Green Synthesis of Spherical Silver Nanoparticles Using Ducrosia Anethifolia Aqueous Extract and Its Antibacterial Activity

Journal of Environmental Treatment Techniques

2019, Volume 7, Issue 3, Pages: 461-466

J. Environ. Treat. Tech.

ISSN: 2309-1185

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

Green Synthesis of Spherical Silver

Nanoparticles Using Ducrosia Anethifolia

Aqueous Extract and Its Antibacterial Activity

†

† a

Mohammad Amin Jadidi Kouhbanani , Nasrin Beheshtkhoo , Pourya

Nasirmoghadas , Samira Yazdanpanah , Kamiar Zomorodian , Saeed Taghizadeh ,

a,e

Ali Mohammad Amani *

^aDepartment of Medical Nanotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of

Medical Sciences, Shiraz, Iran

Department of Microbiology, Faculty of Medical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran

Department of Medical Mycology and Parasitology, School of Medicine and Center of Basic Researches in Infectious

Diseases, Shiraz University of Medical Sciences

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

Medical Sciences, Shiraz, Iran

Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

Received: 30/03/2019

Accepted: 11/06/2019

Published: 30/09/2019

Abstract

The present study introduces a simple, cost-efficient, and eco-friendly method for the synthesis of silver nanoparticles

(

AgNP) using aqueous extract Ducrosia anethifolia and its antibacterial activity. Ducrosia anethifolia aqueous extract was

used both as a reducing and capping agent. Synthesized silver nanoparticles were characterized using several techniques

including UV-Vis spectroscopy, powder X-ray diffraction (XRD), Transmission electron microscopy (TEM), dynamic light

scattering method (DLS), and FT-IR analysis. The results demonstrated that the size range of the synthesized nanoparticles

was 4 to 42.13 nm, with the average size of 11.4 nm and in a spherical shape. Moreover, X-ray diffraction analysis indicated

that the silver nanoparticles were highly crystalline in nature. Examination of antibacterial activity of synthesized on Gram-

positive and Gram-negative bacterial revealed that they had a substantial antibacterial effect.

Keywords: Ducrosia Anethifolia; silver nanoparticle; Green synthesis; antimicrobial performance

hydrothermal synthesis (17), and vacuum deposition or

vaporization (18), and Sol-Gel technique (19).

However, it should be noted that chemical methods

have a poorer performance and that chemical methods are

toxic due to the use of toxic reducing chemicals like

citrate, borohydride, and other organic, toxic compounds

Introduction

Given their unique properties, metal nanoparticles

have a broad array of applications, each of which is

associated with a certain characteristic. (1.4) Moreover, of

such nanoparticles, silver nanoparticles are utilized in a

vast variety of fields such as catalyst in chemical reactions,

solar cells, photography, electronics, food industry,

dentistry, and most importantly, antibacterial effects on

Gram-positive and Gram-negative bacteria (5). This

bioactivity is due to the particle and nanometer size (1-

(

20). In addition, other disadvantages of these techniques

include costliness, high energy consumption, and eco-

toxic nature. Thus, the need for a biocompatible and cost-

efficient method, devoid of the abovementioned

drawbacks, is strongly felt. Biosynthesis methods can

benefit from microorganism cells or plant extracts to

produce nanoparticles. Recently, green synthesis

techniques have turned into an important branch of

nanotechnology (21-29).

This is the reason for the wide application of medical

plants in the synthesis of silver nanoparticles (30-33). In

fact, medical plants have bioactive molecules which make

possible the green synthesis of silver nanoparticles. Such

00) of silver nanoparticles, giving them special physical,

chemical, magnetic, and optical properties which are

different from their bulk structure (8-6). Therefore, silver

nanoparticles are considered as a potent antibacterial

material, comparable to, and even in some cases, stronger

than common antibiotics (9). Currently, there are various

physical and chemical methods of producing metal

nanoparticles (10, 13) which include chemical reduction

(

14), electrochemistry (15), mold-based synthesis (16),

Corresponding author: Dr. Ali Mohammad Amani, Department of Medical Nanotechnology, School of Advanced Medical

Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran. E-mail: Amani_a@sums.ac.ir. Tel: +98

9171324701.

†

These authors contributed equally to this work.

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Journal of Environmental Treatment Techniques

2019, Volume 7, Issue 3, Pages: 461-466

active molecules work as reducing and capping agents for

nanoparticle synthesis, making nanoparticles useable for

biomedical applications (34-35). Synthesis of

nanoparticles, an indicator of the relationship between

biotechnology and nanotechnology, has increased due to

the rising demands for eco-friendly technologies (36).

Various studies have shown that different plant

compositions abound in high levels of strong antioxidants

such as polyphenols, sugar, nitrogen reserves, and amino

acids (37). These compounds function as reducing (38)

and coating agents for NPS-synthesis (39-40). The plant

extract used to synthesize NPS can be useful due to their

lack of need for processes such surface coating, and lower

reaction time (41). Additionally, they do not produce any

detrimental waste (42).

dried plant was grinded so as to yield powders with mesh

20. In the next step, 5g powder was added to 50 ml

distillated water while being completely stirred for 2h. The

solution was cooled at room temperature and filtered

through Grade1 Whatman filter paper. The obtained fresh

extract was used to prepare AgNPs.

