Name:Alina Naeem

3 year Pharm-D
rd

(Institute of pharmaceutical
sciences),Peoples university of
medical and health sciences
Nawabshah.
Topic:Anti-Cancer effect of
silver nano particles

Mail for certificate

an837063@gmail.com
 Links of articles

 Https://onlinelibrary.wiley.com/doi/epdf/10.1002/fsn3.1328
 https://www.sciencedirect.com/science/article/pii/S075333221630395X
 https://innovareacademics.in/journals/index.php/ajpcr/article/download/16027/10
054
 https://www.researchgate.net/profile/Wafa_Abdel-
Fattah/publication/328111631_On_the_anti-
cancer_activities_of_silver_nanoparticles/links/5c57e95e299bf12be3fa93ab/On-the-
anti-cancer-activities-of-silver-nanoparticles.pdf?origin=publication_detail
 https://www.researchgate.net/profile/Valentin_Bhimba/publication/230896020_In_vi
tro_anticancer_activity_of_silver_nanoparticles_synthesized_using_the_extract_of_G
elidiella_sp/links/0fcfd505d581854b1d000000/In-vitro-anticancer-activity-of-silver-
nanoparticles-synthesized-using-the-extract-of-Gelidiella-
sp.pdf?origin=publication_detail
 Topics of articles with author names

 Anticancer activity of silver nano particles from panax ginseng fresh leaves
in human cancer cells.(Veronica Castro Aceituno,Sungeum Ahn and
other)
 Study of anticancer, antimicrobial, immunomodulatory, and silver nanoparticles
production by Sidr honey from three different source(Hameed A.Ghramh,Essa H
Ibrahim,Mona kilany)
 ANTIMICROBIAL AND ANTICANCER ACTIVITY OF SILVER NANOPARTICLES
FROM EDIBLE MUSHROOM (Priya Darshni and Mahalingum)
 INVITRO ANTICANCER ACTIVITY OF SILVER NANOPARTICLES SYNTHESIZED
USING THE EXTRACT OF GELIDIELLA Sp (Valentin Bhimba and Krupa
vatnam)
 On the Anti-Cancer Activities of Silver Nanoparticles (wafa Ismail Abdel
Fattah and Wafaa Gareib)
 Summary:
Nano- particles

Nanoparticles is currently most important researched topic in nano technology.Nanotechnology plays a

vital role in over coming many disease i.e bacterial disease, wound healing ,burns ,cancer
etc.Nanoparticles can be taken from many living organism like bacteria ,fungi,seaweeds,mushrooms,
honey and others plants. The method By which nanoparticles taken from livings called green synthesis.
Many chemicals and physical methods are also used to produce nanoparticles but these metods
are expensive and dangerous to environment.
Silver Nanoparticles:
Silver Nanoparticles produce by the reduction of silver atoms.Silver Nanoparticles having size of 1-
100nm.silver Nanoparticles having particular functions regarding toxicity ,surface plasmon resonance
and electrical resistance. They also act as antimicrobial agent in wound dressings,Anticancer
agents,electronic devices and water treatments.They are also use to detect tumours
Method of extraction

Let we get silver Nanoparticles from honey

 HONEYY SAMPLE COLLECTION AND PREPARATION
Collect honey sample and label them and store them at 4oC.Dilute It with
20% distilled water and use freshly.
 BIOSYNTHESISOF SILVER NANOPARTICLES
. Honey sample is used for biosynthesis of silver nanoparticles.
We take 1ml 2% honey add to 99ml 1mM AgNO3 solution in a Erlenmeyer
flask. The pH of mixture was raised until the color change occurred.
Anti-Cancer activity of silver nano
particles.

