Biomedical potential of silver nanoparticles biosynthesized using gallnut extract

Abstract

The green synthesis of silver nanoparticles (AgNPs) is a good approach to avoiding the drawbacks associated with by-products formed in chemical synthesis. The present investigation was intended to synthesize AgNPs using gallnut extract as reducing agent and evaluate their potential biomedical applications. The ultraviolet-visible spectroscopy provided a preliminary indication of AgNP synthesis. Changing the pH of the reaction mixture from pH 3 to 10 revealed a significant impact of pH on the synthesis of AgNPs with the wavelength shift from red to blue. Transmission electron microscope characterizations showed that the synthesized AgNPs at pH 3-10 were spherical with average sizes of 51, 27, 18, 30, 10, 8, 5 and 4nm. The synthesized AgNPs were further characterized by different techniques such as Fourier transform infrared spectroscopy, scanning electron microscopy, energy-dispersive X-ray spectrometry, X-ray photoelectron spectroscopy and powder X-ray diffraction. The AgNPs biosynthesized using gallnut extract showed higher antioxidant activity (81%) than AgNPs chemically synthesized using sodium borohydride (56%), indicating that AgNP-capping molecules such as tannic acid play an important role in antioxidant function. The biosynthesized AgNPs showed potent anticancer activity on four cervical cancer cell lines, namely, ME180, SiHa, HeLa and CaSki.

Full text

Biomedical potential of silver nanoparticles
biosynthesized using gallnut extract
1Ezhaveni Sathiyamoorthi MTech
PhD scholar, Department of Chemical Engineering, Chungbuk
National University, Cheongju, Republic of Korea
2Bilal Iskandarani MSc
PhD scholar, Department of Chemical Engineering, Chungbuk
National University, Cheongju, Republic of Korea
3Bipinchandra K. Salunke PhD
Postdoctoral Fellow, Department of Chemical Engineering, Chungbuk
National University, Cheongju, Republic of Korea
4Beom Soo Kim PhD
Professor, Department of Chemical Engineering, Chungbuk National
University, Cheongju, Republic of Korea (corresponding author:
bskim@chungbuk.ac.kr)
1 2 3 4
The green synthesis of silver nanoparticles (AgNPs) is a goodapproachtoavoidingthedrawbacksassociatedwithby-
products formed in chemical synthesis. The present investigation was intended to synthesize AgNPs using gallnut extract
as reducing agent and evaluate their potential biomedical applications. The ultraviolet–visible spectroscopy provided a
preliminary indication of AgNP synthesis. Changing the pH of the reaction mixture from pH 3 to 10 revealed a significant
impact of pH on the synthesis of AgNPs with the wavelength shift from red to blue. Transmission electron microscope
characterizations showed that the synthesized AgNPs at pH 3–10 were spherical with average sizes of 51, 27, 18,
30, 10, 8, 5 and 4 nm. The synthesized AgNPs were further characterized by different techniques such as Fourier
transform infrared spectroscopy, scanning electron microscopy, energy-dispersive X-ray spectrometry, X-ray photoelectron
spectroscopy and powder X-ray diffraction. The AgNPs biosynthesized using gallnut extract showed higher antioxidant
activity (81%) than AgNPs chemically synthesized using sodium borohydride (56%), indicating that AgNP-capping
molecules such as tannic acid play an important role in antioxidant function. The biosynthesized AgNPs showed potent
anticancer activity on four cervical cancer cell lines, namely, ME180, SiHa, HeLa and CaSki.
1. Introduction
Developments of eco-friendly nanoparticle synthesis approaches
devoid of lethal chemicals in synthesis protocols are urgently needed.
