Author’s Accepted Manuscript

Green synthesis of silver nanoparticles using

Veerasamy Ravichandran, Sethu Vasanthi,

PII: S0167-577X(16)30937-5
DOI: http://dx.doi.org/10.1016/j.matlet.2016.05.172
Reference: MLBLUE20983
To appear in: Materials Letters
Received date: 3 March 2016
Revised date: 13 April 2016
Accepted date: 28 May 2016
Cite this article as: Veerasamy Ravichandran, Sethu Vasanthi, Sivadasan Shalini,
Syed Adnan Ali Shah and Rajak Haris, Green synthesis of silver nanoparticles
using Atrocarpus altilis leaf extract and the study of their antimicrobial and
antioxidant activity, Materials Letters,
http://dx.doi.org/10.1016/j.matlet.2016.05.172
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Green synthesis of silver nanoparticles using Atrocarpus altilis leaf extract and the study of

their antimicrobial and antioxidant activity

Veerasamy Ravichandrana*, SethuVasanthib, Sivadasan Shalinia, Syed Adnan Ali Shahc,d, Rajak

Harise
a
Faculty of Pharmacy, AIMST University, Semeling, Bedong, Kedah, Malaysia.
b
Faculty of Engineering, The University of Nottingham, Semenyih, Selangor, Malaysia.
c
Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam Campus, Bandar Puncak Alam, Selangor Darul
Ehsan, Malaysia.
d
Atta-ur-Rahman Institute for Natural Products Discovery (AuRIns), Universiti Teknologi MARA, Puncak Alam
Campus, Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia
e
SLT Institute of Pharmaceutical Sciences, Guru Ghasidas University, Bilaspur, India.

*For Correspondence. Faculty of Pharmacy, AIMST University, Semeling – 08100, Kedah, Malaysia. Tel.: 006 04
4298000 Ext. 1029; fax: 006 04 4298007, E. mail: phravi75@rediffmail.com
ABSTRACT

Biological entity is gaining significant prominance in green synthesis of silver nanoparticles as a result of their

potential applications in nanomedicine and material engineering. Herein, we have reported the green synthesis of

Artocarpus altilis silver nanoparticles (BAgNPs) using aqueous leaf extract of Artocarpus altilis. Synthesized colloidal

BAgNPs were confirmed spectrophotometrically at 432 nm and the various reaction conditions were optimized. The

SEM, TEM and DLS analysis confirmed that the average particle size of BAgNPs was34 nm, 38 nm, and 162.3 d.nm,

respectively. Nature and presence of silver were confirmed by XRD and EDX. Further, FT-IR spectra of the

synthesized BAgNPs authorized the presence of phyto constituents as capping agent. The BAgNPs showed moderate

antimicrobial and antioxidant activities. The present study revealed the safer use of eco-friendly green synthesized

BAgNPs in near future in the field of biomedicine, water treatment or purification, and nanobiotechnology.

GRAPHICAL ABSTRACT

The present study revealed the possible safer use of eco-friendly green synthesized BAgNPs in the

field of biomedicine, water treatment/purification and nanobiotechnology.

Keywords: Artocarpus altilis, Green synthesis, Colloidal processing, Nanoparticles

1. Introduction

Nanotechnology with increasing use of nanoparticles or nanoemulsions has progressed as a promising tool for the

treatment of various diseases especially in the field of medicine [1]. In recent years, numerous approaches have been

used to prepare the nanoscale silver particles such as electrochemical, sonochemical and microwave-assisted process,
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but many of these strategies are difficult for purification since the chemicals are hazardous and high energy. To toggle

over these difficulties, biological principles are used in the process of synthesis, meaning the eco-friendly approaches

are established [2]. The advantage of using plant materials in nanoparticles synthesis is it does not need any elaborate

processes such as intracellular synthesis, compound purification steps and the maintenance of microbial cell cultures

[3].

