Artificial Cells, Nanomedicine, and Biotechnology

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Lantana camara Linn root extract-mediated gold

nanoparticles and their in vitro antioxidant and
cytotoxic potentials

Rajendiran Ramkumar, Govindasamy Balasubramani, Ramalingam Karthik

Raja, Manickam Raja, Raji Govindan, Easwaradas Kreedapathy Girija &
Pachiappan Perumal

To cite this article: Rajendiran Ramkumar, Govindasamy Balasubramani, Ramalingam Karthik

Raja, Manickam Raja, Raji Govindan, Easwaradas Kreedapathy Girija & Pachiappan Perumal
(2017) Lantana�camara Linn root extract-mediated gold nanoparticles and their in�vitro antioxidant
and cytotoxic potentials , Artificial Cells, Nanomedicine, and Biotechnology, 45:4, 748-757, DOI:
10.1080/21691401.2016.1276923

To link to this article: https://doi.org/10.1080/21691401.2016.1276923

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ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY, 2017
VOL. 45, NO. 4, 748–757
http://dx.doi.org/10.1080/21691401.2016.1276923

Lantana camara Linn root extract-mediated gold nanoparticles and their in vitro
antioxidant and cytotoxic potentials
Rajendiran Ramkumara, Govindasamy Balasubramanib, Ramalingam Karthik Rajab, Manickam Rajab,
Raji Govindanc, Easwaradas Kreedapathy Girijac and Pachiappan Perumalb
a
Department of Biotechnology, Padmavani Arts and Science College for Women, Salem, India; bDepartment of Biotechnology, School of
Biosciences, Periyar University, Salem, India; cDepartment of Physics, School of Physical Sciences, Periyar University, Salem, India

ABSTRACT ARTICLE HISTORY

The Lantana camara Linn root extract derived gold nanoparticles (Au NPs) were characterized by Received 28 September 2016
Ultraviolet-Visible spectroscopy, X-ray diffraction, fourier transform-infrared, high resolution transmission Revised 2 December 2016
electron microscopy, selected area electron diffraction pattern and energy dispersive X-ray analyses. In Accepted 8 December 2016
DPPH assay, the inhibitory concentration (IC50) of Au NPs and gallic acid was 24.17 and 5.39 lg/ml,
whereas, for cytotoxicity assay, the IC50 of Au NPs was 17.72 and 32.98 lg/ml on MBA-MB-231 and Vero KEYWORDS
cells, respectively. Thus, the Au NPs possess significant in vitro antioxidant and cytotoxic properties Lantana camara; Au NPs;
which could be considered as potential alternate for the development of anticancer drug in future. HR-TEM; DPPH; MDA-MB-
231