Plant extracts,
1
bacteria
2
and fungi
3
are recommended as potential
environmentally friendly substitutes for the physical and chemical
syntheses of metal nanoparticles. In particular, metal nanoparticles
such as silver, gold and platinum can be manufactured through
reduction by means of biological methods. This offers numerous
benefits such as cost-effective production and suitability for
biomedical applications. The approach of using medicinal plants for
silver nanoparticle (AgNP) synthesis is eco-friendly, and the
synthesized AgNPs display virtuous antimicrobial efficacy.
2,4
AgNPs
are valuable due to their utility in applications such as antibacterial
products, anticancer agents, air purifiers, imaging, sensors and
textiles.
5,6
AgNPs are also treasured for diagnostics of disease and
drug delivery treatments.
7,8
Different parameters such as temperature,
pH and reaction time can influence the characteristic of AgNPs in
plant extract-mediated synthesis. Biomolecules present in plant
extracts have a significant share for nanoparticle synthesis.
9
Therefore,
the selection of plant type and quality of extract is very important.
10
Some AgNPs synthesized using plant extracts showed biological
activities such as antimicrobial, antioxidant and anticancer
activities. Cassia fistula leaf extract-mediated biogenic-synthesized
AgNPs showed good cytotoxicity against skin cancer cells (A-431
cell line).
11
Enhanced antibacterial effects were detected for AgNPs
synthesized using the Aloe vera plant compared with antibiotic
drugs.
12
The free radical scavenging potential of Pongamia
pinnata-synthesized AgNPs was evaluated in vitro by using five
different assays.
13
Pimpinella anisum seed extract-synthesized
AgNPs were evaluated for toxicological effects on colon cancer
cells (HT115) against human neonatal skin stromal cells.
14
AgNPs
synthesized using the seed-based extract of P. pinnata showed
antibacterial property and interacted with human serum albumin.
15
The antifungal effectiveness of fungal species Neofusicoccum
parvum and Rhizoctonia solani was found for AgNPs synthesized
by Trifolium resupinatum seed extract.
16
AgNPs synthesized using
the leaf extracts of the species of the Kalopanax plant showed
antimicrobial activity.
17,18
Plant extract-synthesized AgNPs were
advocated to display a variety of biological activities.
19
As the particles with uniform shape demonstrate good functional
properties, increasing research interest has been generated in the
control of the size and shape of AgNPs.
19,20
Tannic acid carrying
numerous phenolic groups is a virtuous reducing agent for AgNP
size control in the range 18–30 nm.
21
Monodispersed spherical
1
Cite this article
Sathiyamoorthi E, Iskandarani B, Salunke BK and Kim BS (2018)
Biomedical potential of silver nanoparticles biosynthesized using gallnut extract.
Green Materials,
https://doi.org/10.1680/jgrma.17.00032
Research Article
Paper 1700032
Received 27/10/2017; Accepted 02/03/2018
ICE Publishing: All rights reserved
Keywords: green chemistry/
nanobioscience/nanoparticles
Green Materials
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AgNPs of controlled size in the range 7–66 nm were formed
utilizing tannic acid as reductant and stabilizer by changing the pH
and molar ratio of tannic acid to silver nitrate (AgNO
3
).
22
Tan nic
acid can effectively promote the equilibrium in nucleation and
growth processes, thereby tuning AgNP size.
22
Moreover, size- and
shape-controlled AgNPs are valuable in plasmonics, catalysts,
bactericides, electronics and optical materials.
17,23–26
Size-
controlled AgNP synthesis is achieved by selecting a reductant with
appropriate reactivity to facilitate processes of nucleation and
growth.
27
Some researchers tried to control the shape and size of
AgNPs by varying the ratio of silver nitrate and the reducing agent
such as sodium borohydride (NaBH
4
), tannic acid and plant
extracts.
9,28,29
Research has also been carried out by some
researchers into the size and shape control of AgNPs using plant
extracts exclusively without additional reducing agents in the
reaction mixture. However, the production of AgNPs with
controlled shape and size had minimal success. More research
efforts are needed to screen different plants and reaction conditions
for the size and shape control of particles.
