IET Nanobiotechnology

Research Article

One-pot green synthesis and structural ISSN 1751-8741

Received on 2nd November 2017
Revised 12th February 2018
characterisation of silver nanoparticles using Accepted on 6th March 2018
E-First on 1st May 2018
aqueous leaves extract of Carissa carandas: doi: 10.1049/iet-nbt.2017.0261
www.ietdl.org

antioxidant, anticancer and antibacterial

activities
Deepika Singh1 , Vikas Kumar2, Ekta Yadav1, Neha Falls1, Manvendra Singh3, Ujendra Komal4, Amita
Verma5
1Department of Pharmaceutical Science, Faculty of Health Sciences, Sam Higginbottom University of Agriculture, Technology & Sciences,
Allahabad, UP 211007, India
2Natural Product Drug Discovery Laboratory, Department of Pharmaceutical Sciences, Faculty of Health Sciences, Sam Higginbottom University

of Agriculture, Technology & Sciences, Allahabad, UP 211007, India

3Department of Computer Sciences, HMFA Institute of Engineering and Technology, Handia, Allahabad, UP, India
4Department of Mechanical & Industrial Engineering, Indian Institute of Technology, Roorkee, Uttrakhand, India
5Bio-organic & Medicinal Chemistry Research Laboratory, Department of Pharmaceutical Sciences, Faculty of Health Sciences, Sam

Higginbottom University of Agriculture, Technology & Sciences, Allahabad, UP 211007, India

E-mail: deepika_singh1888@rediffmail.com

Abstract: Facile green synthesis of silver nanoparticles (AgNPs) using an aqueous extract of Carissa carandas (C. carandas)
leaves was studied. Fabrication of AgNPs was confirmed by the UV–visible spectroscopy which gives absorption maxima at
420 nm. C. carandas leaves are the rich source of the bioactive molecules, acts as a reducing and stabilising agent in AgNPs,
confirmed by Fourier transforms infrared spectroscopy. The field emission scanning electron microscope revealed the spherical
shape of biosynthesised AgNPs. A distinctive peak of silver at 3 keV was determined by energy dispersive X-ray spectroscopy.
X-ray diffraction showed the facecentred cubic structure of biosynthesised AgNPs and thermal stability was confirmed by the
thermogravimetric analysis. Total flavonoid and total phenolic contents were evaluated in biosynthesised AgNPs.
Biosynthesised AgNPs showed free radical scavenging activities against 2, 2-diphenyl-1-picrylhydrazyl test and ferric reducing
antioxidant power assay. In vitro cytotoxicity against hepatic cell lines (HUH-7) and renal cell lines (HEK-293) were also
assessed. Finally, biosynthesised AgNPs were scrutinised for their antibacterial activity against methicillin-resistant
Staphylococcus aureus, Shigella sonnei, Shigella boydii and Salmonella typhimurium. This study demonstrated the
biofabrication of AgNPs by using C. carandas leaves extract and a potential in vitro biological application as antioxidant,
anticancer and antibacterial agents.

