods such as chemical reduction method, physical
reduction method, physicochemical reduction method, biological reduction
method. . The research group F.M. Gutierrez used chemical agents to reduce silver
ions to metal, the resulting silver nanoparticles were smaller than 100 nm in size.
H. Jiang and colleagues used high-energy electromagnetic waves such as gamma
rays, ultraviolet rays, lasers, etc. to reduce silver ions to metal. Under the effect
of electromagnetic waves, there are many transformations of solvents and
additives in the solvent to produce chemical radicals that reduce ions into metals.

CHAPTER 2: TOPIC OVERVIEW

2.1. An overview of AgNPs
Due to the continuous development of nanotechnology in antibacterial and
antifungal properties, currently, many different nanomaterials are used,
including nano silver, nano gold, nano zinc oxide,... However, silver
nanoparticles have gained a distinct impression because of their antibacterial
ability reaching 99.9%, average cost, durability, and not being affected by
oxidizing and reducing agents. Moreover, silver nanoparticles have no effects on
human cells and are safe to use at a dosage of 5mg/kg/day for a person.
Introduction to AgNPs: Silver nanoparticles are nanoparticles of silver
between 1 nm and 100 nm in size. The crystal structure of silver nanoparticles is
face-centered cubic (fcc) (indicated by XRD technique).
Silver nanoparticles are not affected by light, conduct electricity and heat
well, have low sensitivity to the environment, and are not denatured in most
environments.
Compared with nano gold and zinc oxide, nano silver is economically
superior and almost non-toxic to the human body.
Each solution formed after the synthesis of silver nano is measured with a
UV-Vis spectrophotometer, each spectrum will have a particular peak as shown
in Figure 2.1. From the results of UV-Vis spectrum analysis, it will be possible
to determine whether silver nanoparticles are formed after the synthesis process
or not and can preliminarily estimate the size distribution of silver nanoparticles
produced.
 Figure 2.1. UV-Vis spectra of AgNPs solution with different particle diameters
[11].
2.2. An overview of Eclipta prostrata
2.2.1. Properties

Hình 2.2. Parts of Eclipta prostrata Hình 2.3. Eclipta prostrata

Scientific name: Eclipta prostrata L.

Kingdom: Plantae
Order: Asterales
Family: Asteraceae
Genus: Eclipta
Species: E. prostrata
Eclipta prostrata grows commonly in moist places in warm temperate to
tropical areas worldwide such as India, China, Brazil, Vietnam, etc. In Vietnam,
eclipta prostrata grows wildly along rivers, roads, plains, hills, etc. all over the
country.
Eclipta prostrata is an annual herb from 10 cm to 60 cm tall erect or
horizontal. Eclipta prostrata’s body is green or light brown or reddish purple.
Leaves arrange in opposite pairs, the leaf blades are long and narrow in size
2.5x1.2 cm. The margins are entire or serrate, and both sides of the leaves are
densely covered in sniff or soft hairs.
2.2.2. Công dụng và thành phần hóa học
To synthesize silver nano, today there are many different types of extracts
such as eclipta prostrata extract, aloe vera extract, leaf extract by the water lily,
etc. In particular, eclipta prostrata extract has more outstanding properties because
eclipta prostrata is a wild plant growing throughout our country at low cost, has
many effective therapeutic effects, and especially, it is not toxic to human bodies.
Eclipta prostrata is an herbal medicine that cures many types of diseases.
In our country, eclipta prostrata is used in oriental medicine and Vietnamese
traditional medicine to treat various diseases such as hemostasis, fever, sinusitis,
body weakness, kidney stones, fatty liver, and infectious inflammation. Besides,
eclipta prostrata contains vitamin K-like ingredients that help prevent abnormal
uterine bleeding but do not have side effects that increase blood pressure and other
vascular diseases. Due to its cooling and non-toxic characteristics, sweet and sour
taste, eclipta prostrata can detox the liver and kidneys to help cure acne, premature
graying of hair, and urticaria,...
There are such prominent chemical components as [12]: polyphenols like
wedelolactone; luteolin and steroids such as stigmasterol and other organic acids.