Introduction available with nontoxic nature that could replenish the

Nanobiotechnology is a dynamic field of research in materials
science. Nanotechnology has contributed in several applica-
tions, such as therapeutics (Liu et al. 2010), catalysis (Shin
et al. 2009), biosensing devices (Zhou et al. 2011), biomedi-
cine due to its ready manufacturing process (Ragaseema
et al. 2012), quality control, biocompatibility, and indeed its
use in wound healing and antibacterial applications are
already part of clinical practice (Fong and Wood 2006). Of
late, applications of nanoparticles are improving due to their
enriched properties on specific characteristics viz., size, distri-
bution, and morphology. The metal oxide nanoparticles have
been applied for cancer therapy, hyperthermia, drug delivery,
tissue repair, cell labeling, targeting, and immune assays,
detoxification of biological fluids, magnetic resonance imag-
ing, and magnetically responsive drug delivery therapy
(Huang et al. 2007, Iv et al. 2015, Khlebtsov and Dykman
2011). Among all metals, gold nanoparticles (Au NPs) are
being used for tumor detection, angiogenesis, genetic disease
and genetic disorder diagnosis, photo-imaging, and photo-
thermal therapy (Kumar et al. 2016a). Also, gold has lower
toxicity than other metallic nanoparticles, in which, problems
curtailing toward the specificity of targeting the tumor cells
must be fixed, prior the introduction of Au NPs directly to
the clinic. Plant-mediated NPs synthesis has advantages,
including biocompatibility, scalability, and the medical applic-
ability of synthesizing NPs using the universal solvent (water),
as a reducing medium (Noruzi 2015). Plants are readily
demand raised for NPs with suitable applications in the bio-
medical systems (Velusamy et al. 2016). Lantana camara Linn
is an aromatic shrub belonging to the family Verbenaceae,
and is native to tropical America, which was introduced as an
ornamental and hedge plant in India (Sharma & Sharma
1989). Traditionally, it was used to treat bronchitis and arterial
hypertension (Rastogi and Mehrotra 1995), cold, headache,
uterine hemorrhage, chicken pox, eye injuries, whooping
cough, and asthma (Ross 1999). The roots are used for the
treatment of malaria, rheumatism, and skin rashes (Taoubi
et al. 1997). The powdered root of L. camara in milk was
given to children for stomach-ache, as a vermifuge (Kalita
et al. 2012) and also for the treatment of tooth-ache (Kirtikar
and Basu 2006) antipyretic, antispasmodic (Saxena et al.
2012), and antimalarial agent (Weenen et al. 1990). Lantana
oil is used for the treatment of skin itches, antiseptic for
wounds, leprosy, and scabies (Ashish Saraf et al. 2011). The
characteristic features of the root are sweet and bitter tasting,
refrigerant, antifebrile (Kumarasamyraja et al. 2012). Also, sev-
eral triterpenoids, naphthoquinones, flavonoids, alkaloids, and
glycosides isolated from this plant are known to exert diverse
biological activities (Ghodake et al. 2013). Further, it was con-
sidered as an effective biological agent with multiple poten-
tials. Cancer is the most important cause of mortality in the
world. Many cancers initially respond to chemotherapy, but
later develop resistance (Johnston 1997). Currently, available
chemo-preventives and chemotherapeutic agents cause

CONTACT Rajendiran Ramkumar raam.kmr@gmail.com Department of Biotechnology, Padmavani Arts and Science College for Women, Salem, Tamil Nadu,
636011, India
ß 2017 Informa UK Limited, trading as Taylor & Francis Group

