Shiksha Mandal’s

BAJAJ COLLEGE OF SCIENCE, WARDHA

(AUTONOMOUS)

M.Sc. Project

Green synthesis of silver nanoparticles (AgNPs) using

Cinnamomum tamala leaf extract

Submitted by

Vaibhav Dnyaneshwar Kalambe

M.Sc Semester IV

For the award of

Master of Science in Zoology

POST GRADUATE DEPARTMENT OF ZOOLOGY

BAJAJ COLLEGE OF SCIENCE, WARDHA
(Academic Year 2019 – 2020)
Green synthesis of silver nanoparticles (AgNPs) using
Cinnamomum tamala extract

Project report submitted

in the partial fulfilment of the Degree of
Master of Science in Zoology
in faculty of Science
Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur.

Submitted by

Vaibhav Dnyaneshwar kalambe

M.Sc .IV Semester (Zoology)

2019-2020

Supervisor

Dr. Saikat Biswas

(M.Sc., NET-JRF, Ph.D.)
Department of Zoology
Bajaj College of Science
Wardha, Maharashtra

Bajaj College of Science, Wardha- 442001, Maharashtra

 Shiksha Mandal’s
BAJAJ COLLEGE OF SCIENCE, WARDHA
POST GRADUATE DEPARTMENT OF ZOOLOGY
(Academic Year 2019 – 2020)

CERTIFICATE
This is to certify that Mr./Ms./Mrs. VAIBHAV DNYANESHWAR KALAMBE has successfully
completed the M.Sc. Semester IV Project titled Green synthesis of silver nanoparticles (AgNPs)
using Cinnamomum tamala leaf extract in the partial fulfillment of the Degree of Master of
Science in Zoology, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur.
This project report is the record of authentic work carried out by him/her under my supervision.

Date:
Place: Wardha Dr. Saikat Biswas
Assistant Professor
Department of Zoology
Bajaj College of Science, Wardha

Dr. M. R. Chandrakar
Head of the Department
Department of Zoology
Bajaj College of Science, Wardha
 Declaration

I, Vaibhav Dnyaneshwar Kalambe declare that the project report is based on my own work

carried out during the course of M.Sc. in Zoology under the supervision of Dr. Saikat Biswas.

I certify that the work contained in the report is original and has been done by me under the

general supervision of my supervisor. The work has not been submitted to any other Institution

for any other degree/diploma/certificate in this university or any other University of India or

abroad.

(Signature)

Vaibhav Dnyaneshwar Kalambe

Roll No.______________
 INDEX

Sr. No. Contents Page No

1 Abstract 1

2 Introduction 2

3 Literature Review 4

4 Materials and Methods 6

5 Result and Discussion 8

6 Conclusion 12

7 References 13

8 Acknowledgements 17
Abstract:-
In the present study Cinnamomum tamala (Bay leaf) was used to prepare silver nanoparticles.

As the chemical method is more hazardous, green method was followed in synthesizing the

silver nanoparticles. The synthesized AgNPs were characterised by UV spectroscopy

technique. UV visualization show peak in 400 to 450 nm which is in accordance with previous

studies reported in literature. The study revealed antimicrobial activity and H2O2 sensing

capacity of organic tea extract capped silver nanoparticles. These results suggest that organic

tea stabilized silver nanoparticle can be widely used as antimicrobial and in various field of

science.

1
Introduction:-

Nanotechnology is branch of science which aims in studying the matter on atomic and

molecular level. It deals with the preparation of nanoparticles, its characterization, and its

applications. Nanoparticles are small particle which ranges from 1- 100 nm (Arokiyaraj et. al.,

2014). It shows various properties because of it high surface to volume ratio which makes its

suitable for application in human welfare research. Various metals are used for preparation of

nanoparticles and are extensively used in various fields like electronics, environmental studies,

medicinal biology etc. (Harish et al., 2018). Several different alloys and semiconductors are

also used for the preparation of nanoparticles which have similar properties of metal

nanoparticles (Harish et al., 2018). Different methods are adopted to synthesize metallic

nanoparticles. Among all the metals, silver is mostly used to synthesize nanoparticles (Guzel

and Erdal, 2018). The use of silver is well known from the ancient times for its use in medical

treatments (Lawrence Hall of Science, 2013). Nanoparticle are very useful and occurs widely

in nature and are widely studied in the different fields of science like the chemistry, physics

and biology. Silver nanoparticles were used because of their special properties which can be

integrated into biosensor materials, cosmetics and antimicrobial applications.

