Green Synthesis and Characterization of Biocompatible Silver Nanoparticles using Stachys lavandulifolia Vahl. Extract and Their Antimicrobial Performance Study

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 1, Pages: 284-290

J. Environ. Treat. Tech.

ISSN: 2309-1185

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

Green Synthesis and Characterization of

Biocompatible Silver Nanoparticles using Stachys

lavandulifolia Vahl. Extract and Their

Antimicrobial Performance Study

†

2 †

Abbas Zakeri Bazmandeh , Arash Rezaei , Hamid Reza Ghaderi Jafarbigloo , Amir

Mohammad Akbari Javar , Ali Hassanzadeh , Armin Amirian , Mohamad Hadi Niakan ,

Hossein Hosseini Nave⁸and Mohsen Mehrabi *

,9*

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

Shiraz, Iran

Department of laboratory Sciences,School of Medicine,Kerman University of Medical Sciences,Kerman,Iran

Department of Basic Science, Payame Noor University, Iran

Department of Mathematics, faculty of Mathematics, Farhangian University, Kerman, Iran

Department of Tissue Engineering and Applied cell Sciences, Tehran University of Medical Sciences, Tehran, Iran

Thoracic and Vascular Surgery Research Centre, Shiraz University of Medical Sciences, Shiraz, Iran

Trauma Research Center, Shahid Rajaee (Emtiaz) Trauma Hospital, Shiraz University of Medical Sciences, Shiraz, Iran

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

Department of Microbiology and Virology, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran

Department of Medical Nanotechnology, School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran

Received: 22/09/2019 Accepted: 07/12/2019 Published: 20/02/2020

Abstract

Green synthesis of nanoparticles has received extensive attention in the field of nanomedicine as a simple, environment-friendly

and cost-effective alternative in comparison with other physical and chemical methods. Silver nanoparticles (AgNPs) are playing

an important role in the field of nanomedicine and nanotechnology due to their unique and controllable characteristics. In this

research, a biosynthesis approach was used for synthesis of silver nanoparticles (AgNPs). Biosynthesis was carried out by Stachys

lavandulifolia Vahl. extract as green reducing agent for conversion of Ag to Ag . The first visible sign of the synthesis of AgNPs

was the change in color of solution from yellowish to reddish brown. The preparation of AgNPs was performed using several

techniques such as, UV-vis spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), transmission

electron microscopy (TEM), and dynamic light scattering (DLS). Finally, synthesized AgNPs were evaluated for their antimicrobial

activity against four standard bacterial strains using microbroth dilution method. Results of TEM image showed spherical

nanoparticles with high crystalline and monodispersity which is very important in medicinal applications. Additionally, DLS

analysis demonstrated the ultra-fine size of AgNPs with average size of 164.8 nm. FTIR indicated the role of different functional

groups (carboxyl, amine and hydroxyl) in the formation of Silver nanoparticles. A negative zeta potential value of -17.3 mV

determined the stability of the silver nanoparticles. AgNPs showed good antimicrobial activity against both gram-positive and

gram-negative bacteria, with MICs of 4 to 16 μg mL. In conclusion, the results imply that the synthesized nanoparticles using green

nanotechnology could be an ideal strategy to combat multi-drug-resistant pathogenic bacteria.

Keywords: Stachys lavandulifolia Vahl; Silver nanoparticle; Green synthesis; Antimicrobial performance

Introduction

¹Resistance to commonly available antimicrobial agents

2). Serious infections caused by resistant bacteria such as

methicillin-resistant Staphylococcus aureus and

Enterobacteriaceae (3, 4). Nanotechnology has opened a

by pathogenic bacteria has been rising at an alarming rate

and has become a serious global problem for healthcare (1,

Corresponding authors: (a) Department of Microbiology and Virology, School of Medicine, Kerman University of Medical Sciences, Kerman,

Iran. E-mail: Hosseini.nave@gmail.com. (b) Mohsen Mehrabi, Department of Medical Nanotechnology, School of Medicine, Shahroud University

of Medical Sciences, Shahroud. Iran.

†

These authors contributed equally to this article.

