Materials Science & Engineering C 91 (2018) 372–381

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Materials Science & Engineering C

journal homepage: www.elsevier.com/locate/msec

Green synthesized silver nanoparticles: Catalytic dye degradation, in vitro T

anticancer activity and in vivo toxicity in rats
Jayachandra Reddy Nakkalaa, Rani Mataa, Kumar Rajab, Varshney Khub Chandrab,
⁎
Sudha Rani Sadrasa,
a
Department of Biochemistry and Molecular Biology, School of Life Sciences, Pondicherry University, Puducherry, India
b
Department of Veterinary Pathology, Rajiv Gandhi College of Veterinary & Animal Sciences, Kurumbapet, Puducherry, India

A R T I C LE I N FO A B S T R A C T

Keywords: In this study, silver nanoparticles were synthesized (AgNPs) using aqueous rhizome extract of Acorus calamus
Nano silver (ACRE) and evaluated their in vitro anticancer activity and in vivo toxicity in a Wistar rat model. The synthesized
Catalytic activity AgNPs showed good catalytic activity against different organic pollutant dyes. In vitro cytotoxic effects of AgNPs
Anti-cancer activity were assessed in Hep2, COLO 205 and SH-SY5Y cells using MTT assay. Further, the apoptotic changes induced
Bio distribution
by AgNPs in more susceptible Hep2 cells were observed through AO/EB, DCFH-DA, Rhodamine 123, PI/DAPI
Toxicity
staining, oxidative stress markers and Western blotting. In vivo toxicity study revealed substantial alterations in
the levels of serum biochemical markers including AST, ALT, LDH and inflammatory markers such as TNF-α and
IL-6 on day 29 when rats treated with AgNPs as compared to control, however, these levels were restored to
normal at the end of washout period on day 89. No remarkable changes were observed in liver oxidative stress
enzymes. ICP-OES analysis indicated bio-distribution of silver in spleen (5.67 μg/g) and liver (4.98 μg/g) in rats
treated with 10 mg/kg b.w of AgNPs on day 29 and elimination of silver from all organs was observed at the end
of washout period on day 89. Histopathological analysis revealed no significant changes in kidney, spleen, lungs,
heart, testis and brain with 5 and 10 mg/kg b.w of AgNP. However, 10 mg/kg b.w of AgNPs showed moderate
degree of cell swelling and vacuolar degeneration in liver and these alterations were reverted back to normal at
the end of washout period. Findings from this study signify green synthesized AgNPs at low concentrations might
be useful in many ways with ecofriendly nature.

1. Introduction
In recent years, nanotechnology has fabulous growth due to their
various applications in several fields such as material science, chem-
istry, medicine, bionanotechnology, etc. Nanoparticles (NPs) have high
attention because of their great surface to volume ratio, and tre-
mendously small size, which leads to difference of physical and che-
mical properties as compared to same composition of bulk material [1].
Among the NPs, the researchers have shown more interest in silver NPs
(AgNPs) because of their wide range of applications in antimicrobial,
catalysis, medical, optics and energy. These NPs have been commonly
synthesized using electrochemical [2], photochemical [3], microwave
[4], thermal decomposition [5], chemical reduction [6], and radian
assisted process [7] for a long time.
The AgNPs synthesized from various chemical approaches showed
toxicity in in vivo studies. The behavior of AgNPs is influenced by the
size, concentration, coating, and distribution level of the particles [8,9].
Alternately, the microbial and biological system (green chemistry) ap-
proaches are developed to synthesize NPs without the use of harsh and
expensive toxic chemicals [10,11]. The microorganism based NPs
synthesis is rapidly developed due to their eco-friendly formation of
NPs. However, microbial method has disadvantages due to the con-
sumption of more time for maintaining cultures [12]. Nowadays, the
NPs are synthesized using different plant sources due to their natural
availability, rapid formation, and the ecofriendly nature [13]. The
AgNPs were synthesized using Acorus calamus rhizome. Acorus calamus
is also called as sweet flag. In Indian Ayurvedic system this herb is used
to treat brain disorders, chronic diarrhea, liver troubles, rejuvenator for
nervous system, etc. [14]. The rhizome is also used in the preparation of
modern herbal medicine as it has diuretic, laxative, sedative and car-
minative properties [15].
The present study aims to evaluate the catalytic activity of green
synthesized AgNPs on different organic pollutant dyes, in vitro antic-
ancer activity in cancer cells and in vivo toxicity in a Wistar rat model.

⁎
Corresponding author at: Department of Biochemistry and Molecular Biology, Pondicherry University, Pondicherry 605 014, India.
E-mail addresses: dr.ssrlab@gmail.com, sadrassudha@gmail.com (S.R. Sadras).