Nanoparticles used to detect the cancer cells and eliminate or kill them before causing
tumours. So they are using for diagnostic and treatment .They are also used to carry drugs
at Target .They act at the cellwall of cancer cells they also disrupt membrane integrity ,
disrupt normal cell functions.They show anti-proliferative effect on cancer cell lines.
By generating free oxygen radicals silver Nanoparticles induce the apoptotic
pathway in vitro by this way they show anti cancer effect .because of this apoptotic
pathway silver nano particles cause programmed cell death of cancer cells of different
tissues of human body.They also damage cell DNA.They show anticancer effect on
different cell lines such as colon carcinoma,breast carcinoma,breast carcinoma,liver
carcinoma,intestinal etc.
Thank You
Received: 17 September 2019 | Revised: 30 October 2019 | Accepted: 4 November 2019

DOI: 10.1002/fsn3.1328

ORIGINAL RESEARCH

Study of anticancer, antimicrobial, immunomodulatory, and

silver nanoparticles production by Sidr honey from three
different sources

Hamed A. Ghramh1,2,3 | Essam H. Ibrahim1,3,4 | Mona Kilany1,5

1
Research Center for Advanced Materials
Science (RCAMS), King Khalid University, Abstract
Abha, Saudi Arabia Sidr honey is used as food and medicine in many countries. Study of immunomodula-
2
Unit of Bee Research and Honey
tory and anticancer activity of Sidr honey did not tested before. The aim of this work
Production, Faculty of Science, King Khalid
University, Abha, Saudi Arabia was to study the anticancer activity and immunomodulatory as well as antimicrobial
3
Biology Department, Faculty of Science, potential of Sidr honey and its synthesized silver nanoparticles (AgNPs). Sidr honey
King Khalid University, Abha, Saudi Arabia
4
from three sources (two from Kingdom of Saudi Arabia (KSA) and one from Pakistan)
Blood Products Quality Control
and Research Department, National was diluted to 20% and tested for its biological activities and to synthesize AgNPs.
Organization for Research and Control of The results demonstrated that honeys could produce AgNPs (spherical shape), modu-
Biologicals, Cairo, Egypt
5 lated the growth of normal splenic cells, and have antimicrobial activities. Sidr honey
Department of Microbiology, National
Organization for Drug Control and Research has anticancer activity against HepG2 but not Hela cells. Sidr honey can be used as
(NODCAR), Giza, Egypt
antimicrobial agent, but can be used as anticancer agent with care as it stimulated cell
Correspondence growth of some lines (e.g., Hala) and inhibited another (e.g., HepG2).
Essam H. Ibrahim, Research Center for
Advanced Materials Science (RCAMS), King
KEYWORDS
Khalid University, P.O. Box 9004, Abha
61413, Saudi Arabia. AgNPs, anticancer, antimicrobial, Apis mellifera, Sidr honey, splenic cells
Email: essamebrahim@hotmail.com

Funding information
King Khalid University, Grant/Award
Number: RCAMS/KKU/011-19

1 | I NTRO D U C TI O N
In the market, there are wide varieties of honey (e.g., Manuka, Pasture,
Jelly bush, Sidr [Ziziphus spina-christi], Sumra, and Jungle) available,
and these varieties are due to components gathered from different
botanical sources. In reality, honey was used not only as food, but
also as a traditional medicine and also has other several uses. Honey
can be defined as the natural sweet material produced by an insect
(bee) called Apis mellifera after collection of nectar of plants and
other sources, and combining with specific materials produced by the
bee. This finally produced material is deposited, dehydrated, stored,
and left in the honeycomb to ripen and mature. Bees forage different
plants in the same trip, and as a result, honey is always a mixture of
different sources; therefore, no honey is completely similar to an-
other honey (Nouvian, Hotier, Claudianos, Giurfa, & Reinhard, 2015).
Generally, honey contains about 80% carbohydrates (35% glucose,
40% fructose, and 5% sucrose) and the rest (20%) is water with some
other active biomolecules (e.g., amino acids, vitamins, minerals, en-
zymes, organic acids, flavonoids, and phenolic compounds) (Finola,
Lasagno, & Marioli, 2007; Yücel & Sultanoǧlu, 2013).
Health benefits of honey depend on its quality and purity de-
rived from the collected natural substances. Monofloral honey is

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
© 2019 The Authors. Food Science & Nutrition published by Wiley Periodicals, Inc.