Artocarpus altilis (Parkinson) Fosberg (Breadfruit) is a multipurpose forestry tree crop which is primarily

used for its nutritious, rich source of calcium and phosphorus, carbohydrates, minerals and vitamins. Essential amino

acids, sucrose, fatty acids, flavonoids, phenols, steroids, phytosterols and glycosides are the other constituents of A.

altilis [4]. The different parts of breadfruit is used as food, cosmetics medicine, clothing material, treating diarrhoea,

high blood pressure, asthma and enlarged spleen [5]. Herein, we reported the biogenic synthesis of AgNPs from A.

altilis leaf aqueous extract and their biological properties.

2. Materials and methods

2.1. Materials
Healthy leaves of A. altilis were collected from in and around the campus of AIMST University, Kedah,

Malaysia. Silver nitrate (AgNO3), Muller Hinton Agar, Nutrient broth was procured from Himedia Laboratories Pvt.

Ltd., Mumbai, India. The bacterial cultures of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and a

fungus Aspergillus vesicolor were obtained from the Faculty of Applied Sciences, AIMST University, Malaysia.

2.2. Preparation of plant leaf extracts

Twenty five gram of the collected plant leaves surface sterilized with distilled water was weighed and cut into

small pieces. Then the leaves were boiled with 250 mL deionized water at 60˚C for 10 min and cooled. The extract was

filtered with Whatman filter paper No. 1 and stored in a refrigerator at 4C for further use.

2.3. Biosynthesis of silver nanoparticles

Silver nanoparticles were synthesized by following our earlier reported method with slight modification [6].

Biological reduction of AgNO3 was carried out initially as follows: The reaction mixture was prepared by adding 1.5

mL of the plant leaf extracts to 1 mL of 0.01 M AgNO3 solution and the volume was made up to 10 mL with deionized

water in a 25 mL volumetric flask and kept in ambient temperature (25 ± 0.5oC) for 24 h. The color of the solution

changed to brown which confirmed the reduction of silver nitrate to silver ions. The silver nanoparticles formation was

also confirmed by spectrophotometric determination. The synthesized BAgNPs using optimized procedure were

separated by centrifugation at 10,000 rpm for 15 min.

2.4. Characterization of silver nanoparticles

The bio-reduction of Ag+ ions in aqueous extract was monitored by UV–visible spectra (Shimadzu dual beam

spectrophotometer (AV-1800)). The morphology and size of the BAgNPs was measured using FESEM, and EDX
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analysis (FESEM-EDX, Oxford-Instrument INCA400) was performed to determine the elemental composition of

BAgNPs. TEM analysis was recorded in JEOL JEM 2100 high-resolution TEM. The FT-IR spectrum of nanoparticle

powder was recorded on a FTIR-JASCO 4100 Spectrometer at 4000 cm-1 to 400 cm-1. The hydrodynamic particle size

and zeta potential of nanoparticle was determined using Zetasizer Ver. 7.03 (Malvern Instruments Ltd., Worcestershire,

UK). The XRD pattern of nanoparticles was recorded on a Bruker AXS D-8 powder X-ray diffractometer (Shimadzu,

Japan) operated at a voltage of 40 kV and current of 15 mA using CuKα radiation (λ = 1.5406 A°).

2.5. Screening of antimicrobial activity

Muller Hinton Agar plates were used and swabbed with pathogenic organisms from fresh cultures (105-106

CFU/mL) using a sterile cotton swab. With the help of sterile gel puncture, approximately 6 mm diameter of wells was

made in the agar plates. Using a micropipette, the drilled wells were poured with 20 μL of the BAgNps in

concentrations of 50 μg/mL and 100 μg/mL. Separate wells of streptomycin (20 μL, 25 μg/mL) and A. altilis leaf

extract (20 μL) were used in the test as a control for the purpose of comparison. The zone of inhibition was measured

after the plates were incubated at 37ºC for 24 h for bacteria and 25C for 72 h for fungi.