undesirable side effects (Brown 2002) therefore, developing a
biocompatible and cost-effective method of treatment for
cancer is indispensable. Breast cancer is the second leading
cause of cancer death among women in the USA. Every year,
the Times of India estimates 1, 00, 000–1, 25, 000 new breast
cancer cases in India (12 October 2012), and this statistical
survey is projected to an increase in double by 2025. In 2016,
an estimated 1,685 210 new cases of cancer will be diagnosed
in the USA and 595 690 people will die from the disease
(https://www.cancer.gov/about-cancer/understanding/statis-
tics). And also the estimated deaths and new cases among
different age groups of women were 138, 370, and 739 980,
respectively (American Cancer Society 2016). The mortality
rate of patients and the must for cancer therapy coerces the
need for technological breakthrough in terms of easy avail-
ability, cost effectiveness, and safety in terms of side effects.
The existing cytotoxic agents used for the breast cancer treat-
ment are found to be expensive and inefficient because they
induce severe side effects due to their toxicity in non-cancer-
ous tissues (Kim et al. 2007, Yeruva et al. 2008). Therefore, it
is of urgent need to develop novel therapeutic agents that
are biocompatible and cost-effective (Franco-Molina et al.
2010). Though, there have been plenty of reports on synthe-
sis of nanoparticles from L. camara Linn by several research-
ers (Ajitha et al. 2015; Dash et al. 2015; Kumar et al. 2016a, b,
c; Kamarasamyraja and Jaganathan 2013; Mavukkandy et al.
2016; Sivakumar et al. 2012; Thirumurugan et al. 2011), this is
the first attempt to synthesize Au NPs using the root aqueous
extract of L. camara Linn to evaluate its antioxidant as well as
cytotoxic effect on human breast cancer cell line (MDA-MB-
231) by MTT assay, and the possible mechanism for cell death
was addressed through acridine orange and ethidium brom-
ide (AO/EB) dual staining and DNA fragmentation assays.
Materials and methods
Chloroauric acid (HAuCl4) was procured from ACROS,
Belgium, and 3–(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-
tetrazolium bromide (MTT), a yellow tetrazole, was obtained
from Invitrogen, St. Louis, MO. The acridine orange, ethidium
bromide, and all other fine chemicals were obtained from
Sigma–Aldrich, St. Louis, USA. L. camara Linn with healthy
roots were collected from the Periyar University campus
(11.7186
N, 78.0772
E), Salem, India. And the roots were
subjected to nanoparticle synthesis.
Preparation of root extract for nanoparticles
biosynthesis
The roots of the collected healthy plants of L. camara were
excised and washed under running tap water followed by
distilled water until no debris remained. Further, the roots
were chopped and surface sterilized using 70% ethanol, 0.1%
mercuric chloride, and Tween 20 of each at 5 min of interval
followed by a wash with distilled water. The roots were
shadow dried at room temperature and annihilated with a
sterile electrical blender in order to obtain a powdered form.
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 749
The powdered samples were stored in a brown bottle and
protected from exposure to sunlight until further use.
Lantana camara root aqueous broth preparation and
synthesis of Au NPs
Two gram of root fine powder was mixed with 100 ml of deion-
ized water and boiled for 30 min, cooled, and filtered through
Whatman filter paper No. 1. From this, 40 ml of L. camara root
aqueous broth was supplemented with 60 ml of 1 mM HAuCl4
solution, and the mixture was placed in orbital shaker at room
temperature, for the bioreduction of Au3þ to Au0 (Singh et al.
2016a). The dark violet color formed after the addition of
HAuCl4 was characterized in the range 200–700 nm using
Cyberlab UV-100, Spectrophotometer operated at a resolution
of 1 nm. The bio-reduction of Au0 was monitored periodically
by measuring the absorbance of the solutions. The overall opti-
mized reaction condition was observed in 1 mM HAuCl4 solu-
tion with neutral pH. The Au NPs obtained from the reaction