In this research, severalconcentration ranges from 20

ml of 5% to 20% of plant extract was used to synthesis Ag

NPs.Typically, 20 ml fresh filtered extract was interacted

with 5 ml aqueous 0.01 mM AgNO under reflux by

magnetic stirred. After interaction for 30 min, color of

suspensionchanged from light-yellow to brown which this

change indicates formation of Ag NPs. The brown Ag

suspension was cooled at room temperature and kept for

characterization by several techniques.

An aromatic and medicinal plant belonging to the

Apiaceae family, Ducrosia anethifolia grows wild in areas

of Iran-Turan as well as the Gulf of Oman. In Persian

language, this plant is called Moshgak, Roshgak, and

Moshkbu (literally meaning Musk-smelling) and has anti-

inflammatory and pain-relieving effects (43). The main

chemical compositions of this herb include: n-decanal, n-

dodecanal, chrysanthenyl acetate, myrcene-limonene, and

α-pinene. Also, the aliphatic compounds of this plant are

believed to act as an antibacterial agent against Gram-

positive and Gram-negative bacteria (44). The present

study introduced a quick way of producing

3 Result and discussion

3.1 UV-Vis Assessment

After silver nitrate (AgNO3) was added to extracts of

Ducrosia anethifolia, the color of the suspension changed

within some minutes. The extract was yellow by itself, but

it turned brown during the process of silver nanoparticles

synthesis which was due to the surface plasmon resonance

(SPR) of silver nanocrystals as they are dependent on the

size and shape of nanoparticles. As shown by Fig. 1, there

is a sharp peak almost 465 nm which supports silver

nanoparticles synthesis.According to the previous

literature, the observed bond corresponds to abs orption by

colloidal silver nanospheres in 450-500 nm(33, 45-47).

silver nanoparticles using Ducrosia anethifolia plant

extracts and examined their properties and inhibitory

effects on Gram-positive and Gram-negative bacteria.

Materials and method

.1 Instruments

The powder X-ray diffraction (XRD) pattern

measurements of the samples were recorded on a Holland-

Philips X-

radiation

80◦, operating at 40 kV and a cathode current of 20 mA.

Additionally, some specimens of synthesized silver

nanoparticles AgNPs for TEM studies were prepared by

ultrasonic dispersion of the NPs in ethanol, and the

suspensions were dropped onto a carbon-coated copper

grid. TEM was carried out using a (CM30 3000Kv). FT-

IR spectra were recorded to investigate the functional

group on samples which carried out on a Bruker VERTEX

0 v model using the KBr disk method. The size

distribution of Ag NPs was characterized by the DLS

approach, using computerized inspection system

MALVERN Zen3600) with DTS® (nano) software. UV-

Figure1: UV-vis spectroscopy of Ag NPs.

(

3.2 TEM and DLS investigation

Vis spectroscopy (UV-Vis) analyses were taken using a

Varian Cary 50 UV–vis spectrophotometer. Spectra were

recorded in a range of 350-800 nm.

Morphology and size distribution of synthesized

nanoparticles was investigated by TEM images (Fig. 2).

Fig. 2 shows high-quality crystalline structure and

spherical morphology of AgNPs which is matched with

observed peak for nanospheres in UV-Vis spectroscopy.

The size distribution of synthesized silver

nanoparticles was in the ranges from 4 to 42.13 nm with

average size of 11.4 nm. Fig. 3 depicts the particle size

distribution histogram for nanoparticles obtained from

DLS technique. According to the analyses, the silver

nanoparticles had an average size of 117 nm. These

findings are not in agreement with those achieved from

TEM technique. The reason for this difference is that DLS

measures hydrodynamic diameter of nanoparticles as well

as their surrounding cover. Moreover, in an aqueous

environment, it is likely that some nanoparticles connect

to each other and create an aggregation of nanoparticles.

Subsequently, DLS measures the overall diameter of the

.2 Materials and methods

Silver nitrate (AgNO ) was purchased from Merck

Company for this study and was used without any renewed

purification. Fresh samples of Ducrosia anethifolia parts

consisting of leaves and stems were identified and

collected. To remove pollution, all of glassware were

cleaned with dilute HNO and rinsed with distilled water

as well as dried in an oven under air atmosphere.