1 Introduction biofabrication of AgNPs using plant extracts of Ziziphus jujuba,

Production of a novel drug delivery system in recent years, using
the fabrication of metal nanoparticles with specific size and surface
morphology has been pulling the researchers in the field of
nanotechnology [1]. Since 18th centuries, metal nanoparticles are
used for medicinal purpose and have fascinated for consideration in
the field of cancer. Among metal nanoparticles, silver nanoparticles
(AgNPs) are one of most popularised nanomaterial because of its
special physico-chemical properties, biocompatible, low toxicity
and biomedical application such as radiotherapy sensitisation
agent, molecular imaging agents, biological markers, antibacterial,
antifungal, antidiabetic, anti-inflammatory and anticancer property
[2]. They are broadly utilised in food and pharmaceutical
industries, e.g. preparation of creams, ointments and gels to treat
the cut, burn and wound [3]. There are diverse physical and
synthetic techniques are available for the preparation of AgNPs
which includes a sol–gel–sol method, chemical reduction,
photochemical reduction and other biological methods.
Biofabrication or phytofabrication of nanoparticles by using
biological method is an alternate and environmentally friendly
approach for the preparation of AgNPs [4]. The use of plant
extracts is possibly beneficial over the microorganisms due to
apparent progress, cell culture preservation and protection. This
method is preferred over other technique, as it toxic free and acts as
natural reducing and capping agent during the biogenesis of AgNPs
[5]. There are few works of the literature are available on the
Hibiscus cannabinus, Phlogacanthus thyrsiformis and Pelargonium
graveolens.
AgNPs are used as antioxidant, antibacterial agent and effective
drug targeted system for the cancer treatment which has currently
gained extensive attention. It was testified that AgNPs using leaves
extract of Artemisia vulgaris had anticancer property against
human epithelial (HeLa) cancer cell line and human breast cancer
(MCF-7) cell line [6]. By Trojan effect, AgNPs could be
transported into the cancerous cell and stop the activity of RNA
topoisomerase enzyme and transcription of the gene through a
mutual contact. The cancerous cell is more delicate to AgNPs
destruction as compared to normal cells [7]. AgNPs size and
surface characteristics are important tools for biomedical
contemplations. AgNPs which are small in size show an effective
penetration power and target cancer cells [8]. The possible
mechanisms of AgNPs have not been completely known yet but
the researchers are trying to find it.
Recently, exploration of natural and chemical antioxidants
headed the selection and authentication of novel antioxidants from
the traditional plants. Synthetic antioxidants produce various side
effects such as allergic, mutagenic and carcinogenic effects. The
antioxidant property in plant extracts is present due to redox
impending of phytoconstituents. Antioxidants play a pivotal role in
scavenging for oxygen, break down into peroxides and deactivating
the free radicals. It is presumed that nanoparticles possess a higher