https://doi.org/10.1016/j.msec.2018.05.048
Received 13 June 2017; Received in revised form 9 March 2018; Accepted 14 May 2018
0928-4931/ © 2018 Elsevier B.V. All rights reserved.
J.R. Nakkala et al.
2. Materials and methods
2.1. Collection and preparation of extracts
The Acorus calamus rhizome powder was collected from the
Puducherry local market, Puducherry, India. The powder was authen-
ticated by Prof. Parthasarathy, Department of Ecology and
Environmental Science, Pondicherry University, India. The dried
powder was mixed with deionized water and the mixture was filtered
through Whatman No⋅ 1 filter paper. The water from the filtered so-
lution was evaporated using a rotary evaporator followed by lyophilizer
in order to obtain the residue. The phytochemical screening was per-
formed using the residue.
2.2. GC–MS analysis
The phyto-constituents of ACRE were tested using GC–MS (GC and
MS JEOL GC mate supplied with the secondary electron multiplier). The
interpretation of GC–MS spectra was performed by comparing the data
of National Institute Standard and Technology (NIST) database.
2.3. Synthesis of AgNPs
The extract was prepared by mixing the 1 g of the dry powder of
ACRE in100 mL of double distilled water and then kept in boiling water
bath for 15 min. The Whatman No⋅ 1 filter paper was used for filtering
the extract which was then stored at 4 °C for further use. The NPs were
synthesized by mixing different ratios of 1 mM silver nitrate and pre-
pared extract at room temperature. The color change observed in the
mixture is an indicative of formation of NPs. The formation of AgNPs
was further confirmed through periodical observation of absorption of
the solution using UV-visible spectroscopy in the range of 300–700 nm.
The synthesized Acorus calamus silver NPs (ACAgNPs) were collected by
using repeated centrifugation at 18000 rpm for 25 min. The obtained
pellet was further repeatedly washed three times using double distilled
water in order to remove the unbounded biomolecules which may be
the secondary metabolites or the proteins. The purified AgNPs pellet
was dried at room temperature and their characterization was per-
formed by using different techniques.
2.4. Characterization of AgNPs
The formation of ACAgNPs was determined using UV-visible spec-
trophotometer (UV-1700 Shimadzu), with optical density measure-
ments executed at various time intervals between the wavelength
ranges of 300–700 nm. The baseline was corrected using double dis-
tilled water. The micrograph image and elemental composition of the
ACAgNPs were determined by TEM along with EDAX (JEOL 3010). The
sample was prepared by placing dry powdered AgNPs on the carbon
coated copper grid, which have been dried under a mercury lamp for
5 min. The excess powder was removed by using tissue paper. The
functional groups present in the ACRE and ACAgNPs were identified by
FTIR spectroscopy (Thermo Nicolet Nexus 670 equipped with KBr op-
tics and a DTGS detector). The instrument was operated with a re-
solution of 4 cm−1 and scanned between the frequency ranges of
500–4000 cm−1. The average hydrodynamic diameter and poly-
dispersity nature of ACAgNPs were determined by the DLS particle size
analyzer (ZETA Seizers Nanoseries). The zeta potential of the ACAgNPs
in pure water was further analyzed by using electrophoretic light
scattering at 25 °C in 150 V (Malvern Instruments Nano ZS).
2.5. Catalytic activity
The catalytic role of ACAgNPs was assessed using 4-nitrophenol (4-
NP), 3-nitrophenol (3-NP), 2, 4, 6-trinitrophenol (2, 4, 6-TNT) or picric
acid (PA), coomassie brilliant blue (CBB), congo red (CR), eosin Y (EY),
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Materials Science & Engineering C 91 (2018) 372–381
rhodamine B (RB), methylene blue (MB), methyl red (MR), methyl or-
ange (MO), cresol red (CRR), acridine orange (AO), eriochrome black T
(EBT), and phenol red (PR). Briefly, the experiment was performed by
mixing 10 mM of 0.3 mL of 4-NP, 1.7 mL of deionized water, 1 mL of
150 mM NaBH4 and 15 μg/mL of ACAgNPs. The 10 mM of 30 μL picric
acid (PA) or 2, 4, 6-TNT was mixed with 1.97 mL of deionized water,
1 mL of 150 mM NaBH4 and 15 μg/mL of ACAgNPs. Similarly the
10 mM of 100 μL 3-NP, CBB, CR, EY, RB, MB, MR, MO, CRR, AO, EBT,
and PR were individually mixed with 1.9 mL of deionized water, 1 mL
of 150 mM NaBH4 and 15 μg/mL of ACAgNPs. The absorption of the
mixture was monitored periodically with different time intervals be-
tween the ranges of 200 to 800 nm using UV–visible spectro-
photometer.
2.6. Cell culture
The Hep2 (human epidermoid carcinoma), COLO 205 (human colon
adenocarcinoma) and SH-SY5Y (neuroblastoma) cancer cells were
cultured in Dulbecco's modified Eagle medium with L-glutamine and
1000 mg/L glucose (DMEM) which was supplemented with 10% fetal
bovine serum, streptomycin sulfate (0.1 mg/mL) and penicillin G
(100 units/mL) in the humidified environment consisting of 5% CO2 at
37 °C.
2.6.1. MTT [(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium
bromide] assay
The anti-proliferative ability of ACAgNPs on Hep2, COLO 205 and
SH-SY5Y cancer cells was assessed based on the previously reported by
MTT assay [16]. The cell viability was determined by the following
equation and then IC50 values were calculated.
Absorption (test) ∗100
Percentage of viability =
Absorption (control)
2.6.2. Cytomorphological analysis
The cultured more susceptible Hep2 cells were seeded
(1 × 105 cells/well) in a 12 well chamber plate and maintained for 12 h
in a humidified environment consisting of 5% CO2 at 37 °C. The seeded
cells were then treated with ACAgNPs with their respective IC50 values
and then maintained for 24 h. The cytomorphology of the treated and
untreated cells was examined in an OPTIKA (Italia) inverted phase-
contrast microscope.
2.6.3. Acridine orange/ethidium bromide (AO–EB) staining
The induction of apoptosis by ACAgNPs in Hep2 cells was examined
using AO/EB double staining technique. The cultured Hep2 cells were
seeded (1 × 105 cells/well) in a 12 well chamber plate and maintained
for 12 h in a humidified environment consisting of 5% CO2 at 37 °C.
Then the seeded cells were treated with the respective IC50 values of
ACAgNPs and incubated at 37 °C for 24 h. Finally, 50 μL of dye mixture
(AO-EB) was added to the cells and observed under fluorescence mi-
croscope (Nikon Eclipse Ti Japan) to detect any apoptotic cell death.
2.6.4. Detection and quantification of intracellular ROS
In this assay, Hep2 cells (1 × 105 cells/well) were plated in a 6 well
plate and treated with respective IC50 value of ACAgNPs for 24 h.
Following treatment, 10 μM of DCFH-DA was added and then kept for
30 min incubation at 37 °C. The treated and untreated cells were ob-
served under the fluorescence microscopy (Nikon Eclipse Ti Japan) to
detect any ROS production. Similarly, ACAgNPs treated cells were
trypsinized and separately collected in aluminum foil wrapped
Eppendorf tubes for ROS quantification. The DCFH-DA (25 μM) solution
was added to cells followed by incubation for 45 min at 37 °C. The in-
tensity of fluorescence was noted by using the Fluorolog-FL3-11 spec-
trofluorometer (HORIBA JobinYvon, NJ, USA) with respective excita-
tion and emission wavelengths.
J.R. Nakkala et al.
Fig. 1. (a) UV–visible spectral analysis of ACAgNPs at different time intervals (b) TEM micrograph of ACAgNPs (c) DLS particle size analysis of ACAgNPs (d) Zeta
potential analysis of ACAgNPs.
2.6.5. Oxidative stress parameters
The Hep2 cells were cultured in 75 cm2 flasks and treated with
ACAgNPs with IC50 concentration for 24 h. After the treatment, cells
were washed with PBS and collected in ice-cold PBS at 4 °C. The col-
lected cell pellets were then lysed using lysis buffer and centrifuged at
10000g for 10 min at 4 °C. The supernatant was then assembled and
maintained at 4 °C until all the oxidative biomarkers assays were per-
formed. Standard procedures were followed in order to estimate the
liver oxidative stress markers, such as LPO [17], SOD [18], catalase
[19] and GPx [20] and non-enzymatic antioxidant GSH [21].
2.6.6. Assessment of mitochondrial membrane potential (MMP or ΔΨm)
The MMP loss in ACAgNPs treated Hep2 cells were determined by
Rhodamine 123 staining. Herein, the Hep2 cells (5 × 105 cells/well)
seeded separately in a six well plate were incubated for 12 h in a hu-
midified atmosphere having 5% CO2 at 37 °C. Then these cells were
exposed to the IC50 concentration of ACAgNPs for 24 h. Further, the
cells were washed using PBS and fixed in 4% paraformaldehyde and