2.6. In vitro antioxidant assay

The antioxidant activity of BAgNPs was determined by DPPH assay method following the method introduced

by Brand-Williams et al. 1995 with minor modification [7]. The capability to scavenge the DPPH radical was

calculated using the following equation:

DPPH Scavenged (%) = (Ac - As / Ac) x 100

where Ac and As are the absorbance of the control and test sample, respectively, after 30 min measuring at 517 nm.” A

linear regression analysis was performed to determine the IC50 value for the sample [7].

3. Results and discussion

3.1. Synthesis of silver nanoparticles: Process optimization and UV characterization

Aqueous leaf extract of A. altilis were used in the reduction of AgNO3 into Ag0, and the reduction was initially

confirmed by the color chang from colorless to yellowish brown. The UV-visible spectral analysis of the extract with

AgNO3 solution showed maximum absorbance at 432 nm (Fig. S1) is the characteristic surface plasmon resonance

(SPR) peak of BAgNPs. The formation of SPR peak was influenced by various factors such as size, shape and particles

formed [8].

The extract concentration 1.5 mL of 10% A. altilis leaf extract in deionized water (Fig. S2. A), AgNO3

concentration 1.0 mM (Fig. S2.B), reaction time of 60 min (Fig. S2.C), pH 7.0 (Fig. S2.D) and temperature at 70°C

(Fig. S2.E) were the optimized condition for the synthesis of BAgNPs. The pH optimization studies revealed that the

peak intensity was found to be higher at pH 8.0 however pH 7.0 is considered as optimum (the reason is given below in
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stability studies). After optimization of pH and temperature (pH 7.0 and 70ºC), the time period for nanoparticle

formation decreased significantly from 24 h to 1 h.

Synthesized BAgNPs showed good stability at room temperature (25 ± 2C) without aggregation with an

intense SPR peak at 432 nm after 60 days (Fig. S3.A). Figure S3.B shows the stability studies of the silver nanoparticle

at pH 8.0. Another peak was observed nearby at 600 nm, which may be due to the instigation of silver nanoparticles

agglomeration. This may be due to disturbance of the silver nanoparticles capping by high alkalinity.

3.2. Characterization of silver nanoparticles

The SEM image of BAgNPs showed that the particles were spherical in shape and polydispersed with varying

sizes from 25 to 43 nm in diameter with an average size of 34 nm (Fig. 1A). The BAgNPs size measured from the TEM

image was in the range of 20 to 50 nm with the average size of 38 nm (Fig. 1B). Sizes and shapes of metal

nanoparticles are affected by various factors including pH, time of incubation, precursor concentration, reductant

concentration, temperature as well as method of preparation.

In the XRD pattern (Fig. 1C), five prominent diffraction peaks were observed at 2θ = 38.24º, 44.39º, 64.57º,

77.55º and 81.57º, which corresponds to (111), (200), (220), (311) and (222) Bragg’s reflections of the face-centered

cubic (fcc) structure of metallic silver, respectively. Peaks observed in the pattern and lattice parameter ‘a = 4.08 Å’

were in good agreement with reference of fcc structure from Joint Committee of Powder Diffraction Standard (JCPDS)

Card No-087-0720. The average crystallite size of the BAgNPs calculated from XRD spectral data using Scherrer’s

equation [9] was 19.49 nm.

DLS analysis showed that the BAgNPs size was approximately 162.3 d.nm with intercept 0.912, and low

polydispersity index (PDI) of 0.290 (Fig. 2A). The measured size of particles by DLS was moderately bigger as

compared to the SEM and TEM measurements, since the DLS method measures the hydrodynamic radius of particles.

Zeta potential of the nanoparticles was found to be -12.7 mV (Fig. 2B) which confirmed the reasonable stability of the

nanoparticles. The negative potential value shown by biosynthesized AgNPs could be due to the presence of bio-

organic components in the extract as capping agent [10]. Figure 2C shows the elemental profile of synthesized

BAgNPS by EDX studies and confirms the formation of silver nanoparticles. The elemental analysis of the silver

nanoparticles shown in the above said figure revealed the highest proportion of silver followed by C, Cl, and O. These

peaks are from the bio-molecules bound to the surface of the silver nanoparticles. The EDX analysis showed a strong

signal for silver particles in the range of 2.8 - 3.6 keV.