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 1, Pages: 284-290

wide field for the synthesis of new and noble materials for a

variety of applications, especially medical and

pharmaceutical use the term nanoparticle typically refers to

a particle with a size ranging from 1 to 100 nm (5). The

antibacterial activity of nanoparticles is due to their high

surface area to volume ratio, thereby enabling significant

interactions with microbial cell membranes. Silver

nanoparticles (AgNPs) has attracted more attention than

other metal nanoparticles due to exceptional properties such

as surface plasmonic resonance (SPR) and stability, which

can be used as catalyst, sensor, anti-angiogenic, anticancer,

and especially antibacterial agent (5-8). As mentioned, the

exceptional properties of nanoparticles depend on their size

and morphology which are determined by the synthesis

method. The methods for the synthesis of nanoparticles are

mainly grouped into two categories: the physical and

chemical approaches. Although these methods are popular,

they cause considerable environmental and economic issues.

Thus, there is an apparent need for an alternative approach

for the synthesis of nanoparticles. The “biological method”

was developed as a simple approach as an alternative to

physical and chemical methods. Green synthesis method

provides several advantages over chemical methods as it is

eco-friendly, simple, rapid, safe, clean and cost effective.

These features are important in medical applications. A vast

number of biological resources including plants, algae,

fungi, yeast, and bacteria has been studied so far for the

synthesis of nanoparticles (9). The plant-mediated synthesis

of AgNPs can be advantageous compared with other

biological processes as it is relatively fast, does not require

the process of maintaining specific media and aseptic

environments (10).

The formation of AgNPs was monitored using UV–vis

spectrometer in the range of 250–600 nm. Fourier transform

infrared (FTIR) analysis was recorded in the range between

4000 to 400 cm−1 to investigate the functional groups

involved in the synthesis of silver nanoparticles. The

structural characterization and crystalline nature of AgNPs

were analyzed by X-ray diffraction (XRD). The XRD

pattern was recorded using a Holland Philips X-ray powder

diffractometer using Cu K radiation (λ= 0.1542 nm) in the

2θ range of 5°–80°, with operating voltage of 40 kV at a 20

mA current strength. The morphology and average size of

AgNPs were analyzed by transmission electron microscopy

(TEM). The sample for TEM was prepared by ultrasonic

dispersion of the AgNPs in ethanol. Finally, the size

distribution

nanoparticles

was

characterized

through dynamic light scattering (DLS).

2.5 Antibacterial performance tests

The minimal inhibitory concentration (MIC) of the

AgNPs was determined by micro-broth dilution method

using Mueller Hinton broth. Briefly, concentrations with a

starting range of 1024 μg/ml to 2 μg/ml of the AgNPs was

prepared in the wells of a 96-well plate, and bacterial

inoculum with an adjusted bacterial concentration of

1.5×10 colony-forming units/ml (CFU/ml) was added to

this 2-fold dilution series. The plate was incubated at 37°C

for 24 hours. Antimicrobial activity was determined against

four reference strains including two Gram-negative bacteria

[Escherichia coli (ATCC 25922) and Pseudomonas

aeruginosa (ATCC 27852)] and two Gram-positive bacteria

[Staphylococcus aureus (ATCC 25923) and Enterococcus

faecalis (ATCC 29212)]. The ciprofloxacin was used as the

positive control for anti-bacterial screening. The lowest

concentration of AgNPs that was able to completely inhibit

the bacterial growth was considered as the MIC value. The

MIC experiments were performed in triplicate against each

bacterial strain to confirm the value of MIC for each tested

bacterium.

In this work, Stachys lavandulifolia Vahl.

(Family

Lamiaceae) was applied for synthesis of AgNPs as reducer,

surfactant and capping agent. This plant was distributed in

different regions of Asia and Europe. Stachys lavandulifolia

Vahl. has been widely used in traditional medicine for

treatment of various diseases (11). In this study, green

synthesis of AgNPs was done using leaf extracts of Stachys

lavandulifolia Vahl. The prepared nanoparticles were

characterized using several methods such as XRD, UV-vis

along with FTIR spectroscopy, TEM and DLS analysis to

confirm the formation of nanoparticles. After approval of the

structure and purity of AgNPs, antibacterial activity of this

nanoparticles was studied on Gram-positive and Gram-

negative bacteria.

Result and discussion

In this research, the AgNPs were synthesized by green

synthesis method, using Stachys lavandulifolia Vahl. extract

as both reducer and stabilizer agent. The formation of

AgNPs was studied using both visual observation of the

color and UV–visible spectral analysis. The AgNO solution

is colorless (Figure 1a) and there is no absorption peak in the

UV-Vis spectrum (Figure 1d). On the other hand, the color

of Stachys lavandulifolia Vahl. extract was yellow (Figure

1b) and had a weak peak in UV-vis spectrum, that can be

attributed to π→π* transitions of aromatic rings. The color

Materials and methods

.1 Material

In this study silver nitrate (AgNO ) was

.3 Synthesis of AgNPs with Stachys lavandulifolia Vahl.

of solution was changed to brown when the AgNO was

Extract

For synthesis of AgNPs, 20 mL of different

concentrations (5%-20%) of fresh Stachys lavandulifolia

Vahl. extract was added into 5ml of AgNO solution

0.1mM) at room temperature for the reduction of Ag+ ions.