then in 70% of ethanol for 10 min. The 50 μL of rhodamine 123 (10 μg/
mL) were added and then kept for 30 min. The excess dye was removed
using PBS and the cells were detected under a fluorescence microscope
(Nikon Eclipse Ti Japan) at 20× magnification. For quantification, the
treated and untreated cells were trypsinized and separately collected in
aluminum foiled effendrof tubes. Finally, the rhodamine 123 was added
to effendrof tube and kept for 45 min of incubation at 37 °C. The
fluorescence intensity was noted by using a HORIBA JobinYvon, (NJ,
USA) Fluorolog-FL3-11 spectrofluorometer with respective excitation/
emission wavelengths.
2.6.7. Propidium iodide (PI) and 4′, 6-Diamidino-2-phenylindole
dihydrochloride (DAPI) staining
The apoptosis associated changes in the cell nucleus like con-
densation/fragmentation were identified using PI staining. Herein, the
cultured Hep2 cells (1 × 105 cells/well) were plated in a six well plate
and kept for 12 h in the incubator. The cells were then treated with an
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Materials Science & Engineering C 91 (2018) 372–381
appropriate IC50 concentration of ACAgNPs. The treated cells were kept
for 24 h at 37 °C with 5% CO2. After the treatment, cells were washed
using PBS and fixed in 4% paraformaldehyde and then in 70% ethanol.
At the end of the experiment, cells were stained using PI or DAPI and
observed under fluorescence microscope (Nikon Eclipse Ti Japan) at
20× magnification.
2.6.8. Western blot analysis
In this assay, Hep2 cells were exposed to the different concentration
of ACAgNPs and further incubated at 5% CO2 at 37 °C. After 24 h, the
cells were harvested and lysed with lysis buffer in the presence of
protease inhibitor cocktail. The collected lysates were then centrifuged
at the rate of 8000 rpm for 10 min at 4 °C and the concentration of
proteins in supernatant was estimated by Lowry method. Equal con-
centrations of samples were separated by using SDS-PAGE electro-
phoresis. The separated proteins were transferred onto nitrocellulose
membrane and then the membrane was blocked with 5% non-fat milk
solution for 1 h. After incubation, the primary antibody (active caspase
8, 9, 3, pro-lamin, cleaved lamin, cleaved PARP, β-actin) was added and
then kept for overnight at 4 °C followed by the addition of enzyme
horseradish peroxidase (HRP) linked secondary antibody for 2 h at
room temperature (RT). The protein bands were noticed by using
3,3′,5,5′-Tetramethylbenzidine/hydrogen peroxide (TMB/H2O2) solu-
tion or Enhanced chemiluminescence (ECL) and were quantified by
using Image J software.
2.7. In vivo studies
2.7.1. Experimental animals
The male Wistar rats with body weight 200–230 g (12–14 weeks)
were purchased and maintained in the room temperature (22 ± 2 °C)
with 12:12 h lights and dark cycle, according to the recommendations
of the Committee of purpose of Control and Supervision of Experiments
on Animals (CPCSEA), Govt. of India. The rats were kept two weeks for
acclimatization and the work was performed with the permission from
J.R. Nakkala et al.
Fig. 2. (a) Bright field microscopic images of (i) untreated Hep2 cells (ii) ACAgNPs treated Hep2 cells (b) Fluorescent microscopic images of AO/EB staining (i)
untreated Hep2 cells (ii) ACAgNPs treated Hep2 cells (c) Fluorescent microscopic images DCFH-DA staining (i) untreated Hep2 cells (ii) ACAgNPs treated Hep2 cells.
the Institutional Animal Ethical Committee, Pondicherry University
(PU/SLS/IAEC/2014/23).
2.7.2. Experimental procedure
The rats were divided into three groups, each comprising of 6 rats
(n = 6). The total experimental period was 88 days.
Group I rats were fed with normal pellet diet (NPD) and they were
considered as a control group; Group II rats were provided with 5 mg/
kg b.w of ACAgNPs for 28 days orally on alternate days; Group III rats
were treated with 10 mg/kg b.w of ACAgNPs for 28 days orally on al-
ternate days.
At the termination of the experimental period, from each group four
rats were anesthetized with ether and blood was collected in non-he-
parinized test tubes by cardiac puncture. The samples were centrifuged
at 2000×g for 10 min in a refrigerated centrifuge and the serum was
collected in clean centrifuge tubes for biochemical analysis. The re-
maining two rats left for 60 more days (wash out period) to check the
elimination of AgNPs. At 89th day the remaining two rats were an-
esthetized and collected serum in a similar way to the above procedure
for performing experiments.
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Materials Science & Engineering C 91 (2018) 372–381
2.7.3. Body weight
Rats were weighed weekly once in order to check the change in
their body weights throughout the experimental period of 28 days.
2.7.4. Serum markers
At the end of experimental period rats were sacrificed and the blood
was collected. The serum was collected immediately from the blood.
The serum levels of glucose, inorganic phosphorus (IP), aspartate
transaminase (AST), alanine transaminase (ALT), lactate dehy-
drogenase (LDH) and alkaline phosphatase (ALP) were determined by
using diagnostic kits (AGGAPE-diagnostics). Serum cholesterol was
estimated by CHOD-PAP (Cholesterol Oxidase/Peroxidase Amino
Phenazone) method (AGGAPE-diagnostics Pvt. Ltd., Kerala, India).
Serum TG was estimated by Glycerol-3-Phosphate Oxidase – Phenol and
Amino Phenazone (GPO-PAP) method (AGGAPE-diagnostics Pvt. Ltd.,
Kerala, India).
2.7.5. Serum inflammatory markers
The inflammatory markers of TNF-α and IL-6 were estimated by
ELISA kits as stated to the supplier's instructions (TNF-α-EASIA and IL-
J.R. Nakkala et al.
Fig. 3. Oxidant and antioxidant status of Hep2 cells treated with IC50 concentration of ACAgNPs (a) Lipid peroxidase (LPO), (b) Superoxide dismutase (SOD), (c)
Catalase (expressed as μmole of H2O2 degraded/min/mg protein), (d) Glutathione peroxidase (GPx) and (e) Reduced glutathione (GSH). Data is expressed as
mean ± SD of three identical experiments performed in triplicates.
6–EASIA-CE, DIAsource Immuno Assays S.A. - Rue du Bosquet, 2 - B-
1348 Louvain-la-Neuve - Belgium).
2.7.6. Oxidative stress markers estimation
The liver oxidative stress markers were assessed based on the pre-
viously proposed standard procedures for LPO [17], SOD [18], catalase
[19], and GPx [20] and non-enzymatic antioxidant, GSH [21].
2.7.7. Distribution of ACAgNPs in different tissues
The inductively coupled plasma optical emission spectrometry (ICP-
OES) technique was used to detect the bio-distribution of ACAgNPs in
different freeze dried tissues including liver, kidney, heart, brain, lungs,
spleen and testis on the 29th and 89th day. In this assay, 0.5 g tissue
was weighed and digested in 5 mL of ultra-pure nitric acid for 16 h at
90 °C in a water bath followed by the addition of 1 mL hydrogen per-
oxide solution. Finally, the samples were diluted to a known volume
using Milli-Q water and analyzed by ICP-OES. The bio-distribution of
silver in different tissues was expressed as silver quantity/g tissue.
2.7.8. Histopathology
In this procedure, different tissues obtained from kidneys, spleen,
liver, brain, heart, lungs, testes were instantaneously fixed in 10% of
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Materials Science & Engineering C 91 (2018) 372–381
neutral buffered formalin solution. The tissue samples were processed
by using paraffin embedding bath system and thin slices were prepared.
The sections were stained with hematoxylin and eosin (H & E) to
analyze any histopathological changes under a light microscope
(Olympus BX40, Japan).
2.7.9. Statistical analysis
Statistical analysis was done to the all related experiments among
the groups as compared to the control using SPSS software version 20
ANOVA followed Tukey's HSD or by LSD. Student“t” test was used to
analyze the statistical significance between the groups. Significance
difference was set at *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.
3. Results and discussion
The qualitative phytochemical analysis of ACRE showed the pre-
sence of various vital secondary metabolites, such as alkaloids, phenols,
flavonoids, terpenoids and cardiac glycosides. The GC–MS analysis
spectrum revealed that ACRE contains the major constituents of glycine
(N-phenylacetyl) ethyl ester, penta decanoic acid 14-methyl- methyl
ester, 8-octadecanoic acid- methyl ester, isomenthone, 13-hexylox-
acyclotridec 10-ene-2-one, somenthone, and phenol 2 (4 (2-
J.R. Nakkala et al. Materials Science & Engineering C 91 (2018) 372–381