Gallnut is an outgrowth of plant tissues released by the larva of
gall insects such as the Cynipidae family, the gall wasps. As a
result of the unique advantage and market demand, they are
widely used in different industries.
30
The components of gallnut
(Galla chinensis) are 69% gallotannin, 25·7% gallic acid and
5·3% methyl gallate.
31,32
It also exhibits medicinal properties
such as antiallergic, anticancer, antimicrobial, antidiabetic and
antioxidant activities.
33–37
Due to their biological compatibility
and biological activities, AgNPs are important for antioxidant and
antimicrobial agents and treating various cancers.
38–41
Although
some AgNPs have been found to have antioxidant activity, no
research has been conducted to determine the cause of antioxidant
activity. Herein, the potential of gallnut extract containing high
amounts of tannic acid was investigated in the synthesis of
AgNPs and to find conditions for controlling the size of particles.
The antioxidant activity to scavenge 2,2-diphenyl-1-
picrylhydrazyl (DPPH) free radicals of AgNPs biosynthesized
using gallnut extract and AgNPs chemically synthesized using
sodium borohydride was compared for the first time. The
anticancer potential of the synthesized AgNPs was also assessed
using four cervical cancer cell lines (ME180, SiHa, HeLa and
CaSki), with a view to biomedical applications.
2. Materials and methods
2.1 Materials
Gallnut powder was purchased from Jecheon Dongsan Medicinal
Herb (Jecheon, Korea). Silver nitrate (Junsei Chemicals),
potassium carbonate, dimethyl sulfoxide, l-glutamine, fetal bovine
serum (FBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) and Dulbecco’s modified Eagle’s medium
(DMEM) were procured from Sigma-Aldrich. The rest of the
chemicals used were of analytical reagent grade, and experiments
were carried out using deionized water. All glassware was first
rinsed with acetone and then with Millipore water before use.
2.2 Preparation of gallnut extract
The gallnut extract was prepared by boiling 1·7 g of gallnut powder
in sterile distilled water (100 ml) for 1 h. After cooling at room
temperature, the extract was filtered using filter paper (Whatman
number 1) to remove insoluble residues. The filtered gallnut extract
was stored at 4°C and used for further experiments.
2.3 Synthesis of AgNPs
For the biological synthesis of AgNPs, 1 ml gallnut extract solution
was diluted with 99 ml of distilled water in a 100 ml round-bottomed
flask, under constant stirring of 400 revolutions per minute (rpm) at
30°C. After that, 1 ml of 0·1 M silver nitrate was added slowly. The
effect of pH was investigated by adjusting pH in the range 3–10 by
using 0·5 M potassium carbonate. The mixture was stirred for 15 min
and the color change was monitored. Due to the photoactivation of
silver nitrate, the reactions were carried out in darkness. After the
reaction, the purification of AgNPs was carried out by centrifuging at
15 000 rpm for 20 min at room temperature and redispersing pellets
in deionized water. The purified nanoparticles were lyophilized
overnight. For the chemical synthesis of AgNPs, 0·5 ml of a 0·1 M
sodium borohydride solution was added dropwise to 100 ml of
0·05 M aqueous silver nitrate solution at room temperature. The color
change was observed immediately and the solution was stirred at
room temperature for 10 min.