Wedelolactone Luteolin Stigmasterol

CHAPTER 3: MATERIALS AND RESEARCH METHOD

3.1. Tools and chemicals
3.1.1. Tools and equipments
Magnetic stirrer, spectrometer UV-vis, analytical balance, test tube,
measure machine SZ-100, stirrer, petri dish, dryer machine, centrifuge,..
3.1.2. Chemicals
AgNO3, NaOH, CHCl3, H2SO4 98%, I2, KI, FeCl3,H₂SO₄, Eclipta alba
Hassk powder (bột lá cây cỏ mực).
3.2. Experimental method
3.2.1. Liquid distilled from Eclipta alba Hassk leaf
The process of extracting Eclipta alba Hassk leaves is carried out
specifically as follows: weigh m gam of Eclipta alba Hassk leaves into a 250ml
beaker and boil with 100ml of distilled water for a period of time t (min), filter
the extract.
3.2.2. Qualitative analysis of liquid distilled from Eclipta alba Hassk
leaves
3.2.2.1. Steroid
Dissolve 1 ml of the solution in 10 ml of the chloroform solution in the test
tube. Add 98% sulfuric acid (1ml) solution dropwise to the test tube. Shake the
mixture vigorously. The liquid mixture is divided into 2 layers: dark red (top) and
clear (bottom) indicating steroids.
3.2.2.2. Alkaloids
Extract 1 ml of extract. Test with a reagent solution of 1.27 g of iodine and
2 g of potassium iodide in 100 ml of water. Observation of the shape of the red-
brown precipitate indicates the presence of alkaloids.
3.2.2.3. Flavonoids
Extract 2ml of extract and test with 1M NaOH solution. The mixture
gradually turns orange-yellow, indicating that there are flavonoids.
 3.2.2.4. Phenols
Test 2 ml of the extract with 5% FeCl3 reagent. The solution gradually
turned dark green, indicating the presence of phenols.
3.2.2.5. Anthocyanins
Add 2 ml of the mixture of 2M HCl and NH3 reagents to the test tube with
the extract. The red-pink color of the solution rapidly changed to blue-violet,
indicating the presence of athocyanins.
3.2.2.6. Terpenoids
Extract a sample of 2 ml of extract into a test tube with 2 ml of chloroform
and 2 ml of acetic anhydride. Add a few drops of sulfuric acid to the test tube. The
solution gradually turns pink, indicating the presence of terpenoids.
3.2.2.7. Tannins
Draw the extract into the test tube. Add a few drops of 1% lead acetate. The
appearance of a yellow precipitate indicates the presence of tannin.
3.2.3. Process of synthesizing silver nanoparticle solution by liquid
distilled from Eclipta alba Hassk leaves
Take 1.5 ml of the extract slowly into a flask containing 20 ml of 1 mM
AgNO3, cover the flask and stir the solution with a magnetic stirrer for 60 min, at
room temperature. The resulting solution was measured UV-vis, choosing the
highest optical density value.
3.4. Methods of data analysis
Data analysis by statistical method on Microsoft Excel 2019.

CHAPTER 4: RESEARCH RESULT

4.1. Survey results of factors affecting the process of extracting squid grass
leaves
In order to obtain the optimal toner leaf extract for silver nanosynthesis,
we conducted a two-factor survey of extraction time and solid-liquid ratio.

a)

b)
Figure 4.1. UV-Vis spectrum of (a) effect of solid/liquid ratio on ink herb
extract and (b) effect of ethanol on ink herb extract
 The results of UV-Vis spectroscopy analysis (Figure 4.1) showed that
silver nanoparticles were formed with peak absorption peaks in the range of 390
nm – 430 nm, which are peaks corresponding to the surface resonance plasmon
of silver nanoparticles. This shows that the product is nano silver solution. The
results showed that at a solid/liquid ratio of 1 g/100 ml and without ethanol was
the optimal condition for the preparation of the ink grass extract.
4.2. Qualitative results of chemical group composition in squid grass leaf extract