There are different methods to synthesize nanoparticles. Due to the hazardous effect of

chemical synthesis, green synthesis of nanoparticles are generally used now a days. Green

synthesis method involves three main steps (1) solvent medium selection, (2) environmental

reducing agent selection, and (3) non-toxic substances for nanoparticles stability selection. The

synthesis of silver nanoparticles (AgNP) from various plants species like leaf extract of Laurus

nobilis (bay leaf), Cinnamomum camphora (Huang, et al., 2007), tulsi plant leaves

(Ocimumtenui florum) (Logeswari et al., 2012) and tea (Moulton et al., 2010) are commonly

used.

2
In the present study silver nanoparticles (AgNPs) were synthesized using leaves of

Cinnamomum tamala (Bay leaf) extracts. The genus Cinnamomum which belongs in the family

Lauraceae have nearly 260 - 270 species and occur naturally in the Asia and Australia (Sharma

and Nautiyal 2011). Dry leaves of the Cinnamomum tamala are used as flavoring agent for

variety of the food preparations across India. The bay leaf extract was used as a reducing and

capping reagent for the AgNP synthesis, and distilled water serve as reaction medium. The aim

of this study is to prepare the nanoparticles using natural product such as bay leaf extract and

to characterize and check the different antimicrobial effects of the synthesized AgNPs.

3
Review of literature:-

The word “nano-technology” was first introduced by the Japanese scientist Taniguchi in 1974

into a scientific community at the international conference on industrial production (Taniguchi,

1974). Jain et al. (2009) studied extensively on nanoparticles and suggested that the

nanoparticles can be effectively synthesized using simple techniques by following an

environmental friendly method using silver and bay leaves. In recent years, the emergence of

nanotechnology has provided a vast research by intersecting with various branches of science

and creating an impact on all forms of life. Nanotechnology is a field of science which deals

with preparation, manipulation and use of materials ranging in nanometers (Wang, 2018). In

nanotechnology, nanoparticles research is an important aspect due to its innumerable

applications. Nanoparticles have expressed significant advances owing wide range of

application in field of bio-medical, sensors, antimicrobial, catalyst, electronic, optical fibers,

agricultural, bio-labeling and in other areas (Harish et al., 2018). Unlike the conventional

methods to produce nanoparticles, green nanotechnology has become of more interest because

it uses of nanoparticles which are made from herbal or plant extracts and are less hazardous

when interacted with human as it uses less toxic chemical substances (Khandel et al., 2018).

Metallic nanoparticle produced by plant extracts are of the more focus they show extreme anti-

microbial activity and the production of these are not time consuming and cost effective (Harish

et al., 2018). It is now well known that using plants as a sources for manufacturing

nanoparticles is advantageous because it is easily available, easy to handle and non-hazardous.

An extensive work on the achievements in the synthesis and application of antibacterial

nanoparticles was provided by Moritz and Geszke-Moritz (2013).

Studies on greener routes to synthesize nanoparticles of zero valent metals, metal oxides and

salts were reviewed by Kharissova et al. (2013). They discussed various techniques and the

4
merits of various plant based generation of nanoparticles. The potentials of ‘green method’ to

synthesize nanoparticles in the laboratory scale and in natural environment was reported by

Salam et al. (2012). A mini review on the antimicrobial properties of tea (Camellia sinensis L.)

was provided by Hamilton-Miller (1995) where the chemical composition of tea was outlined.

Jain et al. (2009) did extensive study and described that effective and environment friendly

technique for green synthesis of silver nanoparticles from AgNO3 solution through the fruit

extract was used for the synthesis of nanoparticles. This was the first instance when papaya

extract as reducing and capping agent was studied. Geethalakshmi et al. (2010) described a cost

effective and environment friendly technique for green synthesis of silver nanoparticles from

AgNO3 solution using the extract of Trianthema decandra as reducing as well as extract was

capping agent. Vishnudas et al. (2012) explained about the synthesis of silver nanoparticles

from Cardiospermum helicacabum fresh leaf extracts. A novel switch grass (Panicum

virgatum) extract mediated green process was demonstrated by Misra et al. (2012).