Fresh Ducrosia anethifolia extract was preferably used

to reduce aqueous Ag+ solution to Ag NPs. So, the

collected Ducrosia anethifolia was washed with distillated

water to remove any previous contamination, and then

dried in the shade at the room temperature. Afterwards, the

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Journal of Environmental Treatment Techniques

2019, Volume 7, Issue 3, Pages: 461-466

set of nanoparticles. Depending the size of nanoparticles

as well as the magnitude of biological components

connected to various plant extract nanoparticles, the

particle size distribution of nanoparticles accounts for a

highly narrow to a highly broad range in DLS. Similar

findings were reported by G. Singhaj et al who showed

that silver nanoparticles formed from extracts of Ocimum

sanctum differed on average in terms of their particle size

nanoparticles in DLS and TEM techniques, suggesting

that the latter showed the average particle size of 14.3 nm,

while the former the average particle size of 22.3 nm(48).

capable of overlapping with carboxylic acid and amine

groups. Also, the weak peak seen at 2918 cm-1 accounted

for the alkane C–H stretching vibrations of methyl,

methylene, and methoxy groups. Two strong peaks at

1622 and 1616 cm-1 represented the C=O stretching

vibrations due to carbonyl stretching groups in carboxylic

acid, and moreover, the peaks at 1383, 1100 and 616 cm-

1 were assigned to C–H bending, C–OH bond stretching

and aromatic ring, respectively (49). All of the mentioned

bands were considered as principal functional groups in

molecular structures in Ducrosia anethifolia extract which

play a major role in formation of silver nanoparticles

Figure 4: FTIR spectroscopy of prepared silver nanoparticles by

green synthesis

.4 XRD spectroscopy

The peaks caused by X-ray diffraction at 2θ=3.2°,

44.2°, 64.5°, and 77.8°, all shown by Fig. 5. These angles

can be indexed to the 111, 200, 220 and 311 planes of

reflections of silver, respectively. This diffraction pattern

is indicative of natural crystalline structure of silver

nanoparticles and those produced with a face-centered

cubic structure (FCC). These findings are in line with

those obtained from silver nanoparticles produced from

plant extracts. Likewise, there was a series of peaks

witnessed at ranges 10 to 20 which corresponded to the

bioorganic crystalline phase of plant extracts. For

instance, in the previous study conducted on silver

nanoparticles synthesis using Wheat grass extracts, we

showed that peaks caused by X-ray diffraction at

Figure 2: TEM image of synthesized AgNPs using Ducrosia

Anethifolia extract

.3 FTIR analysis

To investigate the functional groups of extracts on the

surface of AgNPs, FTIR analysis was carried out. Several

peaks were observed in FTIR plot (Figure 4) that were

indicate of various functional groups on the surface of

AgNPs. According to Fig. 4, the peaks witnessed at 3500

cm-1 range was associated with the amide group N-H

stretching, whereas the band seen at 3232 cm-1 accounted

for the O-H stretching of the carboxylic acid group.

Furthermore, the peak witnessed at 3500-3200 cm-1 was

corresponded to alcohol/phenol O-H stretching which is

2θ=38.22°, 44.37°, 64.54°, and 77.47°, sugges ting that the

results of the present study are supported (43, 49-53).

Figure 3: DLS of green synthesized Ag NPs by Ducrosia Anethifolia extract

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2019, Volume 7, Issue 3, Pages: 461-466

stress), and iv) AgNPs disrupted the respiration functions

of the cell and permeability through attaching and

penetrating into the cell membrane of bacterial. By

addressing this reasons, the green synthesized AgNPs

demonstrated the best antibacterial activity(54-55-56).

Antibacterial activity of green Ag NPs

The MICs and MBCs obtained from synthetic

nanostructures against various strains of bacteria are

shown in Table 1.

Conclusion

Given the wide application of metal nanoparticles in

Figure 5: XRD pattern of biosynthesized Ag NPs using Ducrosia

Anethifolia extract

industry as well as the desperate need for a low-risk and

safe treatment without any adverse effects on both humans

and environment, it is believed that green synthesis

methods are less costly, highly safer and eco-friendly. In

this study, Ducrosia anethifolia aqueous extracts were

used as the reducing agent for reduction of Ag+ cation to

the AgNO3 to Ag0. Therefore, AgNO3 reaction at the

presence of Ducrosia anethifolia aqueous extracts resulted

in the synthesis silver nanoparticles (AgNPs). Various

methods of identifying the characterization of

nanoparticles such as XRD, TEM, FTIR, DLS, and UV-

Vis have supported successful synthesis of AgNPs.

Likewise, the synthesized nanoparticles were shown to

have substantial anti-bacterial properties, especially

against Gram-positive and Gram-negative bacteria.

Nonoparticles were effective against all strains of

tested Gram-positive and Gram-negative bacteria at

ranges 32-128 μg/ml. Both Enterococcus faecalis and

Pseudomonas aeruginosa showed similar values for MICs

and MBCs.Nanoparticles had no bactericidal activity in

concentration up to 128μg/ml.Mechanism of AgNPs

bacterial inhibition was performed in four ways as

follows: i) Ag+ could form a stable complex with thiols

and phosphate in amino acids and nucleic acids, ii) AgNPs

led to the disruption ATP formation and DNA replication

by producing Ag+, iii) AgNPs was capable of generating

reactive oxygen species (ROS) or oxygen radical species

(

they could damage the DNA by means of oxidative

Table 1: The MICs and MBCs obtained from green Ag NPs

photothermal nanodiagnostics and nanotherapy. Nanomedicine:

Nanotechnology, Biology and Medicine. 2005;1(4):326-45.

Dobrovolskaia MA, McNeil SE. Immunological properties of

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