The color change of the solution from yellow to reddish

brown indicated that the silver nanoparticles were

synthesized.

added to yellow extract (Figure 1c). This change in color

visually indicates the formation of silver Nanoparticles.

Additionally, UV-vis spectroscopy of the colloidal AgNPs

in Figure 1d demonstrates a strong peak in 440 nm due to

surface plasmon resonance (SPR) of silver nanoparticles

formed. Both change of color and SPR band in UV-vis

(

spectra show conversion of Ag in AgNO

solution to Ag

and completion of reduction reaction.

With the objective of finding various functional groups

involved in the synthesis of silver nanoparticles FTIR

analysis was performed. The FTIR spectrum of

.4 Characterization

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 1, Pages: 284-290

biosynthesised AgNPs showed absorption bands at 3432,

band at 3,374 cm indicated stretching of the N–H bond of

amino groups or presence of attached hydroxyl (–OH) group

because of the existence of surface adsorbed alcohols

XRD shows four important peaks at 2θ˚= 38.65, 44.60, 64.85

and 77.47 . This result represents silver crystal formation

which, respectively, corresponds to 111, 200, 220 and 311.

These planes and phase of the Ag nanoparticle are in

agreement with the reference file with JCPD= 01-1167.

Additionally, there are two unassigned peaks at 2θ˚= 28 and

46 denoted by (•) in Figure. 3 which are thought to be related

to crystalline and amorphous organic phases. Hence, the

result of XRD pattern approved the successful synthesis of

pure silver nanoparticles usage plant extract as the capping

agent and surfactant in mild condition. The XRD peaks

revealed that AgNPs formed using Stachys lavandulifolia

Vahl. extract were crystalline and spherical in shape.The

average nanosize was calculated by applying Debye–

Scherrer formula D = K l/β cosθ which was found to be 36.69

nm (14).

−1

073, 1626, 1636, and 634 cm (Figure 2). The absorption

-1

(carbohydrates etc.) and phenols (polyphenols). According

to the result of FTIR analysis, C-H stretching of the methyl

group can be observed at 2073 cm region. The strong peak

at 1636 cm is related to C=O stretching vibrations of

aldehydes, ketones and carboxylic acids (5). Several

investigations have reported that functional groups such as

alcohol, phenol, and amines act as the capping agent for

AgNPs which increases the stability of the nanoparticles (12,

-1

3). Finally, the band at 634 cm is related to several

aromatic amines. The XRD profile of AgNPs synthesized

using the leaf extract of Stachys lavandulifolia Vahl. is

depicted in Figure. 3. The result demonstrated the face-

centered cubic (FCC) for silver crystallites. The diagram of

Figure 1: Color of (a) AgNO

solution; (b) Stachys lavandulifolia Vahl. extract; (c) AgNPs suspension and (d) UV-vis spectroscopy of AgNO ,

Stachys lavandulifolia Vahl. extract, and Ag NPs.

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 1, Pages: 284-290

Figure 2: FTIR spectroscopy of green silver nanoparticles

Figure 3: XRD pattern of biosynthesized AgNPs using Stachys lavandulifolia Vahl. extract

The morphology and size of green synthesized AgNPs

and crystallinity, without any aggregation. Based on the

theory for crystal nucleation and growth, various types of

organic compounds have basically a role in formation of

crystalline nanoparticle as the capping agent. The

polyphenol compounds can control the formation and

growth of the primary core of AgNps in overall reaction

which was clearly illustrated in scheme 1. Besides, the

were studied by TEM. The size and shape of the synthesized

silver nanoparticles significantly affect their antimicrobial

activity. TEM images of the synthesised AgNPs are shown

in Figure 4. TEM images confirm the formation of spherical

Ag nanoparticles (Figure 4a). According to Figure 4b,

spherical nanoparticles have high monodispersity

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 1, Pages: 284-290

particle size of nanoparticles was estimated about 150 nm,

and it was fine and suitable for medical applications.