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© The Institution of Engineering and Technology 2018
Fig. 1  Biosynthesise silver nanoparticle from plant leaves
(a) Leaves of Carissa carandas , (b) Biofabricated silver nanoparticles
content of antioxidant because of adsorption of phytocompounds
from extract directly to the surface of AgNPs [9]. Researchers
focusing on the metallic nanoparticles owing to bacterial
resistance. Silver gained a much consideration among other metals
because of its dynamic property as an antibacterial agent and
generally used in marketed products.
Carissa carandas (C. carandas) Linn. (Karonda) is used as a
therapeutic plant by tribals all over the world and prominent in a
different traditional system of medicine. All parts of the C.
carandas are utilised as a part of folklore medicine [10]. It is an
evergreen deciduous shrub, belong to Apocynaceae family. It is
also knowns as karmada in Hindi and supposes to grow near to the
Himalayas region. Geographically the natural origin of this plant is
Nepal to Afghanistan and also covers some regions of India. It is
distributed all over the hot regions (high temperature) of India and
Srilanka. It is a water scarcity tolerant plant, flourishes well in
tropical and subtropical regions. It has grown in the sandy region
and rocky soils, either grow wild or cultivated in the area of
Punjab. Stem contains a white latex, leaves are conical, long and
green in colour. Fruits are globose and contain seeds, show red
colour after ripening. Flowering occurs in the month of January
and matures in the months of May and June [11]. Traditionally, the
plant has been used in the treatment of scabies, intestinal worms,
pruritus, antiscorbutic, anthelmintic, pain relieving, cancer and
hepatoprotective. Karonda tree is generally grown in the gardens
and agricultural fields [12]. The leaves have triterpenoid compound
as well as tannins, and another isomer of ursolic acid. It also
contains carissic acid, which is responsible for bioreduction of Ag+
ions to AgNPs [13].
C. carandas aqueous extract contains different bioactive
constituents including alkaloids, flavonoids, phenols, tannins,
glycosides, saponins, carbohydrate, proteins and enzymes which
act as a reductant and used as scaffolds during the biofabrication of
AgNPs in the medium [14]. The bioactive molecules act as a strong
antioxidant agent and protect the cell from oxidative stress. Owing
to this property of Carissa carandas AgNPs (CCAgNPs), they are
used in the therapy of cancer, bioimaging, antimicrobial, diabetes,
wound healing, antioxidant and so on.
The aim of this study is to biofabricate and evaluates the AgNPs
utilising aqueous leaves extract of the therapeutic plant, C.
carandas. The cytotoxic effect of bioengineered AgNPs was
evaluated on human hepatic cancer cell lines (HUH-7) and human
renal cancer cell lines (HEK-293) by the MTT assay.
IET Nanobiotechnol., 2018, Vol. 12 Iss. 6, pp. 748-756
© The Institution of Engineering and Technology 2018
2 Material and methods
Silver nitrate was procured from Qualigens fine chemicals,
Mumbai. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) {Sigma-Aldrich
(USA)} and purchased from local suppliers. All other chemicals
were used in the experiment are of analytical grade and used as
such.
2.1 Collection of plant materials
The fresh leaves of C. carandas were collected from the local
region of Mirzapur district as shown in Fig. 1a and kept in a
polythene bag. The leaves were washed with double distilled water
in order to remove extraneous matter and dried under shade for one
month in the absence of sunlight. Then the leaves were grounded
with the help of the blender and kept in the bottle for the further
use.
2.2 Preparation of plant extract
The dried leaves about 10 g were taken into a 500 ml Erlenmeyer
flask which contained 250 ml of distilled water. This solution was
boiling at 80°C with continuous stirring at 200 rpm for 60 min. The
mixture was allowed to cool and filtered through Whatman filter
paper no.1. The filtrate termed as extract and placed in a
refrigerator for further use [15].
2.3 Biosynthesis of AgNPs
To synthesised AgNPs, 80 ml of 1 mM AgNO3 was added to the
20 ml of plant extract in vigorously shaking Erlenmeyer flask at
room temperature. After 60 min, the colour of the solution was
changed from orange to dark brown as shown in Fig. 1b and
indicated the formation of AgNPs. The biofabricated nanoparticles
mixture was subjected to centrifugation in the centrifuge machine
at 12,000 rpm for 10 min. The upper layer of the mixture was
decanted, the precipitate was washed with distilled water two to
three times to remove the particulate matter and dry in an oven.
The AgNPs yield obtained was 63.21% and further used for the
characterisation [16].
2.4 Instrumental characterisation of the AgNPs
2.4.1 UV–visible study: The UV–visible study was achieved
utilising Shimadzu UV-1700 spectrophotometer, working at 1 nm
749
resolution. The samples were suitably diluted with double distilled
water and placed in a quartz cuvette in the range of 200–800 nm.
2.4.2 Fourier transform infrared (FTIR) analysis: FTIR
examination was done in order to determine the biomolecule
presents inside the leaves and nanoparticles. A dried AgNP was
mixed with the potassium bromate pellet to interpret the presence
of functional group on Perkin Elmer FTIR spectrophotometer
which was operated between 4000 and 400 cm−1.
2.4.3 X-ray diffraction (XRD) study: The level of crystallinity of
the bioengineered AgNPs was inspected by XRD study. The
information relating to the X-ray pattern (AgNPs) was achieved by
using PANalytical X pert PRO running (outfitted with a nickel
monochromator) with Cu radiation in the 2θ window of 3°–80°.