Fig. 4. Fluorescent microscopic images of rhodamine 123 staining (i) untreated Hep2 cells (ii) ACAgNPs treated Hep2 cells (b) Fluorescent microscopic images of PI
staining (i) untreated Hep2 cells (ii) ACAgNPs treated Hep2 cells (c) Fluorescent microscopic images of DAPI staining (i) untreated Hep2 cells (ii) ACAgNPs treated
Hep2 cells.

hydroxyethylamino) 2-quinazolinyl (Supplementary data S1). Our results can be corroborated with the earlier report on the formation
of sphere-shaped AgNPs from the Platanus orientalis leaf extract [23].
The average size of the ACAgNPs was found to be 31.83 nm in DLS
3.1. Characterization of AgNPs
particle size analyzer (Fig. 1c). The particle size in DLS appeared bigger
than TEM, because DLS are measured based on the hydrodynamic
The formation of ACAgNPs was observed at room temperature by a
diameter of the particles and provides an intensity weighted average
change in color of the solution from colorless to yellowish brown by
particle size, whereas size obtained from TEM are constructed on the
adding ACRE to 1 mM silver nitrate solution. The color formation might
diameter of dry particle and gives an average particle size [24]. The
be due to the SPR effect and change of Ag+ ions to Ago by the aqueous
zeta potential value of ACAgNPs was −32.3 mV, which indicated that
extract [22]. The aqueous extract contains various secondary metabo-
the synthesized NPs were more stable in nature (Fig.1d). The zeta po-
lites such as alkaloids, phenols, flavonoids, terpenoids and cardiac
tential values of above ± 30 mV can be considered as stable NPs [25].
glycosides, which might be involved in the reduction and capping of
The functional groups present in ACRE which might have involved in
AgNPs. For the optimization process of NPs formation, silver nitrate
the formation of ACAgNPs were determined using FTIR analysis (Sup-
solution was mixed with aqueous plant extract in different ratios, where
plementary data S2). The spectral bands observed in ACRE at 3378,
in optimum formation of NPs was observed in the 5:1 ratio with the λ
2919, 1660, 1400, 1202 cm−1 indicates phenol OeH, alkyl CeH, car-
max at 421 nm. The absorption intensity increased with increasing re-
bonyl C]O, aromatic CeN and CeO or CeOeC respectively. Vibra-
action time. This may be due to the synthesis or increased agglomera-
tional band shifts of 3324, 3234, 1624 and 1373 observed in ACAgNPs
tion of AgNPs. Broadening of peaks was also observed, which indicated
represents aromatic phenol, amine and carbonyl groups of ACRE that
that the synthesized NPs were in poly dispersed nature (Fig. 1a). The
might have involved in the reduction, capping and stabilization of
TEM image revealed spherical shape of ACAgNPs as shown in Fig. 1b.