Size distribution and average size of nanoparticles was

measured using histogram of DLS analysis in Figure 5a. The

measured average size is 164.8 nm which shows the

monodispersity of particles in green strategy for synthesis of

nanomaterials. Also, this size distribution is in agreement

with the SPR peak in UV-vis spectroscopy. This definite size

is matched with TEM image and confirms antimicrobial

activity of AgNPs.

Moreover, zeta potential was performed to study the

dispersion surface charge of AgNPs, which was covered

with bio-molecular of the plant extract and that caused the

nanoparticles stable in colloidal solution. According to the

result in Figure 5b zeta potential diagram shows a negative

charge with a value of -17.3 mV and this value leads to

stability of nanoparticle and prevents the aggregation and

agglomeration of silver nanoparticles and is also in

agreement and comparable to the former studies (15, 16).

In order to evaluate the antibacterial activity of the silver

nanoparticles synthesized using Stachys lavandulifolia Vahl.

extract, we employed microbroth dilution method. For the

bacterial strains E. faecalis 29212, S. aureus 25923, P.

aeruginosa 27853 and E. coli 25922, the MIC values of

silver nanoparticles were 8, 8, 4 and 4 μg mL, respectively.

The antibacterial activity of AgNPs has been well

documented in previous studies (14). However, the

comparison of the results of antimicrobial studies is difficult

as there is no standard method for the determination of

antibacterial activity of AgNPs and different methods have

been used by the researchers. In this study, AgNPs exhibited

excellent antibacterial activity against both gram-positive

and gram-negative bacteria, but the gram-negative bacteria

were more sensitive to the nanoparticles than the gram-

positive bacteria. These results are consistent with those of

other studies, which also showed that Gram-positive strains

exhibited greater resistance against nanoparticles in

comparison with Gram-negative ones. This may be due to

the difference in their cell wall structure. The Gram-positive

bacterial cell wall consists of a thick peptidoglycan layer,

while Gram-negative bacteria have thin peptidoglycan layer

which allows easier penetration of AgNPs into the cell wall

of Gram-negative bacteria (14, 17).

As the results showed, the synthesized AgNPs indicated

a strong inhibition effect on several types of gram-positive

and gram-negative bacteria with penetration in the bacteria

cell due to the small size and high ratio of the surface per

volume which leads to wide application of silver

nanoparticles in the production of various antibacterial

products.

Figure 4: TEM image of synthesized AgNPs a) high-magnification; and b) low magnification

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 1, Pages: 284-290

Figure 5: (a) DLS and (b) zeta potential of synthesized AgNPs using Stachys lavandulifolia Vahl. extract

Modifying Enzymes and Molecular Analysis of the Coagulase

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Nanoparticles Using Plantago major Leaf Extract and Their

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Conclusion

Silver nanoparticles were successfully synthesized using

green approach by Stachys lavandulifolia Vahl. extract as

non-toxic and green reducing agent. After this, nanoparticles

were characterized using various methods, for example

FTIR, XRD, Uv-vis spectroscopy, TEM and DLS; this

analysis approved the successful synthesis of nanoparticles

with high purity. Therefore, XRD pattern demonstrated the

crystalline cubic phase and also spherical shape and

morphology was observed in TEM image with the estimated

size about 32 nm in diameter. Moreover, DLS showed a very

fine size of AgNPs 164.8 nm in diameter which is in

agreement with SPR properties of AgNPs in Uv-vis

spectroscopy. All analyses together showed the fundamental

role of the plant extract as the capping agent and surfactant

to control the size of AgNPs and also as the stabilizer of Ag

in colloidal solution. The biosynthesized nanoparticles also

exhibited a high antibacterial activity against the tested

strains. Therefore, this method is very safe, simple, eco-

friendly and cost-effect for synthesis of biocompatible

AgNPs in a large scale for medical application.

Acknowledgments

This research was financially supported by “Kerman

University of Medical Sciences”. The authors thank

Department of Microbiology and Virology of the School of

Medicine of Kerman University of Medical Sciences for

financial support.

10. Mo Y-y, Tang Y-k, Wang S-y, Lin J-m, Zhang H-b, Luo D-y.

Green synthesis of silver nanoparticles using eucalyptus leaf

extract. Materials Letters. 2015;144:165-7.

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Alimardani V. One-step green synthesis and characterization of

iron oxide nanoparticles using aqueous leaf extract of Teucrium

polium and their catalytic application in dye degradation.

Advances in Natural Sciences: Nanoscience and

Nanotechnology. 2019;10(1):015007.

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