mixture were purified by repeated centrifugation at 2000 rpm
for 10 min followed by dispersion of the pellet thrice in deion-
ized water to remove the water-soluble biomolecules such as
proteins and secondary metabolites. The water-suspended NPs
were dried at 30
C overnight and then kept under vacuum for
24 h to dry the NPs (Jayaseelan et al. 2013).
Characterization of gold nanoparticles (Au NPs)
The water-suspended NPs were subjected to centrifugation at
15 000 g for 10 min, and the resulted pellet was dissolved in
deionized water and filtered through Millipore filter (0.45 mm).
An aliquot of this filtrate containing Au NPs was used for X-
Ray Diffraction (XRD), Fourier transform infrared (FTIR) spec-
troscopy, high resolution transmission electron microscopy
(HRTEM), and energy dispersive X-ray Spectroscopy (EDX) anal-
yses. Dried and powdered NPs were used for XRD analysis. The
spectra were recorded under Rigaku Miniflex II, Japan advance
X-ray diffractometer. Characterization involved FTIR analysis of
the synthesized Au NPs by scanning in the range
400–4000 cm 1 at a resolution of 4 cm 1. These measure-
ments were carried out on a Perkin Elmer spectrum in the dif-
fuse reflectance. The surface morphology of NPs was
examined with the JEOL JEM 2100 high-resolution transmis-
sion electron microscope. The purity and the chemical com-
position of the Au NPs were analyzed by EDX spectroscopy an
inbuilt of JEOL JEM 2100 (Balasubramani et al. 2015a).
In vitro antioxidant assay
DPPH free radical scavenging assay
The DPPH free radical scavenging assay was performed by
modifying the early prescribed method (Balasubramani et al.
2015b). One ml of 0.1 mM DPPH (in methanol) was added to
different concentrations (20, 40, 60, 80, & 100 lg/ml) of gallic
acid, Au NPs, and root aqueous extract of L. camara. The reac-
tion mixtures were stirred well and incubated in the dark for
30 min. The absorbance at 517 nm was measured against a
blank (methanol). Gallic acid was used as the standard, and
750 R. RAMKUMAR ET AL.
the control was prepared as described above without a sam-
ple. The lower absorbance of the reaction mixture indicated a
higher percentage of scavenging activity. The percentage (%)
of inhibition or scavenging of free radicals was determined by
the following formula: % inhibition ¼ (Abscontrol  Abstest/
Abscontrol)  100. The inhibitory concentration (IC50) of the
tested samples was calculated using the software Graph Pad
Prism 6.
In vitro cytotoxicity assay
Cell culture
The MDA-MB-231 cells were purchased from National Centre
for Cell Sciences (NCCS), Pune (India). The cells were stored in
liquid nitrogen. The frozen vials were thawed by gentle agita-
tion in a 37
C water bath immediately after being taken out
from the liquid nitrogen tank. Cells were grown in RPMI 1640
medium containing penicillin (110 IU/ml), streptomycin
(100 lg/ml), and 2 mM glutamine and supplemented with
10% fetal bovine serum. The cells were routinely cultured in
50 cm2 plastic culture flasks at 37
C in a humidified atmos-
phere with 5% CO2 and 95% air. The medium was refreshed
every 24 h, and cells were trypsinized (0.1% trypsin) on reach-
ing 80% confluency (Zhang et al. 2014).
MTT assay
To determine the cytotoxic effect of Au NPs, cell viability
study was done with the conventional MTT-reduction assay
with slight modifications (Kotha et al. 2006). Briefly, MDA-MB-
231 cells were seeded in a 96-well plate at the density of
5  103 cells/well. The cells were allowed to attach and were
grown in a 96-well plate for 24 h, in 200 ml of RPMI with 10%
FBS. Further, media was removed and replaced with suspen-
sion of various concentrations of Au NPs viz., 0.01, 0.1, 1, 10,
and 100 mg/ml (minimum three wells were seeded with each
concentration). Equal concentrations of L. camara root extract
were used as positive control, and the cells were incubated
for 48 h. After the addition of MTT (10 ml, 5 mg/ml), the cells
were incubated at 37
C for another 4 h. Optical density of
the formazan product was read at 495 nm using scanning
