Biosynthesis, Characterization, Antibacterial Activity and Anticancer Effect of Silver Nanoparticles Using Anethum Graveolens Leaf Extract

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 4, Pages: 1625-1629

J. Environ. Treat. Tech.

ISSN: 2309-1185

Journal web link: http://www.jett.dormaj.com

https://doi.org/10.47277/JETT/8(4)1629

Biosynthesis, Characterization, Antibacterial

Activity and Anticancer Effect of Silver

Nanoparticles Using Anethum Graveolens Leaf

Extract

Vahab Alamdari-Palangi , Alireza Shojazadeh , Farnaz Hosseini , Nesa Khalaf , Aria

Dianatinasab *, Mehrdad Ameri *

Department of Medical Biotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences Shiraz, Iran

Student Research Committee, Shiraz University of Medical Sciences, Shiraz, Iran

Department of Medical Nanotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz,

Iran

Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.

Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz,

Iran

Received: 15/07/2020

Accepted: 27/09/2020

Published: 20/12/2020

Abstract

Nanoparticles are used in various fields of science, especially medicine. Advent of nanotechnology has led to significant

development in disease diagnosis, treatment and drug delivery. Silver nanoparticles (Ag-NPs) play an important role in medical

application, which makes them a viable alternative to common antibiotics. Amongst various methods, synthesis of Ag-NPs via green

method has the advantage of being cost effective with no toxic agent. In this study, Spherical shape Ag-NPs with average size of 30 nm

were synthetized using Anethum graveolens leaf extract as a green, cost-effective, non-toxic and environment-friendly source.

Transmission electron microscopy (TEM), particle size analysis (PSA) and Fourier transform infrared (FT-IR) were performed to

characterize synthesized Ag-NPs. The antibacterial activity of the synthetized Ag-NPs was evaluated against gram positive and negative

bacterial pathogens. The minimum inhibitory concentration (MIC) at different concentrations of Ag-NPs were used to evaluate their

antibacterial properties against Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa

pathogens. The results exhibited a desirable antibacterial property of Ag-NPs, suggested its usage as putative antibacterial agents.

Moreover, the anticancer effect of green synthesized Ag-NPs was evaluated against MCF-7 lines and results showed that the cell viability

is depended on the concentration of Ag-NPs. In short, this method provides a simple, cost effective and eco-friendly way to synthesis

Ag-NPs which can be used as a suitable alternative to common antibiotics that use hazardous chemical agents, additionally, with

anticancer effects against MCF-7 cells.

Keywords: Silver nanoparticles, Nanotechnology, Antibacterial agents, Anticancer effect

Nowadays, a great deal of efforts has been put into

Introduction

synthesis of Ag-NPs (8, 9). The Ag-NPs are being applied in

cosmetic and hygiene products such as shampoos, soaps,

toothpastes, anti-septic gels and pomades. Ag-NPs are

generally synthesized using physical and chemical methods,

but these approaches use toxic chemicals, organic solvents and

non-biodegradable materials that are dangerous to human

health and environment. Therefore, non-toxic, biocompatible,

and eco-friendly methods to synthesize Ag-NPs are warranted

Emergence of nanotechnology has attracted researchers in

various fields of science and industries (1). Nanoparticles

bridge the gap between bulk material, atomic and molecular

structures. They have the potential to be used in medicine,

hygiene, pharmacy, dentistry and etc., which is due to their

interesting and unique physical and chemical properties such as

optical, magnetic, mechanical and conductivity properties (2).

Excessive use of antibiotics has led to microbial resistance;

thus, many researchers have focused on development of novel

and effective antimicrobial agents (3, 4). Several studies

showed the antibacterial activity of Ag-NPs against a wide

range of microorganisms (5, 6). Hence, they can be a promising

compound in development of novel antimicrobial agents (7).

(

10, 11). Recent developments showed the critical role of plant

extracts in production of Ag-NPs (12). Plants extracts contain

several phenolic and antioxidant compounds that reduces Ag to

Ag-NPs (13). There are several studies on the synthesis of Ag-

NPs using various plants, such as Cupressus sempervirens Leaf

Corresponding author: (a) Mehrdad Ameri, Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical

Sciences, Shiraz University of Medical Sciences, Shiraz, Iran. Tel: +989386360918, Email address: mehrddameri@gmail.com and (b)

Aria Dianatinasab, Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran. Email address:

dianatinasab.aria@gmail.com

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Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 4, Pages: 1625-1629

(

10) Givotia moluccana leaf (4), Syzygiumcumini fruit (14),

Azadirachta indica aqueous leaf (15), Eriobotrya japonica leaf

16), aloe vera (17), Nigella sativa (18), Pistacia atlantica (13),

Hinton Broth medium. One Ag-NPs sample was used as the

positive control and another with no bacteria as negative

control.