42
2.4 Characterization of AgNPs
The synthesis of AgNPs was observed by taking the ultraviolet
(UV)–visible spectrum between 200 and 800 nm wavelengths of the
reaction medium using a UV–visible spectrophotometer (UV-1601,
Shimadzu, Japan). Fourier transform infrared (FTIR) spectroscopy
spectra were obtained on a Nicolet IR 200 instrument in the range
between 4000 and 400 cm
−1
. The pellet was prepared by mixing
purified AgNP powder and potassium bromide (KBr) and spectra
were recorded against a potassium bromide reference blank. The
purified AgNP powder was coated on the glass substrate to record X-
ray diffraction (XRD; Rigaku, Ultima IV, Japan) spectra using copper
(Cu) Karadiation (l= 1·5418 Å) monochromatic filter in the range
10–80°. X-ray photoelectron spectroscopy (XPS) of AgNPs was
examined using a PHI quantera-II, ulvac-PHI. The morphology of
the AgNPs was investigated using a scanning electron microscope
(SEM; Leo-1530). Energy-dispersive X-ray (EDX) spectra were
obtained from a Leo-1530 instrument coupled with an EDX detector
(Carl Zeiss, Oberkochen, Germany). The morphology and size of
AgNPs were visualized using an energy-filtering transmission
electron microscope (EF-TEM; Libra 120, Carl Zeiss). Before that,
AgNPs were dispersed in 1 ml of ethanol on a hydrophilic carbon-
coated copper grid and then dried under ambient conditions.
2.5 Antioxidant activity assay
The DPPH scavenging assay was performed according to the
modified method described by Clarke et al.
43
In this method, a
96-well plate was used where the synthesized AgNP solution was
diluted from 10 to 100% in a total sample volume of 60 ml. DPPH
ethanol solution (20 ml of 0·2 mM) was added, and the mixture
was shaken and incubated in the dark for 30 min. Absorbance was
2
Green Materials Biomedical potential of silver
nanoparticles biosynthesized using
gallnut extract
Sathiyamoorthi, Iskandarani, Salunke and Kim
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measured at 517 nm against blank ethanol. The DPPH scavenging
potential was compared for gallnut extract and for chemically
synthesized AgNPs. A similar procedure was also performed for
nanoparticles biosynthesized at various pH values.
2.6 Cell viability assay
The MTT assay was used to test cell viability using cervical cancer
cell lines. Briefly, ME180, SiHa, HeLa and CaSki cells were grown
in DMEM medium augmented with 1 mM l-glutamine and 10%
FBS. The seeding of cells was done in 96-well plates composed of
100 mlmediaat1×10
4
cells/ml density, and culturing was done at
37°C overnight in a humidified incubator with 5% carbon dioxide
(CO
2
). The anticancer efficacy of the AgNPs was evaluated by
treating the cultured cells at various doses of AgNPs (10, 25, 50 and
100 mg/ml) for different time periods (24 and 48 h). A microplate
reader was used to record absorbance at 450 nm. For each toxicity,
three end point independent experiments were carried out. For the
determination of cell viability, the ratio of absorbance values for each
treatment was recorded. The inverted microscope images were
captured to monitor cell viability.
3. Results and discussion
3.1 Synthesis of AgNPs and UV–visible absorption
spectroscopy
Gallnut extracts were prepared and used for the synthesis of
AgNPs under facile conditions (Figure 1(a)). The color of the
solution changed within a few seconds from yellow to reddish
brown after the addition of aqueous silver nitrate into the gallnut
extract, giving the preliminary indication of AgNP synthesis. This
is due to the excitation of free electrons in AgNPs. The observed
result is similar to that for AgNPs synthesized using Acalypha
indica leaf extract, which exhibits different colors in the
solution.
44
The UV–visible absorption spectra of AgNPs are
sensitive to various factors including size, shape and interactions
between particles.
45
Gallnut-extract-synthesized AgNPs showed
characteristic UV–visible absorption spectra at 428 nm. This
observation is similar to characteristics of plant-synthesized
AgNPs reported by other researchers.
9,17,18
The bioreduction of silver was postulated as the trapping of silver
ions (Ag
+
) on protein surface due to electrostatic interactions
between Ag
+
and proteins in plant extract.
46
Proteins reduce silver
ions, leading to their secondary structure change and formation of
silver nuclei. The formed silver nuclei successively grow with the
further reduction of silver ions and their build-up in the nuclei,
leading to the formation AgNPs.