Functional group Reagent Phenomenon Results

Steroids CHCl3 Formation of electrolyte (+)

solution 2 layers: dark red
(top) and transparent
(lower)

Alkaloids Wagner Does not form red-brown (-)

(KI+I2) precipitate

Flavonoids NaOH 1M Formation of solutions (+)

yellow-orange

Phenol FeCl3 5% Formations of Navy blue (+)

liquid

Anthocyanins HCl 2M Does not form Purple Blue (-)

and NH3 solution

Terpenoids CHCl3 Does not form pink (-)

and solution
(CH3CO)2O

Tannins Pb(C2H3O2)4 Formations of gold (+)

precipitation

Table 4.1. Qualitative results of chemical functional groups present in squid

leaf extract
 4.3. Survey results of factors affecting silver nanofabrication

a)

b)

c)
Figure 4.2. UV-Vis spectrum of (a) influence of extract volume ratio and silver
nitrate solution volume, (b) influence of silver nitra solution concentration and (c)
influence of stirring time from solution mixture to silver nanoparticle formation.
Based on Figure 4.2 and Appendices 2.1, 2.2, 2.3, the results show that at
an extract volume ratio and a silver nitrate solution volume of 5:20, a silver nitrate
solution concentration of 1 mM and a magnetic stirring time of 30 minutes are the
most optimal conditions for silver nanosynthesis.

a)
 b)
Figure 4.3. UV-Vis spectrum of (a) influence of temperature and (b) pH on
silver nanoparticle formation
Based on Figure 4.3 and Appendices 2.4, 2.5, the results show that at a
magnetic stirring temperatureof 600C and an ink grass extract with pH=8 is the
most optimal condition for silver nanosynthesis.
From the results of the survey, factors such as the influence of light,
volume of squid leaf extract, concentration ofAgNO3 solution, reaction time,
temperature, pH to AgNPs particle formation are summarized as in Table 4.2.
Table 4.2. Optimal Conditions for AgNPs Synthesis with Lemongrass Leaf Extract
Factors Value
Volume of Inkweed Leaf Extract 5 ml
Synthesis time 30 mins
Reaction temperature 600C
Solution concentration AgNO3 1.0 mM
pH 8
4.4. Results of analysis of physicochemical properties of silver nanotubes
synthesized by ink weed
4.4.1. Infrared spectrum (FT-IR)

a)

b)
Figure 4.4. FT-IR spectra of squid grass leaf extract and silver nano solution
synthesized by squid grass leaf extract
 Observed in Figure 4.4, the FT-IR spectra of the squid leaf extract and the
silver nano solution synthesized by the squid leaf extract have the characteristic
peaks at the wave number of 3448.59cm−1, 2093.96cm−1, 1638.53cm−1,
1080.29cm−1 corresponds to the -NH, -O-H, C=O, C-OH. The wide band
appearing at wave number 3448.26 cm−1 characterizes the tensile oscillations of
the -OH group in alcohol or phenol and the -NH group in the secondary amine.
The spectral peak at wave number 2093.96 cm−1 corresponds to the tensile
vibration of the O-H group in the carboxylic group. The peak at wave number
1638.25 cm−1 is characteristic of the C=O bond in aromatic ketones or in the
COOH group. The spectral peak at wave number 1080 cm−1 characterizes the C-
OH tensile oscillations of secondary alcohols or polyols such as flavonoids
present in the extract of squid leaves. This shows that the presence of hydroxyl
groups acts as a reducing agent for Ag+ to Ago and as a stabilizer to reduce the
oxidation process coagulation of silver nanoparticles. The FT-IR spectra of the
squid leaf extract and the silver nanoparticle solution were not significantly
different. Compared with squid leaf extract, the synthesized silver nano sample
gives a lower signal intensity, which can be explained by the decrease in the
concentration of surface -OH groups in the silver nanosample solution after the
deionization reaction Ag+ to Ago.
4.4.2. Dynamic Laser Scattering Spectroscopy (DLS)
The results of measuring particle size distribution by DLS of silver
nanoparticles synthesized from squid grass extract are presented in Figure 4.5.