The possible groups responsible for the interaction between the capping agents and silver nanoparticles were

confirmed by FTIR. The FTIR spectrum of BAgNPs (Fig. S4) showed intensive peaks at 3173 cm-1 (aromatic C-H

stretching), 1605 cm-1 (amide I bond of proteins due to carbonyl stretch in proteins), 1397 cm-1 (C-C bond of aromatic
 6

ring or amide group-II), 1100 and 1218 cm-1 (stretching vibrations of C-N aromatic and aliphatic amines) and 657 cm-1

(C-H of alkynes). The obtained results indicated that the C-C and C-N vibration stretches in BAgNPs are from

polyphenols of leaf extract of A. altilis which might be involved in the formation of BAgNPs by acting as capping and

stabilizing agents.

3.3. Antimicrobial and antioxidant activity of BAgNPs

Zone of inhibition of silver nanoparticles revealed that E. coli and P. aeruginosa were more susceptible to the

BAgNPs while the S. aureus showed reasonable susceptibility towards the BAgNPs and the fungus A. vesicolor

showed a minimal level of susceptibility towards BAgNPs (Fig. 3A).

The DPPH assay indicated the maximum antioxidant activity of 79.79% at 100 μg/mL for BAgNPs. The

antioxidant assay results are shown in Fig. 3B. The DPPH assay demonstrated potent radical scavenger properties of

BAgNPs having an IC50 value of 51.17 μg/mL (Fig. 3C) were compared with the IC50 value of ascorbic acid 42.70

μg/mL. This antioxidant ability of BAgNPs could be attributed by the functional groups adhered to them.

4. Conclusion

A. altilis leaf extract was used successfully to synthesize silver nanoparticles. However, by using the present

method, aggregation of AgNPs could not be completely avoided. These BAgNPs seemed to exhibit bacterial inhibitory

effect comparable to that of streptomycin and antifungal inhibitory effect similar to that of amoxicillin against human

pathogens and also free radical scavenging activity comparable to that of ascorbic acid. Thus, the plant-mediated

nanoparticles can be used against human pathogens as good therapeutic agent and also for the treatment of diseases

caused by free radicals in the near future.

Acknowledgements

Authors are pleased to acknowledge AIMST University, University of Nottingham and Universiti Teknologi MARA,

Malaysia for providing the necessary facilities to carry out this work.

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Figure caption

Fig. 1. (A) SEM image of with 400 nm resolution, (B) TEM image of BAgNPs with 100 nm resolution (C) XRD
pattern of BAgNPs

Fig. 2. (A) Size distribution of BAgNPs obtained from dynamic light scattering, (B) Zeta potential of BAgNPs, (C)
EDX spectra of BAgNPs,

Fig. 3. (A) Antimicrobial activity (Zone of inhibition) of BAgNPs against human pathogens ▪ Extract, ▪ 50 μg/mL of
BAgNPs, ▪ 100 μg/mL of BAgNPs, ▪ Standard drug, the values are with ± SD (n = 3) (B) DPPH radical scavenging
activity of ascorbic acid and BAgNPs ▪ BAgNPs, ▪ Ascorbic acid, the values are with ± SD (n = 3) (C) IC50 value of
BAgNPs radical scavenging activity
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Fig. 1
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Fig. 2

Fig. 3

Highlghts

 Green synthesis of silver nanoparticles using aqueous Artocarpus altilis leaf extract
 Synthesized colloidal silver were confirmed spectrophotometrically at 432 nm
 X-ray diffraction confirmed the nature of the BAgNPs is crystalline
 Energy dispersive spectrometer authorized the presence of silver
 BAgNPs showed moderate antimicrobial actions than plant leaf extract
 DPPH assay results showed that the BAgNPs had good antioxidant activity