46
It has been reported that the
key mechanism behind the plant-mediated synthesis of AgNPs is
a plant-assisted reduction due to phytochemicals. The primary
phytochemicals are ketones, terpenoids, amides, flavones,
carboxylic acids and aldehydes.
47
Water-soluble phytochemicals,
including organic acids, quinones and flavones, are responsible
for the instantaneous reduction in silver ions in the reaction
(a)
2
1
Absorbance: arbitrary units
0
200 300 400 500 600 700 800
Wavelength: nm
(b)
pH3 (426 nm)
pH4 (420 nm)
pH5 (418 nm)
pH6 (416 nm)
pH7 (414 nm)
pH8 (413 nm)
pH8 (410 nm)
pH10 (409 nm)
3
2
1
0
200 300 400 500 600 700 800
Wavelength: nm
Tannic acid
Gallnut
(c)
Absorbance: arbitrary units
Figure 1. (a) Photographic representation of gallnut dried fruit, gallnut powder and gallnut extract; (b) UV–visible spectra for the
synthesized AgNPs from gallnut extract at pH 3–10; and (c) UV–visible spectra for gallnut extract and tannic acid
3
Green Materials Biomedical potential of silver
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gallnut extract
Sathiyamoorthi, Iskandarani, Salunke and Kim
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mixture.
48
In the majority of cases, reducing agents in plant
extracts also serve as capping and stabilizing agents, thereby
eliminating the need for external capping and stabilizing agents.
49
The reduction of silver ions has been found to depend on the type
of plant extract used as a reducing agent.
50
3.2 Effect of pH
pH plays a significant role in the synthesis and stability of
AgNPs. The pH of the solution affects the formation rate of
nanoparticles and therefore their final size. Furthermore, AgNPs
along with other metal nanoparticles are unique by having surface
plasmon resonance (SPR). The absorption peak detected in
UV–visible absorption spectra allows approximate particle size
estimation. This is due to the direct proportionality between the
wavelength of maximum absorption of AgNPs and their size.
51
Figure 1(b) displays the UV–visible absorption spectra of AgNPs
in the range 200–800 nm. The maximum absorption peaks for
AgNPs synthesized at pH 3–10 occurred at 426, 420, 418, 416,
414, 413, 410 and 409 nm, respectively, and the wavelength
shifted from red to blue.
52
The absorbance of AgNPs increased
with increasing pH from 3 to 10 probably due to the increase of
particle numbers in the solution. The absorption spectrum is due
to strong SPR, the resonance absorption of photons by AgNPs.
Since the SPR band depends on the size and refractive index of
the solution, the observed absorption band is dependent on size.
53
The shift in peak wavelength indicates that the size of the
particles decreases as the pH of the solution increases. As
the diameter of the particles increases, the energy required
for excitation of the surface plasmon electrons decreases, and
as a result, the absorption maximum shifts toward the
longer-wavelength region. In the UV–visible spectroscopy of
gallnut extract and tannic acid, similar peaks at the wavelength
around 271–276 nm were observed (Figure 1(c)). Peak intensity
suggests that gallnut contains a higher amount of tannic acid.
Tannic acid has been reported to synthesize different types of
metal nanoparticles.
54
Tannic acid present in gallnut can play an
important role in the synthesis of AgNPs.
3.3 Transmission electron microscopy
Transmission electron microscopy (TEM) micrographs suggest
that at pH 3–6, the size and shape of AgNPs are irregular
(Figures 2(a)–2(d)) compared to pH 7–10 (Figures 2(e)–2(h)). This
indicates the poor balance between nucleation and growth processes
in acidic conditions compared to neutral and basic conditions. The
low formation rate of AgNPs was also observed at acidic pH
compared to neutral and basic conditions. It was reported that the
formation of AgNPs using Solanum xanthocarpum berry methanol
extract was lower in acidic conditions and higher in basic
conditions.
55
Larger particles were formed at pH 5, while highly
dispersed and smaller nanoparticles were formed at pH 9.