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J.R. Nakkala et al. Materials Science & Engineering C 91 (2018) 372–381

Fig. 5. (a) Western blot analysis shows the expression of β-actin, cleaved caspase 8, caspase 9, caspase 3, Lamin and PARP in Hep2 cells treated with various
concentrations of ACAgNPs for 24 h (b) Densitometric analysis of ACAgNPs treated Hep2 cells. Protein levels were quantified using densitometry analysis and
expressed in relative band intensity. Values represent mean ± SD (n = 4). * indicates value significantly different from the control at P ≤ 0.05.

Table 1
Serum biochemical parameters on 29th (before washout) and 89th day (after washout) rats treated with 5 and 10 mg/kg b.w of ACAgNPs as compared to control rats.
Data is expressed as mean ± SD from four rats in each group on the 29th day and two rats in each group on the 89th day (washout period). (AST- Aspartate
aminotransferase, ALT- Alanine transaminase, LDH-Lactate dehydrogenase, ALP- Alkaline phosphatase (ALP), TG- Triglycerides). * specifies a value significantly
different from the control at P ≤ 0.05.
Parameters Control 5 mg/kg 10 mg/kg Control Washout Washout
5 mg/kg 10 g/kg

Glucose 105.8 ± 6.2 102.8 ± 5.8 93.8 ± 4.0 93.7 ± 4.0 88.2 ± 4.8 87.8 ± 3.5
AST 119.5 ± 3.1 147.7 ± 2.9* 164.3 ± 3.5* 122.9 ± 2.6 120.9 ± 2.6 123.9 ± 3.2
ALT 62.5 ± 4.5 82.7 ± 2.4* 89.1 ± 1.7* 64.5 ± 3.0 68.3 ± 2.9 67.3 ± 0.9
LDH 502 ± 18.5 799.3 ± 12.8* 780 ± 23.9* 459.5 ± 18.5 458.8 ± 9.2 467.2 ± 8.8
ALP 281.4 ± 3.8 280.9 ± 13.8 275.5 ± 17 257.0 ± 7.0 243.4 ± 9.4 246.7 ± 13.1
TG 68.8 ± 4.4 59.1 ± 4.4 72.2 ± 2.2 76.1 ± 3.1 66.2 ± 3.9 89.7 ± 1.4
Cholesterol 71.3 ± 3.3 67.8 ± 2.7 60.6 ± 4.8 83.2 ± 4.6 83.0 ± 4.3 92.1 ± 3.6