2.4.4 Field emission scanning electron microscope (FESEM)
coupled with energy dispersive X-ray spectroscopy
(EDX): FESEM pictures of bioengineered AgNPs were taken to
determine its size and surface morphology. The suspension was put
on the coverslip, dried under vacuum and placed on a FESEM Carl
Zeiss SMT AG (Germany) worked at an accelerated voltage of 20  2.7.1 Preparation of cell culture: Human hepatocellular
kV and filtering in between 2° and 30° for its examination. The
FESEM instrument was attached to the EDX device to determine
the elemental composition of AgNPs.
2.4.5 Transmission electron microscope (TEM): TEM images
revealed the size and shape of biosynthesised AgNPs. A few drops
of a suspension of AgNPs was put on a carbon coated copper grids
worked at the voltage of 200 kV, dried at RT and analysed by
Hitachi TEM instrument.
2.4.6 Thermogravimetric (TG) analysis: Effects of high
temperature on biosynthesised AgNPs were assessed utilising a TG
machine (EXSTAR TG/DTA 6300). For TG examination,
powdered AgNPs (3.0 mg) was set in an alumina container and
heated from 35 to 700°C for 10°C/min under nitrogen climate in a
heating chamber.
2.5 Quantification of bioactive compounds
2.5.1 Total phenolic contents assay: Total phenolic contents of
C. carandas extract (CCE) and AgNPs were measured by utilising
the Folin–Ciocalteu method. In a reaction mixture, 0.1 ml of CCE
and CCAgNPs were blended with 2.8 ml deionised water. This
mixture was blended with 2 ml of 2% sodium carbonate and 0.1 ml
of 0.1 N Folin–Ciocalteu reagents. The reaction mixture was
subjected to incubate for 30 min at RT and the absorbance of the
sample was recorded at 750 min UV/visible spectrophotometer
[17].
2.5.2 Total flavonoid contents assay: Total flavonoid contents
of CCE and AgNPs were measured by utilising the aluminium
chloride colorimetric technique. In this method, 0.5 ml of CCE and
CCAgNPs were blended with 1.5 ml of ethanol, 0.1 ml of 10%
aluminium chloride and 2.8 ml of double distilled water. The blend
was left for 30 min at room temperature and the absorbance was
measured at 415 nm [18].
2.6 In vitro antioxidant assay
2.6.1 DPPH free radical scavenging assay: This activity
utilised to determine the in vitro antioxidant property of silver
nitrate, plant extract and AgNPs. Different dilutions of samples
were prepared and 1 ml (0.1 mM) solution of DPPH which was
previously dissolved in methanol was added to the samples. The
samples were mixed vigorously and subjected to incubate for 30 
min. 100 μl of methanol (without extracts) mixed with DPPH
solution to prepare a control. The absorbance was noted at 517 nm
by using a UV spectrophotometer and compared with a positive
reference (ascorbic acid). The percentage of inhibition was
calculated by using the following formula [19]:
750
ODc − ODs
% inhibition = × 100,
ODc
where ODc is the absorbance of control and ODs is the absorbance
of sample.
2.6.2 Ferric reducing antioxidant power (FRAP) assay: This
assay was done to determine the in vitro antioxidant activity of
plant extract, silver nitrate and AgNPs of the various
concentrations. This activity was assayed by the reduction of a
ferric 2, 4, 6-tripyridyl-triazine complex (Fe3+-TPTZ) to the
ferrous form (Fe2+-TPTZ). In the reaction mixture, 2.5 ml of TPTZ
solution (10 mM TPTZ in 40 mM HCl) and FeCl3 (20 mM) in 25 
ml of acetate buffer (0.3 M and pH 3.6), was mixed with 0.5 ml of
the different test samples. In the same way, control was prepared
without added an extract. All samples were subjected to incubation
at 37°C for 30 min, the optical density was noted at 593 nm and
compared with a positive reference [19].
2.7 In vitro cytotoxic activity
carcinoma (HUH-7) and human kidney (HEK-293) cell lines were
procured from National centre for cell science (NCCS), Pune,
India. Cells were kept in DMEM (Dulbecco's Altered Bird
Medium) and 10% foetal bovine serum, 100 units/ml penicillin,
100 mg/ml streptomycin, 0.14% sodium bicarbonate and 0.1 mM
sodium pyruvate. Cells were developed in an incubator in a CO2
atmosphere at room temperature and 95% humidity.
2.7.2 Study of anticancer property: MTT reagents were utilised
to measure the in vitro anticancer effect of bioactive AgNPs of the
leaves of C. carandas on HUH-7 and HEK-293 cell lines. MTT
reduces to form blue formazan by mitochondrial dehydrogenase,
which demonstrates mitochondrial activities and subsequently cell
growth. 3 × 103 cells per well were seeded in 96 well plates in 100 
ml cell culture medium and allowed to incubate for 24 h. After 24 
h cells were treated with different dilution of the biofabricated
AgNPs. After this, 5 mg/ml of MTT reagent was mixed with a
fresh medium into test, and control wells and again subjected to an
incubator for 4 h. MTT was dissolved in DMSO and formed
formazon products. The absorbance of a mixture was recorded at
540 nm against blank and calculated as [20]
X
% of cell inhibition = Y − × 100,
Y
where Y is the absorbance of the control (untreated cells) and X is
the mean absorbance of treated cells with AgNPs.
2.8 Antibacterial activity of AgNPs: Biofabricated AgNPs were
examined by the agar well-diffusion technique against various
pathogenic bacteria (methicillin-resistant Staphylococcus aureus
(MRSA), Shigella boydii (S. boydii), Shigella sonnei (S. sonnei)
and Salmonella typhimurium (S. typhimurium). The experimental
bacteria were kept on the nutrient broth medium (3.0 g beef extract,
5.0 g/l peptone and a final pH of 6.8 ± 0.2; Sigma-Aldrich,
Germany) at room temperature for 48 h. The microscopic
organisms were homogeneously swapped onto singular plates by
utilising the sterile cotton swabs. Wells of 3 mm breadth were
drawn on Mueller–Hinton agar (3.0 g beef infusion, 1.5 g starch,