This suggests that there had been an extensive number of work in generation of silver

nanoparticles using green method and there are number of methods in synthesizing silver

nanoparticles, but, there are very few reports where a mixture of organic tea was used for

generation of silver nanoparticles.

5
Materials and methods:-

Materials

For the study following chemicals and solutions were used: Cinnamomum tamala (bay leaf),

distilled water, deionized water or battery water, AgNO3, methylene blue solution, H2O2.

Experimental methods

Preparation of Cinnamomum tamala extract

10 gm of bay leaf (Cinnamomum tamala) was cleaned, chopped and boiled in 100 ml distilled

water for 10 minutes. Then the extract was obtained by filtration through filter paper.

Preparation of 1mM AgNO3 solution

Solid AgNO3 solution weighed as 0.008 g and dissolved in 50 ml distilled water (Deionised

water or battery water). The solution was stored in dark colour bottle.

Synthesis of nanoparticle

1 ml of bay leaf extract and 14 ml of AgNO3 was mixed and heated for 35 mins on hot plate

kept at 5 to 6 grid scale (Mentioned on Hot plate; about 80ºC). The color change was observed

from light brown to dark brown, this change in colour indicates that nanoparticles are formed.

Optimization of time for nanoparticle preparation

1 ml organic tea extract and 14 ml AgNO3 solution mixed properly and heated at different

time interval (5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min) on hot plate kept at 5 to

6 grid scale. The colour change was observed from light brown to dark brown spectra were

recorded for every time of interval in UV-vis spectrophotometer. The time at which single

sharp peak observed that indicates nanoparticles are formed.

6
Stability test for nanoparticles

A stability test was done after nanoparticles were synthesized. After preparation of

nanoparticles, the solution containing them was scanned by UV spectrophotometer in the range

of 300 – 600 nm for 7 days to check the peak value.

Hydrogen peroxide sensing

To check hydrogen peroxide sensing properties of organic tea stabilized silver nanoparticles,

serial dilution (1%, 2% ,5% ,10% ,15%) of hydrogen peroxide solution were made using 30%

H2O2 solution and dilution was done with 20mM phosphate buffer with pH 7.4. The diluted

solution was added to silver nanoparticles and spectra was scanned using UV spectroscopy in

ranges of 300- 600 nm after incubation time of 15 min.

Antimicrobial test for Nanoparticle

Antimicrobial activity of bay leaf stabilized silver nanoparticle was done with E coli bacteria.

For this experiment, nutrient agar plates were prepared (7 gm in 1000ml distilled water). After

agar was solidified, the bacterial inoculum was spread in the petri dishes. Wells were created

in the solidified medium and different volumes (100- 400μl) of nanoparticles was poured the

wells and the plates were left for incubation at 37ºC for 24 hours allowing the growth of

bacteria. Next day result was observed, and inhibition zones were measured.

7
RESULTS AND DISCUSSION:-

Synthesis of Silver nanoparticles (AgNPs)

The reduction of silver nitrate using the bay leaf extract was determined by the colour change

in the reaction mixtures which changes from colorless to yellowish brown. Simultaneously,

UV-Visible spectra recorded for the reaction mixture of reduced silver nitrate by bay leaf

extract. The dispersed AgNPs displayed a absorption maxima at a range of 420 to 450 nm

characteristics to the surface plasmon resonance (SPR) band of silver nanoparticles (Chandra

et al., 2020). We also estimated the incubation time required for synthesis of nanoparticle at

80℃. Influence of incubation time is most important factor for preparation of nanoparticle.

After heating at different time of interval (5min, 10 min, 15 min, 20 min, 25 min, 30 min, and

35 min) the colour change was observed which indicates formation of nanoparticles. It was

observed that the optimum incubation of time obtained was at 35 min on hot plate at 5 to 6 grid

scale (80℃) which indicates that is optimum reducing condition for silver to convert itself into

nanoparticle (Fig. 1).