56
Figure 2(i) shows the graphical representation of average particle
size with pH. The average sizes were 51, 27, 18, 30, 10, 8, 5 and
4 nm, respectively, for AgNPs synthesized at pH 3–10. Although
particle sizes were lower at higher pH at 8–10, more evenly
dispersed particles were observed at pH 7 in the study. Almost
70% of particles were in similar size and shape at pH 7 compared
to other pH conditions. The spherical shape of AgNPs at pH 7
can be attributed to the balance between the nucleation and
growth processes as well as to the silver precursor (silver ions)
reduction rate increase.
57
AgNPs formed at pH 7 were further
characterized by other techniques.
The previous analysis and other references indicate the formation
of a good crystalline structure of the synthesized nanoparticles,
with a distance of 0·23 nm between the lattice planes matching
the (111) lattice of the face-centered cubic silver (Ag).
9,58,59
The
crystal structure of the NPs was further demonstrated by the
selected area (electron) diffraction pattern with bright circular
rings corresponding to the (111), (200), (220) and (311) Bragg’s
reflection planes.
60,61
3.4 FTIR spectroscopy
FTIR spectrum peaks for gallnut extract occurred in 2345, 1613,
1535, 1448, 1321 and 1201 cm
−1
. For tannic acid, the peaks were
60
40
20
0
Average particle size: nm
(i)
345678910
pH
(a) (b) (c) (d)
(e) (f) (g) (h)
pH 3
pH 7 pH 8 pH 9 pH 10
pH 4 pH 5 pH 6
50 nm 50 nm 50 nm
50 nm
50 nm
50 nm50 nm
50 nm
Figure 2. (a–h) TEM images of AgNPs synthesized at different pH
(pH 3–10); (i) graphical representation of AgNPs synthesized at
different pH values against average particle size (nm)
4
Green Materials Biomedical potential of silver
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gallnut extract
Sathiyamoorthi, Iskandarani, Salunke and Kim
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found at 2377, 1612, 1535, 1448, 1322 and 1202 cm
−1
. The peaks
for silver nitrate were at 2305, 1634, 1357 and 828 cm
−1
. AgNPs
biosynthesized by using gallnut extract showed peaks at 2373,
1634, 1545, 1460, 1365 and 1224 cm
−1
(Figure 3). The peaks
presented in gallnut extract (2345 cm
−1
) and AgNPs (2373 cm
−1
),
represent the C–H asymmetric stretching. The bands at 1613 and
1535 cm
−1
are due to the C=C stretching and protein secondary
amine bending vibration. The band at 1321 cm
−1
signifies
carboxylate group symmetrical stretch. The broad band at
1201 cm
−1
is due to C–H and C–O stretching modes. The peak at
1634 cm
−1
suggests that the O–H and C=O groups were adsorbed
on the surface of AgNPs and involved in the reduction process.
The band at 1364 cm
−1
suggests the silver ion binding to
carboxylate and hydroxyl groups, respectively. The peak at
1224 cm
−1
signifies the polyol C–O group, demonstrating the role
of polyols in the reduction of AgNPs. FTIR studies reveal that the
carbonyl groups of amino acid, peptides and proteins can bind to
metal and coat the particles and stabilize the AgNPs against
agglomeration. This demonstrates that the proteins and tannins
from the gallnut extract similar to phytochemicals present in gum
kondagogu may be involved in the formation of AgNPs.
62
3.5 SEM, EDX, XPS and XRD
The morphology of AgNPs was analyzed by SEM, which revealed
the presence of spherical particles (Figure 4(a)). EDX measurement
(Figure (4b)) showed the presence of silver, confirming the synthesis
of AgNPs. The composition analysis indicated the presence of 93·85
weight% and 71·82 atom% of silver. Figure 4(c) shows the XPS
spectrum of the capped AgNPs with gallnut. The characteristic
feature showed the silver electron valence state Ag
0
.