Table 2
Serum oxidative stress markers, Lipid peroxidase (LPO), Superoxide dismutase (SOD), Catalase (CAT-expressed as μmole of H2O2 degraded/min/mg protein),
Glutathione peroxidase (GPx), Reduced glutathione (GSH) and inflammatory markers like TNF-α – Tumor necrosis factor- α, IL-6 Interleukin-6 of rats treated with 5
and 10 mg/Kg b.w of ACAgNPs as compared to control on day 29th and 89th (Washout period). Data is expressed as mean ± SD from four rats in each group on 29th
day and two rats in each group on 89th day (washout period). *designates value significantly different from the control at P ≤ 0.05.
Parameters Control 5 mg/kg b.w 10 mg/kg b.w Control Washout Washout
5 mg/kg b.w 10 mg/kg

LPO 17.6 ± 0.57 17.0 ± 1.0 16.7 ± 0.7 17.6 ± 0.5 17.2 ± 1.0 17.8 ± 0.7
SOD 29.3 ± 3.9 25.9 ± 2.3 27.4 ± 1.6 29.3 ± 3.9 27.0 ± 3.9 29.1 ± 2.2.
CAT 212.2 ± 22.7 208.1 ± 14.1 197.1 ± 7.4 122 ± 22.7 224.9 ± 11.5 205.7 ± 9.9
GPx 27.6 ± 2.5 25.6 ± 2.0 26.3 ± 2.6 27.6 ± 2.5 25.6 ± 2.5 24.6 ± 2.5
GSH 44.9 ± 1.4 53.9 ± 3.0 52.3 ± 1.7 44.9 ± 1.4 49.8 ± 0.7 44.5 ± 1.7
TNF-α 6.5 ± 0.75 68 ± 0.3* 74.5 ± 1.08* 6.5 ± 0.75 9.5 ± 0.7 9.0 ± 0.7
(pg/ml)
IL-6 77 ± 1.4 213 ± 7.0* 330.5 ± 3.5* 64.5 ± 1.0 70 ± 0.7 71 ± 1.0
(pg/ml)

ACAgNPs. The GC–MS analysis showed the presence of bioorganic
compounds in ACRE such as glycine (N-phenylacetyl) ethyl ester, 13-
hexyloxacyclotridec 10-ene-2-one, somenthone, and phenol 2 (4 (2-
hydroxyethylamino) 2-quinazolinyl, which supports the functional
groups revealed in FTIR spectrum that are involved in the NPs forma-
tion and capping.
3.2. Catalytic activity
The catalytic dye degradation relays on the transfer of electrons
between the donor and the acceptor. However, large differences in
redox potential among the donor and the acceptor could deter the
passage of electrons between them [26,27]. Silver, platinum and gold
NPs acts as an effective catalyst based on the intermediate redox po-
tential value between the donor and the acceptor may aid in the
transfer of electrons, which could cause an electron relay system [28].
In aqueous solution the different organic dyes such as 4-NP, 3-NP, PA or
2,4,6-TNP, CBB, CR, EY, RB, MB, MR, MO, CRR, AO, EBT, and PR
showed absorption maximum respectively at 318 nm, 331 nm, 357 nm,
588 nm, 495 nm, 530 nm, 554 nm, 664 nm, 522 nm, 464 nm, 440 nm,
490 am 545 nm and 426 nm. The dyes absorption maxima was not al-
tered after addition NaBH4, might be due to the presence of high kinetic

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J.R. Nakkala et al. Materials Science & Engineering C 91 (2018) 372–381

Fig. 6. H&E stained histopathological images of rats (a) liver (b) kidney (c) spleen (d) brain (e) heart (f) lungs (H&E × 200), (a) (i) Control (ii) ACAgNPs (5 mg/kg
b.w) (iii) ACAgNPs (10 mg/kg b.w) (iv) Washout ACAgNPs (5 mg/kg b.w) (v) Washout ACAgNPs (10 mg/kg b.w).