17.5 g peptone, 17.0 g/l agar; pH 7.4 ± 0.2) by the utilising stopper
borer. AgNP (80 μl) was transferred to each well, and three
antibiotics, which serve as a control (gentamicin, ampicillin and
neomycin) were utilised as controls. After a 24 h period of
incubation, the inhibition zones (mm) were noted [21].
3 Result and discussion
3.1 Structural characterisation by absorbance spectroscopy
The UV–visible spectrum of the synthesised AgNPs was delineated
in Fig. 2. The absorption spectrum depicts the formation of AgNPs,
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formed by silver nitrate and the chemical constituents found in the
leaves of C. carandas [22].
It was concluded from Fig. 2 that AgNPs possess free electrons,
which show mutual vibration in reverberation with the light due to
surface plasmon resonance. The sample has been obtained from a
plant source contains a different type of phytocompounds. It is an
essential instrument which determines synthesised nanoparticle
Fig. 2  UV–visible spectrum of AgNPs
Fig. 3  FTIR spectra
(a) C. carandas leaves, (b) AgNPs
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because the valence of silver from Ag1+ in AgNO3 modifying to
Ag0 in the prepared nanoparticles [23]. The formation of metal
nanoparticles involved the features of the nucleated particles. The
other organic compounds which are present in the aqueous extract
interact with silver ions and peak move to the shorter wavelength.
With the time, the absorption peak shifted to higher wavelength
and showed absorbance maxima at 420 nm. A comparable
absorption spectrum at 433 nm was envisaged during
biofabrication of AgNPs utilising Zizyphus xylopyrus bark extract
leaf extract [24].
3.2 FTIR spectroscopy
FTIR analysis of CCAgNPs shows the possible association
between silver and the bioactive moieties involved in the formation
and the stability of AgNPs. The spectra reveals the intense peak at
3426, 2905, 1610, 1444, 1381, 776, 343 cm−1 in AgNPs and 3370,
2960, 1620, 1444, 1317, 777, 409, 342 cm−1 in plant extract.
Figs. 3a and b show the FTIR spectrum of CCE and CCAgNPs.
The vibration band found at 3426 cm−1 in CCAgNPs spectra
assigned the OH stretching of the phenolic group, a similar result
was also obtained during biosynthesis of AgNPs of Helicteres
isora root extract [25]. In the same case, the vibration bands at
2905 cm−1 assigned to the stretching vibration of aliphatic C–H
group, 1610 cm−1 attributed to the bending vibrations modes of
aromatic ring C=C [26], 1444 cm−1 due to aliphatic C–H group,
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Fig. 4  XRD diffraction pattern of AgNPs
Fig. 5  TG analysis of AgNPs
1381 cm−1 due to aromatic stretching of C–N (the peptide bonds of
proteins) [27], 776 cm−1 denoted to bending of the aromatic
carbon–hydrogen bond, respectively [28], observed in the past
reports.
From the FTIR study, it can be demonstrated that the functional
groups show the unique features of phenols, aromatic amine and
carbonyl groups of CCE. As reported by reference, carbonyl
groups and hydroxyl groups in phenols, have a noteworthy
capability to chelate silver ions and permits them to mount and
prevent the AgNPs against accumulating in solution [29]. The
shifting of peaks up and down reveals the synthesis of the AgNP.
The characteristic features of the biomolecule in CCE are
responsible for the formation and stabilisation of AgNPs [30].
3.3 XRD analysis
XRD analysis was performed to determine the geometric structure
and gapping between atoms of AgNPs. Fig. 4 delineates the XRD
pattern of the incorporated AgNPs by the leaves of CC. It was
suggested from the results that crystalline planes indicate the peaks
of AgNPs. It shows main characteristic peaks for silver, which are
found at 38.12°, 44.18°, 64.53°, 77.48°, 81.07° and might be
corresponding with (111), (200), (220) and (311) alluded from the
JCPDS card no. 040783. Subsequently, AgNPs suggests the face-
centred cubic (FCC) structure which is same as silver structure.
The comparable crystallographic result was developed during
biosynthesis of AgNPs using the seed extract of Cydonia oblong
[31].
752
3.4 TG analysis
A TG graph of CCAgNPs was represented in Fig. 5. The stepwise
decomposition process of AgNPs is shown in the graph. The TG
was used to study the thermal stability of AgNPs which start to
degrade at about 225°C. There was a steady weight loss was found
at 700°C. The weight loss up to 250°C due to the evaporation of
the solvent molecule (water moisture) present on the surface of
nanoparticles [32]. The weight loss from 250 to 700°C represents
the loss of carbohydrate and protein decomposition present in the
nanoparticles. The total loss of weight of AgNPs up to 700°C was
97.41% [33]. It suggests that biomolecules are present in the plant
extract act as a capping agent for the reduction of silver ion and
stable the AgNPs in the solution.
3.5 FESEM with EDX analysis
FESEM provide images of samples at the submicron level and
provide higher resolution pictures. The images were distinct and
spherical in shape. The size of AgNP ranged between 28 and 60 
nm (Figs. 6a and b). Biosynthesised AgNPs using the aqueous seed
extract of night jasmine yielded obtained nanoparticles in the size
of 50–80 nm [34].
The qualitative and quantitative elemental detection of AgNPs
was confirmed by using EDX. It shows a higher amount of silver
content at 3 keV which confirms the synthesis of AgNPs (Fig. 6b).
The biofabrication of AgNPs using Saraca indica leaf extract
demonstrated similar result for EDX at the 3 keV [35]. The high
peaks in the region showed various valence conditions of Ag in the
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© The Institution of Engineering and Technology 2018