multi well spectrophotometer. The results were given as
mean of three independent experiments.
Acridine orange/ethidium bromide dual staining
Acridine orange/ethidium bromide (AO/EB) dual staining was
carried out to detect the morphological evidence of apop-
tosis in Au NPs-treated cells. Twenty-five microliters of treated
and untreated cell suspension (5  106 cells/ml) was stained
with 1 ml of acridine orange and ethidium bromide dye mix
(100 mg/ml of acridine orange and ethidium bromide pre-
pared in PBS separately) (Winnicka et al. 2007). Then the sam-
ples were examined under fluorescent microscopy (Nikon
Eclipse TS 100).
Statistical analyses
The data analyses were done using the SPSS Statistical
Software Package version 20.0. All data were expressed as
mean ± SD. One-way analysis of variance (ANOVA) was per-
formed with Tukey’s HSD test for the in vitro cytotoxic effects
of Au NPs. Two-way ANOVA was performed with Bonferroni’s
test by comparing the DPPH scavenging ability of Au NPs
versus root extract and gallic acid. In all analyses, a probabil-
ity level of P < 0.05 was used as the significance of differen-
ces between values. The difference between two groups was
considered to be significant when the “P” value was less than
0.05 and highly significant when the “P” value was less than
0.01 or 0.001.
Results
The boiled root of L. camara retained pale yellow color after
30 min. The reaction was rapid, when the colored root broth
was mixed with 1 mM HAuCl4 and the mixture was turned
into dark purple color within 10 min (Figure 1). The span of
overall bio-reduction reaction was within 2 h, in which the
Au3þ reduced into Au
ions in optimum condition, and the
formation of Au NPs was confirmed. The UV-visible spectra of
synthesized Au NPs showed the strong surface plasmon res-
onance corresponds to the peak at 552 nm, during different
time intervals of bio-reduction (Figure 2). The exact nature
and size of the Au NPs formed were studied through XRD
analysis. Figure 3 shows strong and narrow diffraction peaks
indicated that the product have well crystalline. The XRD
peaks at 38
, 44
, 64
, and 77
can be indexed to the (1 1 1),
(2 0 0), (2 2 0), and (3 1 1) Bragg’s reflections of cubic struc-
ture of metallic gold respectively and the crystallinity of the
Au NPs was confirmed Joint Committee on Powder
Diffraction Standards (JCPDS no. 04–0784). The broadening of
Bragg’s peaks indicates the formation of NPs. Nearly mono-
dispersed Au NPs with controllable size and uniform shape
can be easily obtained in the simple aqueous reduction
method. The mean size of Au NPs was calculated using the
Debye–Scherrer’s equation by determining the full width half
maxima of the (1 1 1) Bragg’s reflection. The intense IR bands
are observed at 3408.49 cm 1 is due to N–H and O–H
stretching. A peak was observed around 2920 and 2853 cm 1
that could be assigned to the C–H stretching vibration of
alkyl and aldehyde groups. The bands observed at 1607 cm 1
assigned to the stretching vibrations of the C ¼ C, respectively
(Figure 4). The band located at 1258 and 1063 cm 1 was due
to C-N stretching of aromatic amines and aliphatic amines.
The typical peak at 619 cm 1 was observed in functionalized
Au NPs pattern close to 600 cm 1, which signify the presence
of R–CH. The functional groups N–H and O–H and C ¼ C, the
biomolecule present in the plant extract, facilitated the reduc-
tion of Au ions to Au NPs. Thus FT-IR analysis clearly shows
that bio-molecules present in plant’s root aqueous extract
which could be responsible for the formation of Au NPs.
Representative HRTEM images were recorded at a magnifica-
tion of 10 000 revealed the size, shape, and distribution of
synthesized NPs shown in (Figure 5(a,b)). The high-resolution
study of the nanoparticles using HRTEM predominates with
spherical morphologies with an average size of around
11–32 nm. Most of the nanoparticles were roughly circular
with smooth edges and few were triangle with sharp edges.
The nanoparticles obtained are highly crystalline as shown in