63
The XRD of
the AgNPs suggested that sharp peaks were observed in the silver
region (Figure 4(d)).
64
As per the powder diffraction card of the Joint
Committee on Powder Diffraction Standards silver file number 04-
0783, the high intense peak was observed in (111) reflection. The
majorfourstrongBraggreflections at 38·199, 44·379, 64·657 and
77·584° correspond to the planes of (111), (200), (220) and (311),
respectively, which can be indexed according to the facets of the
face-centered cubic crystal structure of silver.
65
The average
crystallite size Dof AgNPs was estimated from the diffractogram
using the Debye–Scherrer formula, D=0·94l/bcos q, where lis the
wavelength of the X-ray used in the diffraction and bis the full
width at half maximum of a peak.
66
The calculated average
crystallite of AgNPs from the four peaks is 11·8 nm. The value of
the interplanar spacing between atoms dwas calculated using
Bragg’slaw,2dsin q=nl, where nis the order of the diffraction
pattern (n= 1 in this case). The calculated dvalues are 2·356, 2·041,
1·442 and 1·230 Å for the (111), (200), (220) and (311) planes,
respectively, and matched with standard silver values. The lattice
constant was estimated using the formula a=d(h
2
+k
2
+l
2
)
1/2
,
where h,kand lare Miller index parameters. The average value of
the four avalues calculated from the four dvalues obtained from the
data for four peaks is 4·0804 Å. This is in good agreement with the
standard value for silver, 4·0857 Å.
67
Thus, XRD analysis showed
that AgNPs with well-defined dimensions can be synthesized by the
reduction of silver ions using gallnut extracts.
3.6 Antioxidant activity
The DPPH scavenging results showed an effective free radical
scavenging potential independent of the nanoparticle solution
dose and nanoparticle size (Figures 5(a) and 5(b)). The typical
scavenging activity of biosynthesized AgNPs is 81%, similar to
73% of gallnut plant extract. AgNPs chemically synthesized using
sodium borohydride showed a much lower scavenging activity of
56%. This indicates that AgNP-capping molecules such as tannic
acid play an important role in antioxidant function. Other plant
extracts showed different DPPH scavenging potential, where
59·3% was reported for AgNPs synthesized using Bergenia ciliate
plant extract,
68
and about 70% for AgNPs synthesized using
Iresine herbstii leaf aqueous extract.
69
Therefore, AgNPs
synthesized using gallnut extract showed clear superiority as an
antioxidant to remove DPPH free radicals, which can be attributed
to AgNP-capping molecules such as tannic acid, which improves
the free radical scavenging ability of AgNPs.
3.7 Cell viability of cervical cancer cell lines
The cytotoxic activity effects of AgNPs biosynthesized using gallnut
extract on four cervical cancer cell lines are depicted in
Figures 6(a)–6(d). The dose-dependent reduction in cell viability was
observed. The photomicrographic images (Figures S1–S4 in the
online supplementary material) reveal cell morphological changes.