Fig. 6  FESEM with EDX
(a), (b) FESEM images of AgNPs, (c) EDX spectrum
AgNPs. The peaks represent other elements are normally
distinguished in the specimens gathered on a carbon-coated copper
grid or might be the presence of bioactive in the leaves extract [36].
3.6 TEM analysis
TEM examination provides data about the size and development of
AgNPs. This technique is used to decide the morphology and the
exact size of synthesised AgNPs. The images verified that
morphology of fabricated AgNPs irregular and polydispersed in
nature (Figs. 7a and b) [37]. Moteriya et al. [38] reported irregular
and polydispersed appearance during biofabrication of AgNPs
using Cassia roxburghii leaf extract.
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Fig. 7  TEM
(a), (b) TEM images of AgNPs
3.7 Total flavonoid and total phenolic contents
It was observed that high concentration of flavonoid contents and
low concentration of phenolic contents in the extract of CC when
compared to CCAgNPs as represented in Figs. 8a and b. The
antioxidant property of the plant is due to the presence of total
flavonoid and phenolic contents [39]. The flavonoid and phenols of
CC extract reduced the silver nitrate solution to nanoparticles.
3.8 In vitro antioxidant activity
The antioxidant property of CCE and AgNPs was assessed by
DPPH free radical scavenging tests. The outcomes of antioxidant
property were represented in Fig. 8c. DPPH is a steady and very
strong free radical for assessment of the antioxidant property of
any therapeutic agents [40]. The antioxidant activity was observed
to be higher in AgNPs when contrasted with CCE. AgNPs
demonstrated most elevated level than CCE against DPPH.
A comparable effect was found in the FRAP test for AgNPs and
plant extract. Reduction in the levels of FRAP assay was observed
in both test samples as same happen in DPPH-scavenging
activities, at the concentration of 100 and 150 µg/ml. As appeared
in Fig. 8d, the reduced level of AgNPs and CCE reversed
(increased) with increasing serial dilution (>150 µg/ml). Ascorbic
acid obtained the highest reducing power when compared to both
test samples.
Reactive oxygen species (ROSs) are a chemically reactive
substance which contains oxygen. ROSs are used for cell signalling
and homeostasis which is by-products of metabolism of oxygen.
Due to oxidative stress, there is a destruction of cell structure. The
antioxidants component guards the cells against the impeding
properties of ROS. The DPPH radical scavenging activity of green
synthesised AgNPs might be attributed to their capability to give
electrons to counteract the unsteady DPPH free radicals during
reaction [41]. The principle of FRAP analysis is grounded on the
capability of antioxidants to reduce Fe3+ to Fe2+ by using TPTZ to
give blue Fe2+-TPTZ compound at 593 nm [42]. In agreement with
our results, Mahendran and Kumari testified FRAP assay of
AgNPs from Nothapodytes nimmoniana (Graham) Mabb. fruit
extracts [43]. The result of the DPPH assay and the FRAP assay
753
Fig. 8  Phytochemical assay and antioxidant activity
(a) Total phenolic content of AgNPs, (b) Total flavonoid content, (c) DPPH free scavenging radical activity, (d) FRAP assay