Food Sci Nutr. 2020;8:445–455.  www.foodscience-nutrition.com | 445

446 | GHRAMH et al.
defined as that type of honey which has a high value in the mar-
ketplace due to its distinctive flavor and other attributes resulted
being predominantly from the nectar related to one plant species
(Cotte, Casabianca, Chardon, Lheritier, & Grenier-Loustalot, 2004).
Sidr monofloral honey is found in the desert areas of Yemen, Saudi
Arabia, and Pakistan's Potohar region (Al-Waili, Salom, Butler, & Al
Ghamdi, 2011).
Honey has the power to kill microorganisms, and this power
is attributed to the high osmolarity and pH, hydrogen peroxide,
as well as the phytochemical nature of honey (Molan, 2015). The
antimicrobial potential depends on several factors like the type
of honey, geographical location, and the botanical nature (Jull
et al., 2015). It was reported that honey has an inhibitory effect
against about 62 species of bacteria (aerobes and anaerobes, gram
positives and negatives) (Hussain et al., 2015; Patton, Barrett,
Brennan, & Moran, 2006). Sidr honey is widely used as a medica-
tion to treat liver diseases, ulcers of the stomach, lung infections,
malnutrition consequences, digestion problems, constipation, in-
fections of eyes, infections following burns, wounds and surgery,
and general health and vitality. Sidr honey is known to have a
strong antioxidant and antibacterial activities (Alandejani, Marsan,
Ferris, Slinger, & Chan, 2009). Saudi market has numerous honey
kinds (produced locally and imported). Some of them are used as
folk medicine.
Cancer is one of the major scaring diseases to human. Treatment
using chemotherapy is the widely used approaches to treat, but
long-term use of this technique may lead to drug resistance. Some
workers (Ma, Dong, & Ji, 2010; Sarkar, Banerjee, & Li, 2007) re-
ported resistance to anticancer agents such as including doxorubi-
cin, camptothecin, cisplatin, 5-fluorouracil, and taxol. Because of this
resistance and bad side effects of chemotherapeutic agents, search
for safer and effective drugs is mandatory. Honey has several bioac-
tive molecules such as caffeic acid, caffeic acid phenethyl ester, and
flavonoid glycons which have been shown to have inhibitory effects
on tumor cell division (Rao et al., 1993). Honey was reported to have
a moderate antitumor and antimetastatic effects in tumors of some
strains of mouse and rat (Gribel' & Pashinskiĭ, 1990). Bee honey was
shown to inhibit bladder cancer (Swellam et al., 2003) and potenti-
ate the antitumor effects of chemotherapeutic drugs (Saunders &
Wallace, 2010).
Nobel metal nanoparticles such as gold and silver got an high level
of interest because of their multipurpose applications in several fields
like biology, medicine, industry, etc. (Yokoyama & Welchons, 2007).
The physiochemical characteristics of silver nanoparticles (AgNPs)
made it point of interest for many researchers (Sharma, Yngard, &
Lin, 2009). Nanoparticles can be prepared chemically and physically
(Hanžić, Jurkin, Maksimović, & Gotić, 2015; Maleki, Simchi, Imani, &
Costa, 2012; Okitsu, Yue, Tanabe, Matsumoto, & Yobiko, 2001), but
green synthesis using plants (He et al., 2018; Kumar & Yadav, 2009;
Makarov et al., 2014), yeast, bacteria, and fungi (Singh, Kim, Zhang,
& Yang, 2016) now more used because these methods are nontoxic,
clean, and eco-friendly. AgNPs have many biological properties such as
anticancer, antimicrobial, antifungal, antiviral, anti-inflammatory (He et
al., 2018; Jeyaraj et al., 2013; Monteiro et al., 2012; Wong & Liu, 2010;
Zhang, Liu, Shen, & Gurunathan, 2016), anti-parasite (Marimuthu
et al., 2012), and insecticidal potentials (Moorthi, Balasubramanian,
& Mohan, 2015). In addition, silver nanoparticles have been used in
industry like in paints, detergents (Gottesman et al., 2011), clothing
(Perelshtein et al., 2008), and pharmaceutical preparations (Martinez-
Gutierrez et al., 2010). Preparation of nanoparticles using plant extract
is valuable due to the ease of preparation methods and with low bio-
hazardous contents (He et al., 2017).
In this work, we tried to study the immunomodulatory and anti-
cancer activity of Sidr honey that did not tested before. The power
of the Sider honey to synthesize nanoparticles and the antimicrobial
activity were studied too. The results showed that the three types of
Sidr honey have anticancer activity and immunomodulatory poten-
tials as well as antimicrobial potential. Sidr honey could synthesize
silver nanoparticles (AgNPs).
2 | M ATE R I A L S A N D M E TH O DS
2.1 | Honey samples collection and preparation
In this study, 3 honey samples were collected and categorized as
shown in Table 1. Samples were labeled and stored at 4°C till used
in biological activity studies. Honey samples were diluted at 20% in
distilled water and used fresh every time when used.
2.2 | Biosynthesis of silver nanoparticles
Honey samples were used to synthesis silver nanoparticles (AgNPs)
following the method shown by Ghramh, Al-Ghamdi, Mahyoub, and
Ibrahim (2018). In brief, 1 ml 20% honey was added to 99 ml 1 mM
AgNO3 solution in an Erlenmeyer flask. The pH of the mixture was
raised until the color change occurred.
2.3 | Characterization
All characterization methods for AgNPs prepared by honey samples
and active biomolecules found in honey samples before and after
the synthesis of AgNPs were done following the same methods and
instruments described by Ibrahim, Kilany, Ghramh, Khan, and ul
Islam (2018).
TA B L E 1 Codes and types of collected honey samples
Sample code Honey type Honey bee species
H1 Sider (Rijal Ulma, Apis mellifera
Saudi Arabia) jemenitica
H2 Sider (Rijal Ulma, Apis florea
Saudi Arabia)
H3 Sider (Pakistani) Apis mellifera ligustica
GHRAMH et al. | 447