2
5 min
10 min
ABSORBANCE

15 min
1 20 min
25 min
30 min
35 min
0
250 350 450 550
WAVELENGTH (NM)

Figure 1: Graphical representation showing the spectral analysis during the synthesis of bay
leaf stabilized AgNPs at different time intervals

8
Stability of synthesized nanoparticles

The synthesized AgNPs were kept at room temperature for five days and the absorbance was

measured each day. It was observed that the nanoparticle can remain stable for 2 days (Fig. 2).

3.5

2.5 Day 1

2 Day 2
ABSORBANCE

1.5 Day 3

1 Day 4

0.5 Day 5

0
250 350 450 550 650
-0.5
WAVELENGTH (NM)

Figure 2: Graphical representation showing the absorbance spectrum of synthesized AgNPs

for four days.

Hydrogen peroxide sensing capacity of AgNP

The ability of SNPs to detect the presence of H2O2 in a sample was affirmed by adding serial

dilution (1%, 2%, 5%, 10%, 15%) of hydrogen peroxide solution with green synthetically

generated AgNPs. The diluted solution was added to silver nanoparticles and spectra was

scanned using UV spectroscopy in ranges of 300- 600 nm after incubation time of 15 min and

it was observed that the AgNPs has the ability to sense the H2O2 solutions (Fig. 3). The H2O2

sensing capacity of AgNPs were also suggested by previous reports (Tagad et al., 2013).

According to Mohan et al. (2014), the addition of SNPs to H2O2 resulted in the formation of

free radicals that initiated the degradation of the SNPs. Subsequently, Ag0 was oxidized to Ag+

and a decrease in absorbance was observed. These findings suggest that the AgNPs can used

to detect the concentration of H2O2 in samples.

9
 1% 2% 5% 10% 15%

3.5

2.5
ABSORBANCE

1.5

0.5

0
300 350 400 450 500 550 600
WAVELENGTH (NM)

Figure 3: Graphical representation showing the sensing of different concentrations of H2O2 by

AgNPs synthesized using the bay leaf extract.

Antimicrobial activity of AgNPs

Antimicrobial activity of bay leaf extract stabilized silver nanoparticle was to found positive

with E. coli and the maximum inhibition zone was obtained with the maximum concentration

of nanoparticle (Fig. 4A-D). Since, the antibacterial ability of the silver ion is well known (Liu

et al., 2010; Jung et al. 2008), AgNPs also showed antimicrobial properties. Several

mechanisms are proposed for the antimicrobial properties of AgNPs. It is known that AgNPs

kill the bacteria by attaching with the bacterial wall or by formation of free radicals (Kim et

al., 2007; Yu et al., 2013). Patil et al. (2012) suggested that the cell death due to AgNPs might

be due to the cytoplasmic membrane disorganization and the consequent leakage of various

biomolecules such as amino acids, protein and carbohydrates. It has also been reported that

silver ions are released from AgNPs and that interact with thiol groups of some enzymes

causing the killing of the bacteria (Feng et al., 2010). Krishnaraj et al. (2009) reported that the

change in membrane permeability caused by the action of silver nanoparticles is a function of

10
conductivity and the high conductivity of cells treated with AgNPs was due to the release of

cellular components present inside the cells.

Figure 4A-D: Antibacterial activity of silver nanoparticles synthesized using bay leaf extract.
(A, B) Figure depicting culture plates before introduction of AgNPs. (C, D) Figure showing
the culture plates after introduction of AgNPs with a prominent zone of inhibition.

11
Conclusions:-
Green method for synthesizing silver nanoparticles was successfully performed using bay

leaf extract. The various biomolecules in the mixture was responsible for the formation and

stability of the synthesized silver nanoparticles. The sensing capacity of The AgNPs towards

H2O2 was also carried out. The synthesized silver nanoparticles were also able to inhibit

microbial growth, thus the antimicrobial property of AgNPs were confirmed.

12
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 Acknowledgement

The success of any project depends largely on the encouragement and guidelines of many

others. I would like to take this opportunity to express my gratitude to the people who have

been instrumental in the successful completion of this project. I would like to show my greatest

appreciation to the Principal, Bajaj College of Science, Wardha for providing all the necessary

permissions. I can’t say thank you enough for the support and help of the Head of the

Department, Department of Zoology for constant support and encouragement. I am grateful to

all the teaching and non-teaching staff of the Department of Zoology for their help and support

during the period of my work.

17