(

Nyctanthes arbortristis (19), Orange and pineapple (20), and

Pinus densiflor (21). Anethum graveolens (dill) is an annual

herb and since antient time it has been exploited in ayurvedic

medicines. Anethum graveolens is a member of Apiaceae

family and rich in polyphenols, anti-oxidants and minerals (22).

It is helpful in patients with hyperlipidemia (23) and as a

component in gripe water, it can alleviate colic pain in babies

and flatulence in young children (24). We used Anethum

graveolens aqueous leaf extract to synthesize and evaluate

antibacterial activity of Ag-NPs.

2.5 Anticancer effect

To evaluate the anticancer effect of synthesized synthetized

nanoparticles, five different concentration of Ag-NPs (6.25,

12.5, 25, 50, 100 µgr/ml) were exposed to MCF-7 cells for 24

hours. MCF-7 cell line which is a breast cancer cell line was

purchased from Pasteur institute of Iran. At first, cancerous

cells were cultured in RPMI1640 medium containing 10% fetal

bovine serum (FBS), 1% penicillin/streptomycin. Optimum

condition for cell growth were considered as 5% CO , 95%

The main purpose of this study was to green synthesis of

Ag-NPs, using Anethum graveolens leaf extract as an

inexpensive, simple and non-hazardous BioSource. The

antibacterial properties of resultant Ag-NPs were investigated

against Staphylococcus aureus, Enterococcus faecalis,

Escherichia coli, and Pseudomonas aeruginosa pathogens.

Moreover, the anticancer effect of green synthesized Ag-NPs

were evaluated against MCF-7 cells.

humidified atmosphere air and 37 C temperature. After

subculture and cell count, we added 10000 cells to each well of

a 48-well plate, additionally, cells were treated with different

concentration of Ag-NPs. In addition to samples treated with

silver nanoparticles, an untreated cell sample was considered as

a control. After 24 hours of incubation (MTT) assay was

performed to evaluate the effect of Ag-NPs on MCF-7 cells.

Therefore, supernatant of each wells was extracted and while

cells are attached at the bottom of wells, MTT was added to

each well. After 4 hours of incubation in 37 C and 5% CO

Materials and methods

contents of each wells were removed and 50 µl of DMSO was

added to the wells. Consequently, after 30 min of incubation,

the plate was read at 570nm with an ELISA reader and the

percentage of cell cytotoxicity was calculated using the optical

density and the below formula:

.1 Chemicals

Silver nitrate (AgNO3, 99%) was purchased from Sharlo

(

Spain). Before usage, the glassware was washed with HCl and

deionized water. Ultrapure deionized water was used for

synthesis reactions and leaves extraction.

Cell viability = (OD value for test/OD value for

.2 Leaf extract preparation

A. graveolens leaves were collected from the countryside

control)ꢀ×ꢀ100

of Shiraz, Fars, Iran and an herbalist confirmed the species. The

fresh plant leaves were washed with distilled water for several

times (diH2O) to remove any dusts and mods. The leaves were

shade dried at room temperature. To prepare leaf extract, 5 g

dried leaf powder was boiled in 100 ml deionized water for 15

min. Then mixture was filtered through a Whatman filter paper

No.1. The filtered extract was stored at 4oC, which was used

for synthesis reaction.

3 Results

3.1 synthesis of Ag-NPs

After adding AgNO3 solution to the A. graveolens leaf

extract, the color of mixture changed to dark brown. This

alteration indicates the silver ions reduction to Ag-NPs.

3.2 Characterization of synthetized Ag-NPs

The TEM micrograph and PSA data of the synthesized Ag-

NPs by A. graveolens extract are depicted in figure 1. TEM

micrograph showed spherical Ag-NPs was surrounded by a thin

layer of biological matrix from A. graveolens leaf extract. The

particle size distribution of the biosynthesized Ag-NPs was in

the range about 30 nm. The PSA analysis showed that most Ag-

NPs had a size less than 100 nm. It is worth noticing that PSA

measures hydrodynamic diameter of nanoparticles, always

reported larger size than TEM image. The FTIR analysis are

.3 Ag-NPs synthesis

For green synthesis, 1 ml silver nitrate (AgNO3·6H2O, 0.1

M) was added to 9 ml leaves extract under vigorous stirring at

room temperature. After 24 h, the final mixture was centrifuged

at 12000 rpm for 5 min. The obtained Ag-NPs was washed with

deionized water and dried in an oven at 50°C.