Table 1 MTT assay of CCAgNPs on HuH-7 and HEK-293

cell lines
Concentration, % Cell inhibition
µg/ml AgNPs Silver AgNPs Silver
(HUH-7 nitrate (HEK-293 nitrate
cell line) (HUH-7) cell line) (HEK-293)
50 82.34 46.79 79.21 40.23
30 76.47 38.28 69.89 33.78
25 67.29 24.12 52.45 20.38
20 34.98 13.24 28.78 11.90
10 12.57 5.42 9.56 3.67
control 0 0 0 0
Fig. 9  In vitro cytotoxic activity of AgNPs

demonstrated the antioxidant potential of AgNPs when compared
to ascorbic acid (standard). The influential DPPH free radicals
activity and FRAP assay of biosynthesised AgNPs accepted
previously supported our outcomes in the current investigation.
3.9 Cytotoxic impact of AgNPs
The anti-proliferative impact of AgNPs was appraised against
HUH-7 and HEK-293 cell lines, by utilising MTT test. The
reactions are exhibited as the percent cell inhibition, at varying
dilutions of CCAgNPs and silver nitrate from 10 to 50 µg/ml for
24 h, and compared with control. CCAgNPs displayed powerful
anti-proliferative activity against both cell lines. It is obvious from
Figs. 9a and b and Table 1 that levels of % cell inhibition of
HUH-7 and HEK-293 cell lines were elevated as there was an
increment in the concentration of AgNPs. The IC50 cell inhibition
of CCAgNPs against HUH-7 and HEK-293 cell lines was found at
10.29 and 22.14 µg/ml. The IC50 cell inhibition of silver nitrate
against HUH-7 and HEK-293 cell lines was found at 57.32 and
46.60 µg/ml. The results of in vitro anticancer activity for HUH-7
and HEK-293 were found different, as variation in concentration.
From the consequences, the cancer cell lines reveal higher
sensitivity to anticancer drugs. The debilitated survival cell lines
demonstrate a potential cytotoxic impact of CCAgNPs.
The potential application of biosynthesised AgNPs seems to be
tremendously favourable due to their pivotal role as an
antiangiogenic agent. Kim and Choi [44] explored the underlying
mechanism of anticancer activity of biosynthesised AgNPs on
human cell lines. They specified that AgNPs were engrossed by a
different process such as pinocytosis, endocytosis and phagocytosis
on human cells. AgNPs interact with cellular materials of the cell,
destructs DNA of the cell and leads to cell death. In other reports, it
was suggested that AgNPs which implicates commotion of the
mitochondrial respiratory chain and disruption of ATP synthesis
and cause DNA damage [45].
The dose-dependent enhancement was found on cell inhibition
after CCAgNPs treatment. The exploration revealed that the cell
inhibition of cell lines increased with increasing concentration (10–
50 μg/ml) of CCAgNPs after a time duration of 24 h. This study
approved the dose graded tactic to the assessed toxicity of AgNPs
on the HUH-7 and HEK-293 cell lines. The cancer cell lines were
treated with CCAgNPs at varying concentrations shows significant
morphological alteration (unique characteristics of apoptotic cells),
such as disruption of cell membrane integrity, shrinkage of the cell
and low cell concentration. Biosynthesised AgNPs using
methanolic leaves extract of Euphorbia helioscopia were found to
be effective against human epithelial cells (HeLa) and L929 cells
[46].