barrier between the reciprocally deterring negative ions BH4− and
organic dyes. The 4-NP, 3-NP, PA or 2,4,6-TNP, CBB, CR, EY, RB, MB,
MR, MO, CRR, AO, EBT, and PR organic dyes absorption intensity was
decreased respectively within 8, 9, 16, 5, 10, 8, 3, 12, 15, 3, 6, 60, 16,
and 18 min by the addition of ACAgNPs. The time dependent de-
gradation/reduction of organic pollutant dyes spectral peaks in the
presence and absence of ACAgNPs was showed in supplementary data
S3.
3.3. Anticancer activity
Results obtained from the MTT assay indicated decreased cell via-
bility in all the cancer cell lines treated with ACAgNPs. The IC50 values
of ACAgNPs were found to be 29.5 μg/mL, 99.4 μg/mL and 149.4 μg/
mL for 24 h, while 16.4 μg/mL, 73 μg/mL and 97.2 μg/mL for 48 h
respectively for Hep2, COLO 205 and SH-SY5Y cells (Supplementary
data S4). Based on these results more susceptible Hep2 cells were se-
lected due to their low IC50 values for further studies to assess the mode
of action of ACAgNPs. Cyto-morphological analysis of Hep2 cells
showed distinctive features of cell death, including rounding up of
nuclei and shrinkage of cells following treatment with IC50 concentra-
tions of ACAgNPs (Fig. 2a). Hep2 cells treated with ACAgNPs exhibited
orange colored apoptotic bodies an indication of late apoptosis
(Fig. 2b). Similar observations were reported in a previous study where
Chitosan nanocarrier AgNPs induced late apoptosis in human colon
cancer cells as (HT-29 cells) [29]. A majority of anticancer drugs trig-
gers apoptotic cell death in cancer cells by altering the levels of oxi-
dants/anti-oxidant status. Hence, ROS levels in Hep2 cells treated with
ACAgNPs were detected by DCFH-DA staining. The increased intensity
of green fluorescence was observed in ACAgNPs treated Hep2 cells as