Figure 1. L. camara Linn root extract treated with HAuCl4 resulted Au NPs (dark purple).
Figure 2. UV-visible spectra of Au NPs synthesized by L. camara root extract.
Figure (5c) by clear lattice fringes and the upper inset pattern
of SAED confirmed the crystalline nature of synthesized Au
NPs through bright circular Bragg’s rings corresponding to
their reflections. Figure (5d) shows the spot-profile EDX of Au
NPs showed strong signals for gold atoms along with weak
signals from carbon and oxygen. These weak signals could
have been arisen from X-ray emission from macromolecules
like proteins/enzymes bound to the NPs or in the vicinity of
the particles.
The DPPH radical scavenging activities of root aqueous
extract and Au NPs increased gradually in a dose-dependent
manner. The significant differences among the treated con-
centrations and the tested samples (Au NPs vs. root extract
and gallic acid) were observed. The percentage inhibition of
the aqueous root extract, Au NPs, and the control (Gallic
acid) was represented in Figure 6. The root aqueous extract,
Au NPs, and gallic acid showed 67.06, 88.95, and 95.60% of
scavenging effect at the sample concentration of 100 lg/ml,
respectively. The inhibitory concentrations (IC50) of root aque-
ous extract, Au NPs, and Gallic acid were found to be:
60.58 ± 0.13, 24.17 ± 0.07, and 5.39 ± 0.16 lg/ml, respectively.
Thus, the root extract-mediated Au NPs possess considerable
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 751
Figure 3. X-ray diffraction patterns of synthesized Au NPs.
scavenging activity, when compared to gallic acid. The cyto-
toxicity assay was carried out with different concentrations of
Au NPs which resulted in the decreased percentage of
growth on breast cancer cell line (MDA-MB-231) and normal
cell line (Vero). The L. camara Au NPs exhibited significant
inhibition in MDA-MB-231 and normal Vero cell line at a
dose-dependent manner. The percentage growth of both the
cells was gradually decreased in the increased concentration
of Au NPs. The significant differences were found, among all
different treated concentrations of Au NPs on MDA-MB-231
(F ¼ 494.765; df ¼10, 22; P < 0.05) and Vero cells (F ¼ 281.710;
df ¼10, 22; P < 0.05), respectively (Figure 7). The growth
inhibitory rate (IC50) of synthesized Au NPs against human
breast cancer (MDA-MB-231) cells and normal Vero cells was
found to be 17.72 lg/ml and 32.98 lg/ml after 48 h of incuba-
tion. An Apoptotic morphological change caused by Au NPs
was studied using acridine orange/ethidium bromide differen-
tial staining method. The stained cells were characterized to
viable (light green), early apoptotic (bright green fluorescence
and condensed chromatin), late apoptotic (orange
752 R. RAMKUMAR ET AL.
fluorescence), and nonviable cells (red-colored fluorescence)
(Figure 8). The Au NPs-treated cells showed condensed
nuclei, membrane blebbing, and apoptotic bodies. In con-
trast, the control cells showed intact nuclear architecture.
However, very few apoptotic bodies were noticed in HAuCl4-
treated cells. The biologically synthesized Au NPs induced cell
Figure 4. FT-IR spectrum of synthesized Au NPs of L. camara root.
Figure 5. HR-TEM of Au NPs synthesized from root extract of L. camara. (a) and (b) the shapes and different sizes of Au NPs; (c) EDX analysis of Au NPs of L. camara;
(d) SAED pattern showing that the nanoparticles are crystalline.
death via apoptosis; DNA laddering assay was performed on
agarose gel. A clear fragmented DNA ladder was observed in
Au NPs-treated MDA-MB- 231 cells whereas HAuCl4-treated
cells did not show such clear fragmented DNA ladder
(Figure 9). In addition, the untreated (control) cells did not
show any prominent DNA ladder on the agarose gel.
Therefore, the data obtained from this study confirm that Au
NPs induced cell death through apoptosis.
Discussion
The weed, L. camara Linn, has various medicinal
applications which were contributed with earlier synthesized
nanoparticles by several researchers (;;; Ajitha et al. 2015;
Dash et al 2015; Kumar et al. 2016a, b, c; Mavukkandy et al.
2016; Kamarasamyraja and Jaganathan 2013; Sivakumar et al.
2012; Thirumurugan et al. 2011). Their results described about
multi-potentials of L. camara which opened up its use in bio-
medical applications. Therefore, the present study focused on
the in vitro antioxidant and cytotoxic effects of Au NPs syn-
thesized from root aqueous extract. L. camara on human
breast cancer (MDA-MB-231) and normal (Vero) cells.
 ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 753