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© The Institution of Engineering and Technology 2018
Table 2 Antibacterial activity of AgNPs against various
types of bacteria
Bacteria Tetracycline Ampicillin Neomycin AgNPs
Inhibition zone,
mm
MRSA 18 ± 1.62 0.0 ± 0.0 7 ± 0.1 22 ± 0.9
S. sonnei 15 ± 1.01 6 ± 0.2 12 ± 0.62 20 ± 0.16
S. boydii 21 ± 0.9 22 ± 0.61 19 ± 0.21 25 ± 0.34
S. typhimurium 18 ± 0.6 25 ± 0.28 26 ± 0.31 28 ± 0.71
The lower IC50 values of HuH-7 and HEK-293 represent the
prospect of CCAgNPs to be a potential anti-proliferative agent. It
was concluded from MTT assay that, data of mortality acquired
from the present research displays the incidences of dead cells are
notably more than in cancer cell lines and synthesised AgNPs kills
both types of cells, even though, the degree of destroying cells was
higher in cancer cell lines. The detected outcomes unquestionably
favoured the implication that CCAgNPs provide new expectation
as anticancer agents.
3.10 Antimicrobial activity of AgNPs
From long times, silver metals and silver derived compounds have
been used as antibacterial agents and used to store water to provide
silver elements in the body. We investigated biosynthesised AgNPs
as potential antimicrobial agents. It was used to test the
antimicrobial activities towards gram-positive as well as gram-
negative bacteria displaying inhibition zones. On the basis of the
inhibition zone results, AgNPs displayed significant antimicrobial
activity against MRSA (22 ± 0.9 mm), S. sonnei (20 ± 0.16 mm), S.
boydii (25 ± 0.34 mm) and S. typhimurium (28 ± 0.71 mm) as
compared to standard drugs. The consequences of antibacterial
activities of biofabricated AgNPs appraised by the agar well-
diffusion technique are portrayed in Table 2.
This excellent capability of the biofabricated AgNPs using
aqueous leaves extract of C. carandas will be key apprehension for
the purpose of clinical and environmental protection. The
antimicrobial activity of AgNPs on gram-negative as well as gram-
positive bacteria may be substantial importance. The incubation
period of the pathogenic microorganisms with biosynthesised
AgNPs was observed to lower the colony forming bacteria. The
inhibition rate was also raised after treating with AgNPs and
reduction was observed in MRSA, S. sonnei, S. boydii and S.
typhimurium, respectively. The improved inhibition of AgNPs
because of its capability to bind the peptidoglycan content presents
in the cell wall of bacteria and disrupt the membrane of bacteria. It
forms pits in the membrane which cause leakage of cellular matter
in the culture media [47]. The destruction produced by AgNPs
caused inhibition of respiratory chain dehydrogenases and leads to
cell death. When AgNPs comes in the contact of the cell, it affects
the protein and phospholipid content that leads to shrinkage,
putrefaction and cell death [48].
Several approaches have been suggested to enlighten the
inhibitory effects of AgNPs on bacteria. It is also presumed that
silver shows a high affinity towards sulphur and phosphorus, which
is the main component for the antibacterial effects. The bacterial
cell membrane contains a sulphur contain proteins in large quantity,
AgNPs reacts with sulphur-containing proteins present in the cell
and affects the viability of cells [49]. It was also proposed that
AgNPs released silver ions in the bacterial cell and interact with
the phosphors molecule of DNA and inactivate the replication of
DNA. It also interacts with sulphur-containing amino acids and
inhibits the functions of enzymes and resulting in the death of
bacterial cell [50].
4 Conclusion
Cancer kills near about seven million people every year worldwide.
In present times, reliable and potent drugs are available from a
plant source which are non-toxic, economic, fast and eco-friendly.
Facile green synthesis of AgNPs shows a significant effect against
IET Nanobiotechnol., 2018, Vol. 12 Iss. 6, pp. 748-756
© The Institution of Engineering and Technology 2018
bacteria and cancerous cells and could be used as a pioneer drug in
future. This investigation may offer an innovative opportunity for
the survey of traditional plants for the fabrication of AgNPs to
discover a therapeutic anticancer, antioxidant and antimicrobial
agent. This study demonstrates the biosynthesis of AgNPs utilising
the aqueous extract of leaves of C. carandas. It can be concluded
from the study that it can be explored for it potential effect in the
field of pharmaceutical and biomedical applications.
5 Acknowledgments
The authors were highly thankful to SAIF, Punjab University, and
Chandigarh for providing analysis of all characterisation, Maratha
Mandal Dental College, Belgaum for carried out a cytotoxic
activity and Shalom Institute of Health and Allied Sciences, Sam
Higginbottom University of Agriculture, Technology and Sciences,
Allahabad (UP) for providing research facilities.
DS, MS, NF, UK, and EY performed the experimental study.
VK and AV analysed the biochemical parameters. All the authors
contributed to the proofreading.
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