.4 Ag-NPs characterization

Ag-NPs size and morphology were evaluated by TEM

−1

exhibited in figure 2. The band at 3417 cm indicates the

combined peaks of the amine or amide and –OH group

stretching vibration in the plant extract. The methyl group gives

(

Philips, CM 10; HT 100 Kv) and PSA. Functional groups of

the phytochemicals, involved in the formation of Ag-NPs were

analyzed using FTIR (Perkin- Elmer, Two).

-1

its symmetric tensile vibrations of about 2863 cm and

−1

asymmetric traction in 2922 cm . The peak at 1100 cm was

−

related to C-O stretching. The peak at 1628 cm (sp 2 -

hybridized C = C) can be assigned to the skeletal vibrations of

the plant extract. Likely, the peak intensities in 1383 cm is

.5 Antibacterial activity

The synthesized Ag-NPs were tested for antimicrobial

−1

activity by evaluating MIC minimum inhibitory concentration

MIC) against gram-positive (Staphylococcus aureus ATCC

5923 and Enterococcus faecalis ATCC 29212) and gram

due to C–N stretching in the extract of the plant capped silver

(

−1

nanoparticles. The peaks at 2322 and 2355 cm are related to

N-H amine and amide group. This indicates the binding of Ag

with O and N of –OH and –NH2 groups in the extract of the

plant.

negative (Escherichia coli ATCC 25922 and Pseudomonas

aeruginosa ATCC 27853) pathogens. The bacteria were

cultured in Muller-Hinton agar and fresh colonies were

suspended in sterile water to reach their tarnish equivalent to

(

.5 McFarland. MIC with different concentration of Ag-NPs

4, 8, 16, …, 2048 µgr/ml) was evaluated against 50 µl of 1.5

106 CFU/ml bacterial suspension and cultured in Muller-

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Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 4, Pages: 1625-1629

size of 10-30 nm. Raj et al. (26) synthesized Ag-NPs in a

diameter ranging 2-20 nm, and Arunachalam et al. (27)

reported the biosynthesis of spherical Ag-NPs with a size range

of 9-12 nm.

Figure 2: The FTIR spectra of synthesized Ag-NPs is illustrated. The

standard peaks related to amine, amide and –OH groups appear at 3417

-1

cm . The peaks at 2863 cm (methyl), 2922 cm (methyl), 1100 cm

C-O), and 1628 cm^-¹(C = C) can be observed. The C-N stretching

(

caused an intensive peak at 1383 cm . The N-H amine and amide group

give the peaks at 2322 and 2355 cm^-¹

Table 1: Minimum inhibitory concentrations of Ag-NPs (µgr

-1)

ml are evaluated against candidate bacteria

Strains

MIC

E. coli

E. faecalis

P. aeruginosa

S. aureus

512

Figure 1: a) The approximate size and shape of synthetized Ag-NPs are

shown in TEM micrograph. b) The PSA analysis of the Ag-NPs showed

that the most of Ag-NPs have size less than 100 nm

.3 The antibacterial activity of obtained Ag-NPs

The antibacterial activity of the synthesized Ag-NPs against

Staphylococcus aureus, Enterococcus faecalis, Escherichia

coli, and Pseudomonas aeruginosa photogenes were evaluated.

This study is a report about the synthesis of Ag-NPs by A.

graveolens extract, their antibacterial activity and anticancer

effect against some pathogenic bacteria and MCF-7 cell line

respectively. In our study, TEM micrograph and PSA data were

used to investigate the morphology, size and shape of Ag-NPs.

-1

The lowest concentration of Ag-NPs (µgr ml ) which

prevented the visible growth of bacteria are reported as MIC in

table 1. The results showed noticeable effect of synthesized Ag-

NPs on the growth of these pathogens. The MIC of Ag-NPs

against Escherichia coli and three other organisms were

evaluated 64 µgr ml^-¹and 512 µgr ml , respectively.

Consequently, it can be concluded that Escherichia coli was

more sensitive than the other tested pathogens against

synthetized Ag-NPs.

-1

.4 The anticancer effect Ag-NPs on MCF-7 cell line

The anticancer effect of five concentration (6.25, 12.5, 25,

0, 100 µgr/ml) of the synthesized Ag-NPs were evaluated

against MCF-7 cell line using cell culture and MTT assay. After

exposing each concentration of Ag-NPs with MCF-7 cells for

4 h, cell viability was calculated using the optical density.

MCF-7 cell treatment with Ag-NPs at 6.25, 12.5, 25, 50, 100

µgr/ml concentration in 24 h reduced cell viability 60.40 ± 0.83

Figure 3: Cell viability of MCF-7 cells against different concentration

of Ag-NPs after 24 h. results are reported as % cell viability compared

with control sample

, 48.70 ± 0.49 %, 35.54 ± 0.78 %, 21.78 ± 0.39 %, 11.14 ±

.13 % respectively and cell viability for control sample

calculated 100 % after 24 hours. (Figure 3).