Recently, researchers have successfully synthesized the Au suggested the use of medicinal plants as resources. Similarly,
NPs using the root extract from the medicinal herbal plant the present findings utilized the L. camara’s root aqueous
Panax ginseng (Singh et al. 2015; Singh et al. 2016a) which extract for the Au NPs synthesis by their medicinal value

Figure 6. The DPPH scavenging assay of root extract, Au NPs, and Gallic acid.
DPPH scavenging ability of Au NPs vs. root extract and ascorbic acid. Values are mean
of independent determinations. Two-way ANOVA, significant different from Au NPs:
“” p < 0.001. Figure 7. Percentage growth curve of MDA-MB-231 against the Au NPs.

Figure 8. Cytotoxicity (a) Untreated, (b) treated MDA-MB-231 cells at 48 h, (c) Live cells (AO-Et-Br staining), and (d) damaged cells.
754 R. RAMKUMAR ET AL.
Figure 9. DNA fragmentation assay.
Where, M, Marker; UT, untreated cells at 48 h; T- MDA-MB-231 cells exposed with GNPs.
inculcated earlier. The bio-reduction of Au3þ to Au0 was
achieved within 10 min after adding HAuCl4 solution. Au NPs
are known to exhibit a purplish color in the reaction mixture
which is due to the excitation of the surface plasmon vibra-
tions (SPR). The colloidal Au NPs showed intense color due to
SPR arising from combined oscillation of electrons induced
by an electromagnetic field (Toderas et al. 2009). Generally,
the synthesized Au NPs are confirmed by the terminated
ruby red within 10 min as reported earlier by Sheny et al.
(2011). In our present study, the optimum time required for
the completion of reaction was rapid, i.e., in 8 min, which
supports early finding (Jayaseelan et al. 2013) for the pre-con-
firmation of Au NPs. As the earlier report of Mittal et al.
(2013), the present study supports for the bio-reduction of
metal ion into base metal which was quite rapid, readily, and
easily scaled up. The spectrophotometric wavelength ranges
of 500–550 nm are normally used in characterizing the Au
NPs (Karuppaiya et al. 2013). The peak of absorption spec-
trum at 552 nm was quite clear like that of the earlier find-
ings of several researchers, which could be attributed to the
blue-shifted peak range of Au about 560 nm (Aromal and
Philip 2012; Subramaniam et al. 2016). Au NPs synthesized
from root extract of L. camara showed three prominent
Bragg reflections viz., (111), (200), and (220) that were analo-
gous on the basis of the face-centered cubic nature of Au
(Punuri et al. 2012) with Joint Committee on Powder
Diffraction Standards (JCPDS no. 04–0784), which indicates
the formation of nanoparticles. From the result of selected
area energy-dispersive study, the upper inset pattern of syn-
thesized Au NPs accompanied the earlier findings of Philip
(2009) that endorses the crystalline nature of gold through
circular rings analogous to the Bragg’s reflections. Plant
extracts may act both as reducing agents and stabilizing
agents during the synthesis of nanoparticles (Kumar and
Yadav 2009). The findings of several researchers have demon-
strated the functional groups that act in capping, bioreduc-
tion, and stabilization of NPs. Presently, the Au NPs
synthesized from L. camara root extract containing biomole-
cules, which have been highlighted that the stabilization of
inert surfaces, might be due to the electrostatic barriers on
the surfaces of NPs as reported earlier by El-Batal et al. (2013)
and Murugan et al. (2015). The different functional groups
with their respective stretches obtained in the presently syn-
thesized Au NPs are in line with the previous findings of
Varshney et al. (2010). Recent studies have shown that bio-
molecules such as protein, phenol, and flavonoids present in
the plant extract play an important role in the reduction of
metals ions and capping of the nanoparticles (Mittal et al.
2013). The size ranges (between 11 and 32 nm) of Au NPs
obtained were similar to the earlier report of Ghosh et al.
2011 from Dioscorea bulbifera, and the observed differences
in size of NPs could be due to the reduction of ions to gold
being formed at different times (Rajasekhar et al. 2012). The
size of the NPs may be smaller at higher concentration of
extract owing to the phytochemicals (present in the extract)
that can effectively stabilize the NPs (Paul et al. 2013). The
different size and shape might occur due to the aggregates
of leaf aqueous extract. The EDX attachment on the HRTEM
provided elemental composition analysis in view as well as
spot analyses of minute particles and thus confirms the pres-
ence of specific elements. Figure (5a) is a representative plot
of the spot EDX analysis in which 98.73% signals the gold
(Au) and the rest is oxygen (O). The HRTEM-EDX analysis dis-
played the well-known signature spectra for gold and thus
convincingly evidenced the presence of this noble metal gold
(Au). These results are in accordance with other reports on
the EDX analysis of gold structures synthesized using extracts
derived from Diospyros kaki (Song et al. 2009). The SAED pat-
terns revealed that the particles are crystalline in nature. The
fringe spacing for the colloids are correspond to the spacing
between the (111) plane of face-centered cubic (fcc) nature
of gold. The SAED pattern shows the diffraction ring from
inner to outer which can be indexed as (111), (200), and
(220) reflections, respectively of fcc gold. The earlier report of
Philip (2009) has interestingly noted that the particles with
deviated fringe spacing are not almost in contact and they
are separated from each other with invariable distance as
obtained in this present SAED pattern. In DPPH assays, lower
IC50 values showed higher free radical scavenging activity. Au
NPs showed significant free radical scavenging activity
(IC50–24.17) against DPPH radicals, over the standard antioxi-
dant gallic acid (IC50–5.39) which has higher inhibitory effects
(95.60%). The root aqueous extract comprises a noticeable
amount of hydrogen donor molecules, which might reduce
the free radicals in DPPH scavenging assays as in agreement
with earlier report (Murugan et al. 2016). Also, the scavenging
ability might endorsed by the phenolic contents in plants
probably due to their redox potentials, which could act as
reducing agents and singlet oxygen quenchers (Al-Shmgani
et al. 2016, Saravanakumar et al. 2016). The IC50 values
obtained by the several researchers in earlier for the Au NPs
on MDA-MB-231 cells were 50 and 56.65 lg/ml (Adeyemi and
Whiteley 2015, Krishnaraj et al. 2014). In this line, the pres-
ently synthesized Au NPs revealed the IC50 value of 17.72 lg/
mL on MDA-MB-231 cells which was 2-fold decrease in con-
centration. The cytotoxic effect of Au NPs could be exhibited
due to the physical and chemical interactions of gold ions
with functional groups of intracellular components viz., pro-
teins, nitrogen bases, and phosphate groups (Moteriya and
Chanda 2016). Of late, Singh et al. (2016b) have been