Membrane lyses of cancer cells and nuclear damage in all four
cervical cancer cell lines were observed. In comparison to the
controls, the cell number reduction was witnessed for high doses of
AgNPs (25, 50 and 100 mg/ml). From observations, a heterogeneous
model of cell toxicity with the inclusion of a combination of different
death modes depending on various parameters such as cell line,
exposure time, AgNP concentration and capping agents can be
D
Transmittance
C
AgNPs
Silver nitrate
B
A
Tannic acid
Gallnut
4000 3500 3000 2500 2000 1500 1000 500
Wavelen
g
th: nm
Figure 3. FTIR spectra of gallnut, tannic acid, silver nitrate and
AgNPs synthesized at pH 7
5
Green Materials Biomedical potential of silver
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gallnut extract
Sathiyamoorthi, Iskandarani, Salunke and Kim
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Mag - 100·00 K X 100 nm
(a)
80 000
60 000
40 000
20 000
0
02468101214161820
Ag
Element Wt% Atom%
keV
C K
O K
Al K
Si K
I K
Ag L
Total
(b)
100 100
93·85
1·67
2·06
11·51
10·65
0·49
0·08
1·84 4·28
71·82
0·24
1·50
8000
6000
4000
2000
385 380 375 370 365 360
Intensity: arbitrary units
Binding energy: eV
(c)
Intensity: arbitrary units
(122) (210)
(111)
(200)
(231)
(220)
(311)
(222)
10 20 30 40 50 60 70 80 90
2θ: °
(d)
Figure 4. (a) SEM image, (b) EDX, (c) XPS and (d) XRD for AgNPs synthesized at pH 7
100
75
50
25
0
DPPH scavenging potential: %
Biologically
synthesized
AgNPs
Gallnut
extract
Chemically
synthesized
AgNPs
(a)
100
75
50
25
0
678910
pH
DPPH scavenging potential: %
(b)
Figure 5. (a) DPPH radical scavenging percentage for AgNPs biosynthesized using gallnut extract, gallnut extract alone and AgNPs
chemically synthesized using sodium borohydride; (b) DPPH radical scavenging percentage for biosynthesized AgNPs at different pH values
6
Green Materials Biomedical potential of silver
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gallnut extract
Sathiyamoorthi, Iskandarani, Salunke and Kim
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suggested. Similar kinds of anticancer potential of AgNPs were
reported by other researchers.
70,71
A suitable complex antioxidant enzyme network may be involved
according to the hypothetical mechanism of AgNP biosynthesis.
72
Current research on the antioxidant potential of gallnut provides a
hypothesis that antioxidant molecules in the gallnut extract are
involved in the biological synthesis of AgNPs. Previous studies have
shown that plants containing phenols and flavonoids have high
antioxidant capacity and therefore biosynthesis of nanoparticles.
73
Electrostatic interactions between positively charged nanomaterials
and target cancer cells are described in the literature. Cancer cells
usually exhibit a high concentration of anionic phospholipids on their
outer leaflet, compared to normal cells that exhibit zwitterionic
phospholipids.
74
This condition plays an important role in the cellular
uptake of positively charged nanoparticles in cancer cells compared
to normal cells. Because AgNPs are positively charged (+20·1 mV
zeta potential), it is expected that they are more toxic to cancer cells
than to normal cells.
75
4. Conclusion
The economical, simple and green biosynthesis of AgNPs was
studied using gallnut extract for the first time. Color change from
light yellow to dark brown indicated the reduction of silver ions to
Ag
0
and spectra at 426–409 nm in UV–visible spectroscopy
confirmed the synthesis of AgNPs at different pH values. Out of the
different pH values tested, pH 7 was found to be good for the
synthesis of AgNPs with uniformity. FTIR studies indicated the role
of plant tannins and polyphenolic compounds in the metal reduction
and capping of AgNPs. The XRD results suggested the crystalline
nature of the biosynthesized AgNPs. The antioxidant activity to
remove DPPH free radicals was compared between biosynthesized
and chemically synthesized AgNPs. AgNPs biosynthesized using
gallnut extract showed a higher antioxidant activity than AgNPs
chemically synthesized using sodium borohydride, indicating that
gallnut extract plays an important role in improving antioxidant
ability. Cell viability analysis showed potent anticancer effects of
gallnut-synthesized AgNPs on different cancer cell lines depending
on dose, exposure time and cell line. Due to the high antioxidant and
anticancer activities of gallnut extract-synthesized AgNPs, this study
opens opportunities for further research into the exciting use of
gallnut extract for AgNP synthesis toward potential applications in
the field of biomedical nanotechnology.
Acknowledgements
This research was supported by the Small and Medium Business
Administration (C0505005 and S2492537).
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Per cent cell viability
Per cent cell viability
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100
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