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compared to untreated cells, which suggest increased ROS levels in
treated cells (Fig. 2c).
The spectrofluorometric analysis carried out to quantify ROS levels
in ACAgNPs treated Hep2 cells; there was a 66% increase in the in-
tensity of green fluorescence (Supplementary data S5). In order to as-
sess the oxidative stress mediated damage, the levels of oxidant markers
including LPO and anti-oxidant enzymes- SOD, GSH, GPx, and catalase
were measured in Hep2 cells treated with ACAgNPs. Results indicated
1.2 fold increase of MDA level in Hep2 cells after treatment with
ACAgNPs as compared to control cells. Decreased activity of natural
defense enzymes like SOD, GPx, catalase and depletion of non-enzy-
matic GSH levels were also observed, respectively 2.7, 2.5, 1.1 and 0.53
folds in ACAgNPs treated cells as compared to control (Fig. 3). Similar
observations have been reported earlier in zinc oxide NPs treated
HepG2 cells where induction of apoptosis was mediated through the
alteration of the oxidant/anti-oxidant balance [30].
Interruption in mitochondrial membrane potential (MMP) is one of
the most basic intracellular actions after the stimulation of apoptosis
[31]. It is well known that mitochondria is the main site for production
of ROS inside the cell and any condition causing depletion of anti-
oxidants and excess formation of ROS trigger mitochondrial damage.
The increase in ROS levels has been shown to cause loss of MMP, which
is a main characteristic of apoptosis. In this study, MMP was de-
termined using Rhodamine 123 staining. Rhodamine 123 is a lipophilic
cationic dye which can easily pass into the intact mitochondria and
markedly accumulate in the inner mitochondrial membrane and exhibit
high green fluorescence. ACAgNPs treated Hep2 cells exhibited less
green fluorescence intensity indicating loss of MMP while the untreated
cells exhibited high green fluorescence implying healthy mitochondria
(Fig. 4a). The spectrofluorometric analysis indicated that the ACAgNPs
treated Hep2 cells showed 28% decrease in the fluorescent intensity as
compared to untreated cells (Supplementary data S6). In acute myeloid
leukemia cells, treatment with PVP-coated AgNPs induced generation
of ROS and subsequent loss of MMP [32]. To further investigate the
apoptosis-associated nuclear changes in Hep2 cells treated with
ACAgNPs, PI and DAPI staining was performed to assess changes such
as nuclear condensation and fragmentation. The ACAgNPs treated Hep2
cells showed condensation/fragmentation of nucleus by staining with
PI or DAPI (Fig. 4b–c), indicating apoptotic changes in treated cells and
normal smooth nuclei in untreated control cells.
The induction of apoptosis generally occurs through two major
signaling pathways namely extrinsic and intrinsic that is regulated
through caspase 8 and caspase 9 respectively [33]. The extrinsic
pathway mediated apoptosis is initiated by binding of membrane re-
ceptor to an extracellular ligand such as FAS, which progress to bind
adapter proteins connected death domain (FADD) in the intracellular
receptor part and this death inducing signaling complex (DISC) recruit
procaspase 8 and initiate activation of caspase 8 [34]. Once caspase 8 is
activated, it activates the signaling cascade and consequent cell death
by triggering downstream execution caspases, such as caspase 3 [35].
On the other hand, intrinsic pathway is initiated by the accumulation of
ROS, which in turn lead to failure in continuing the MMP and release of
cytochrome C [36]. The released cytosolic cytochrome C combines with
pro-caspase 9, dATP and apoptotic protease activating factor 1 (Apaf-1)
to form apoptosome complex [37]. The apoptosome complex activates
caspase 3 through the downstream signaling cascade and the activated
caspase 3 cleaves over hundred substrates, including lamins, PARP, and
plentiful DNA associated proteins that identify and triggers signals for
DNA strand break [38]. The active caspase 3 cleaves PARP, and the
cleaved product of PARP is a hallmark of apoptosis [39]. To reveal the
molecular mechanism involved in induction of apoptosis by ACAgNPs
in treated Hep2 cells expression of caspase 8, caspase 9, caspase 3,
lamin and PARP were determined. It was observed that A549 and Hep2
cells showed increased expression of active caspases- 8, 9, and 3, lamin
and PARP with increasing concentration of ACAgNPs (Fig. 5a). Densi-
tometry analysis exhibited fold increase in the expression of active
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Materials Science & Engineering C 91 (2018) 372–381
caspases- 8, 9, 3, lamin and PARP respectively 3.8, 1.9, 4.1, 1.1, and 2.5
after 24 h in Hep2 cells treated with ACAgNPs (45 μg/mL) as compared
to control cells (Fig. 5b). These findings reveal that both intrinsic and
extrinsic apoptotic pathways are involved in cell death of Hep2 cells
when treated with ACAgNPs.
3.4. In-vivo toxicity
The small quantity of silver is commonly nonhazardous [40].
However, the toxicity of AgNPs is most controversial till date due to its
possible risk when used in biological system. It is necessary to evaluate
the in vivo toxicity of green synthesized AgNPs in animal models for
further successful use in the future applications in the field of biology
and medicine.
3.4.1. Body weight
Body weight of the rats treated with ACAgNPs was recorded at
various time intervals during the experimental period. It was observed
that the body weight of treated rats was similar to that of control group
(Supplementary data S7). Similar findings have been reported with
PVP-coated AgNPs and silver ions [41] which showed no significant
changes in body weight.
3.4.2. Biochemical parameters
The serum biochemical parameters such as glucose, inorganic
phosphorous, SGOT, SGPT, ALP, LDH, cholesterol, triglycerides and
anti-oxidant enzymes like LPO, SOD, CAT, GPx, and GSH were analyzed
at the end of the experimental (29th day) and washout period (89th
day). A significant increase in the serum AST, ALT, and LDH levels were
observed in ACAgNPs treated rats on day 29th as compared to control
group rats but no significant difference was observed in between 5 and
10 mg/kg b.w treated group rats. However, increased AST, ALT, and
LDH levels on day 29th were reverted to normal levels after washout
period (89th day) (Table 1). In a previous study, liver markers including
AST, ALT, and ALP levels were found to be negatively altered in rats
treated with 10 mg/kg b.w of Ag/Au nanocomposites [42].
The ACAgNPs treated rats did not show any significant alteration in
other biochemical parameters such as glucose, inorganic phosphorous,
cholesterol, triglycerides, and anti-oxidant enzymes like LPO, SOD,
CAT, GPx, and GSH as compared to control rats (Table 2). Previous
study showed that the GPx, and SOD levels were decreased, while total
anti-oxidant capacity (TAC) levels were positively altered in Wistar rats
administrated (intra peritoneal) with 5 mg/kg b.w of AgNPs [43].
3.4.3. Inflammatory markers
The levels of inflammatory cytokines, TNF–α and IL-6 in ACAgNPs
treated rats were found increased as compared to control rats on day
29. There were no significant differences observed in the levels of
TNF–α between 5 and 10 mg/kg b.w of ACAgNPs treated rats. On the
other hand the IL-6 levels were significantly differed between 5 and
10 mg/kg b.w of ACAgNPs treated rats. However, these levels were
restored to normal after washout period on day 89 (Table 2). Previous
report also indicated alterations in the levels of inflammatory markers
when treated with NPs [44,45].
3.4.4. ICP-OES analysis
ICP-OES analysis was used to detect the distribution of AgNPs in
different vital organs such as liver, kidney, spleen, lungs, heart, testis
and brain at the end of the experimental period on day 29 and at the
end of the washout period on day 89. ACAgNPs were not detected in the
liver, kidney, spleen, lungs, heart, testis and brain in rats treated at
5 mg/kg b.w concentration. On the other hand, at 10 mg/kg b.w con-
centration, ACAgNPs were found in liver and spleen with concentration
of 5.67 μg/g in spleen and 4.98 μg/g in liver (Supplementary data S8).
However, it was found that silver was completely eliminated from the
liver and spleen at the end of washout period. The major silver
J.R. Nakkala et al.
deposition in the liver may be due to its detoxification function in the
biological systems. Previous studies also report that the accumulation of
silver may due to the formation of thiol‑silver complexes in liver be-
cause the sulfur containing groups commonly present in liver, which
have high affinity to silver [46,47].
3.4.5. Histopathology
The rats treated with 5 mg/kg b.w of ACAgNPs exhibited normal
hepatic parenchyma whereas 10 mg/kg b.w treated rats exhibited his-
topathological alterations like moderate degree of cell swelling and
vacuolar degeneration. However, these histopathological alterations
were not observed in rats after washout period. The other vital organs
such as kidney, spleen, lungs, heart, testis and brain did not show any
histopathological changes by treatment with 5 and 10 mg/kg b.w of
ACAgNPs (Fig. 6).
4. Conclusion
The green based silver NPs showed good in vitro catalytic and anti-
cancer activities. The in vivo data suggest that the NPs alter some of the
serum biochemical markers in treated rats on day 29 but the levels were
restored to normal after washout period on day 89. Complete elim-
ination of silver from rats was observed on day 89. This study provides
the preliminary guidance towards the development of multifaceted
applications of green synthesized AgNPs.
Acknowledgements
The authors acknowledge the DST, Government of India, New Delhi,
for financial support in the form of DST-FIST. The authors thank DBT-
IPLS (BUILDER) program for Fluorescence microscope facility and the
Central Instrumentation Facility (CIF), Pondicherry University. The
authors also express sincere thanks to IIT Madras and ILS, Ahmadabad
University for instrument facility. The first author (JR Nakkala) conveys
special thanks to CSIR, India for the financial assistance in the form
junior research fellowship (JRF) and senior research fellowship (SRF).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.msec.2018.05.048.
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