reported that the biologically synthesized nanoparticles are
quite potent and highly preferable in terms of effectiveness
as compared with physiochemically synthesized nanopar-
ticles. Therefore, the L. camara-mediated Au NPs might also
considered being a significant bio-controlling agent for can-
cer cells. Apoptosis of the Au NPs-treated cells was escorted
by a fall in the percentage of cells in G0/G1 phase and a raise
in the percentage of G2/M phase cells, representing cell cycle
arrest at G2/M (Lee et al. 2011). The ROS can act as signal
molecules promoting cell cycle progression and can induce
oxidative DNA damage (Hu et al. 2009). DNA fragmentation is
broadly considered as a characteristic feature of apoptosis
(Allen et al. 1997). Induction of apoptosis can be confirmed
by two factors such as irregular reduction in size of cells, in
which the cells are reduced and shrunken, and lastly DNA
fragmentation. The present study, therefore, illustrated the
anticancer property of L. camara-mediated Au NPs, which
may be employed as a drug in nanomedicine to treat
breast cancer in future. In breast cancer, nanoparticle-based
therapies are paving the way in diagnosis, imaging, screen-
ing, and anti-proliferation. But the challenges met in the
field of pharmacology, toxicology, immunology, large-scale
manufacturing, and regulatory issues were disadvantaged
severely in translating such advances from the bench to the
bedside. However the presently synthesized Au NPs might
act as a considerable biological agent on breast cancer
treatment.
Conclusions
To sum up the present investigations, the in vitro antioxidant
and cytotoxic properties of Au NPs synthesized from root
extract of L. camara were confirmed. Stable and spherical-
shaped Au NPs have been achieved from a simple, economic,
nontoxic, and efficient green method. The crystalline nature
of NPs is confirmed from bright rings in the SAED pattern,
clear lattice fringes in the HRTEM images and peaks in the
XRD pattern. From FT-IR spectrum, it is found that biomole-
cules responsible for capping and stabilization of Au NPs are
flavonoids. Au NPs-treated cells exhibited dose-dependent
cell death and membrane leakage. From the MTT assay, the
synthesized Au NPs exhibited preponderant activity on
human breast cancer (MDA-MB-231) cells and normal Vero
cells. Au NPs also document the induction of apoptosis and
fragmentation of DNA. Finally, the chance of using Au NPs
synthesized from root aqueous extract to inhibit the growth
and their cytotoxicity for potential therapeutic treatments can
bids a new way to combat cancer diseases.
Acknowledgements
The authors are grateful to their host institutes for providing necessary
facilities to carry out this work.
Disclosure statement
All the authors declare that they have no conflict of interests including
any financial, personal, or other relationships with other people or
organizations.
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 755
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