According to PSA analysis, the spherical nanoparticles

with average size of 30 nm have desirable size distribution. The

FTIR analysis was performed to identify the functional groups

of biological compounds in the A. graveolens leaf extract that

was involved in the reduction of Ag ions to Ag-NPs. FTIR

analysis indicates the binding of Ag with O and N of –OH and

Discussion

The study results indicated that A. graveolens leaf extract

can efficiently reduce the silver ions to Ag-NPs in a diameter

range of 30 nm, and prevented the aggregation of nanoparticles.

Srinithya et al. (25) biosynthesized spherical Ag-NPs with a

–NH2 groups in the plant extract.

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2020, Volume 8, Issue 4, Pages: 1625-1629

Various studies showed antimicrobial activities of Ag-NPs

against both Gram-negative and Gram-positive bacteria (7). In

the present study, the antibacterial effect of Ag-NPs was

evaluated against pathogenic strains such as E. coli, E. faecalis,

P. aeruginosa and S. aureus. According to the results,

synthesized Ag-NPs exhibited excellent antibacterial effect

against these pathogenic bacteria. Based on MIC value reported

researches, it is suggested to evaluate anticancer effects of

green synthesized Ag-NPs against different cell lines, and anti-

inflammation studies can be implemented. Moreover, the

antibacterial activity of the synthetized Ag-NPs should be

investigated against other pathogenic bacterial strains.

Aknowledgment

in table 1, it can be concluded that Escherichia coli (64 µgr ml

The authors wish to thank Mr. H. Argasi at Research

Consultation Center (RCC) of Shiraz University of Medical

Sciences for his invaluable assistance in editing this

manuscript. This study was supported by a grant from the

Research Council of Shiraz University of Medical Sciences.

)

was more sensitive than the other tested pathogens (512 µgr

-1

ml ) against synthetized Ag-NPs. These results are in line with

previous studies. For example, Singhal et al. investigated

antimicrobial properties of synthesized silver nanoparticles by

Ocimum sanctum leaf extract. The MIC of biosynthesized

silver nanoparticles was also measured using pathogenic

Ethical issue

Authors are aware of, and comply with, best practice in

publication ethics specifically with regard to authorship

-1

bacteria such as E. coli (0.314 µgr ml ) and S. aureus (1.25 µgr

-1

ml ). Hence, results showed that Escherichia coli was more

sensitive than S. aureus (28). Martınez-Castanon et al. (29)

Synthetized 29 nm silver nanoparticles and reported MIC 13.02

and 16.67 µgr ml for E. coli and S. aureus, respectively. It

seems these differences might be due to particle size and

bacterial strains (29, 30).

(

avoidance of guest authorship), dual submission, manipulation

of figures, competing interests and compliance with policies on

research ethics. Authors adhere to publication requirements

that submitted work is original and has not been published

elsewhere in any language.

-1

This inhibitory effect was due to different mechanisms. For

example, Ag-NPs can be attached to the bacterial cell wall and

infiltrate it. Therefore, these nanoparticles can produce reactive

oxygen species (ROS) and free radicals in the bacterial cell,

causing apoptosis and cell death (28). Anticancer effect of

green synthesized silver nanoparticles with different leaf

extracts have been reported recently (31, 32). Thus, we

prepared five different concentration of synthesized Ag-NPs

Competing interests

The authors declare that there is no conflict of interest that

would prejudice the impartiality of this scientific work.

Authors’ contribution

All authors of this study have a complete contribution for

data collection, data analyses and manuscript writing.

(

6.25, 12.5, 25, 50, 100 µgr/ml) to evaluate their anticancer

effect against MCF-7 cell line. Cell culture and MTT assay

were used for this purpose. MTT assay is a colorimetric assay

and reduction of MTT (yellow) to formazan (purple blue) is the

basis of test. This reaction is mediated by a mitochondrial

enzyme termed succinate dehydrogenates. The results of cell

exposure to various concentration of Ag-NPs after 24 h

indicated that the cell viability is depended on the concentration

of silver nanoparticles. Highest concentration of Ag-NPs (100

µgr/ml) caused lower cell viability (11.14 ± 0.13 %). A key

strength of the present study was synthetizing Ag-NPs, using

green synthesis, which is eco-friendlier and more cost effective

in comparison to commonly used methods. However, there are

some drawbacks and limitation when using this method to

produce metal nanoparticles. Due to plant impurities, the size

and size distribution was not controllable. The nanoparticles

produced using green synthesis are sometimes less stable than

the ones synthetized using chemical methods (33).

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