Figure 4.5. The results of measuring the size distribution by DLS of AgNPs
particles synthesized by squid leaf extract
Silver nanoparticles were synthesized by squid leaf extract with an
average particle size of 33.2 nm.
4.4.3. Surface morphology analysis of synthesized silver nanoparticles
by scanning electron microscopy (SEM)
Figure 4.6 shows that silver nanoparticles synthesized by lemon basil leaf
extract have spherical shape and uniform size.
Figure 4.6. SEM image of silver nanoparticles synthesized from squid leaf extract

Thus, the flavonoids, phenolic compounds, etc. present in the extract of the
leaves of squid grass have played two roles in the synthesis of silver nano: both
as a silver ion reducing agent and as a coating for the nanocrystalline nuclei.
silver, preventing them from clumping together. That means that when the
extract of squid leaves is added to AgNO3 solution, a complex reaction occurs
between flavonoids, phenolic compounds... with Ag+ ion, flavonoids-Ag
complex, phenolic-Ag... formed as an intermediate. Then, the flavonoids,
phenolic... will denuclearize Ag+ ions into silver atoms, and then form silver
nanoparticles suspended in solution. These silver nanoparticles are coated with
organic compounds found in squid grass extract, thereby maintaining the
stability of the silver nano colloidal solution.
4.5. Antibacterial activity of silver nano-solutions synthesized by extracts of
squid grass leaves
To investigate the antibacterial activity of the silver nano solution synthesized
by the squid leaf extract using the sterile loop method with three concentrations
of 150 ppm on the strains of Bacillus spizizenii, Staphylococcus aureus,
Salmonella typhi and Escherichia Coli, the negative control is an extract of the
leaves of squid grass and the positive control is the antibiotic Ampicillin.

a) b) c) d)
Figure 4.6. Antibacterial results of silver nano-solutions synthesized by extract of
squid leaves on bacterial strains (a) Escherichia coli, (b) Staphyloceus arueus, (c)
Bacillus spizizenii and (d) Salmonella typhi

Table 4.3. Antibacterial ring diameter of silver nanoparticle solution

Template Antimicrobial Ring Diameter (mm)
 Bacillus Staphylococcus Salmonella Escherichia
spizizenii aureus typhi Coli
Squid grass extract 0 0 0 0
14,75 ± 13,26 ±
AgNPs 15,96 ± 0.5 15,10 ± 0.6
0.3 0.4
The bactericidal mechanism of silver nano-solutions involves their
interaction with the -SH groups of the oxygen-transporting enzyme molecule and
inactivation of this group of enzymes leads to inhibition of bacterial cell
respiration. The research results showed that the silver nano solution synthesized
by the extract of squid leaves has antibacterial ability on Escherichia coli,
Staphyloceus arueus, Bacillus spizizenii and Salmonella typhi.

CHAPTER 5: CONCLUSIONS AND RECOMMENDATIONS

CONCLUSION
In this study, we have:
- Successfully synthesized a solution of silver leaf ink grass.
- The conditions for the synthesis of silver nanoparticles have been
determined.
- Measurement results of SEM, DLS, EDX of the sample showed that silver
nanoparticles were successfully synthesized by green chemistry method, silver
nanoparticles were spherical in shape, with uniform structure. The silver
nanoparticle size measured by DLS is 33.2 nm.
- Nano silver solution synthesized from squid leaf extract has high
antibacterial ability against Bacillus spizizenii, Staphylococcus aureus,
Salmonella typhi and Escherichia Coli.
RECOMMENDATIONS
Due to the limited time to do the experiment and the limited equipment
room of the laboratory, we hope that further studies will apply silver nanoparticles
in practice.
THEME DEVELOPMENT DIRECTIONS
Research and development of biological products with medical applications
based on the properties of nano Silver.
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