2.4 | Antimicrobial potential test
Well diffusion assay was adopted according to Kilany (2016) using
gram-positive bacteria (Bacillus subtilis), gram-negative bacteria
(Escherichia coli and Pseudomonas aeruginosa), and the fungal strain
Candida albicans as a model of fungus. The 6-mm wells were asepti-
cally bored into agar, and 40 µl of honey or honey containing AgNPs
from each sample was aseptically pipetted into the wells. Penicillin
(10 µg) was used as positive control.
at density of 4 × 10 4/ml was prepared according to Algarni et al.
(2019). A 100 μl culture media containing 20% honey or 20% honey
containing AgNPs were added to 100 μl of the cell suspension (4,000
cells/well). Control untreated cell culture was included. Plates con-
taining the cells were incubated for 72 hr at 37°C in 5% CO2 (CO2
Incubator, Memmert, Gmbh). Number of viable cells was measured
using Vybrant® MTT Cell Proliferation Assay Kit (Thermo Fisher
Scientific) (Ibrahim et al., 2018).

2.6 | Anticancer activity test

2.5 | Effects of different honey samples on normal
rat splenic cell proliferation
Adult male Sprague Dawley rat weighing 239 g was kindly given by
the animal house at King Khalid University. Single-cell suspension
The cell lines HepG2 and Hela were used to test the anticancer po-
tential of honey and honey containing AgNPs. The cells were main-
tained and grown in supplemented minimal essential medium (MEM),
fetal calf serum (10%), penicillin/streptomycin (100 U/ml/100 μg/ml),

F I G U R E 1 Silver nanoparticle
synthesis by 20% Sidr honey. Where A, C,
and E stand for color absorbance of H1,
H2, and H3 alone, respectively; B, D, and
F stand for color absorbance of H1, H2,
and H3, respectively, after the synthesis
of AgNPs; i, iii, and v stand for color of
H1, H2, and H3, respectively, and ii, iv,
and vi stand for color of H1, H2, and H3,
respectively, after the synthesis of AgNPs
448 | GHRAMH et al.

and l-glutamine (2 mM) in the CO2 incubator. After reaching conflu-

ency, cells were trypsinized (2% trypsin-EDTA) to prepare single-cell
suspension. Single-cell suspension was adjusted to 1 × 105/ml, and
then 100 µl (10 4 cells) was plated into each well of 96-well plate and
incubated overnight in the CO2 incubator. The medium in the plate
was decanted and 200 µl media containing 20% honey and 20%
honey containing AgNPs. The plate was incubated for an additional
24 hr in the CO2 incubator. The media in wells were replaced with
a fresh 100 µl/well culture medium. The viability of the cells was
determined the exact way as above.

3 | S TATI S TI C A L A N A LYS I S

All data were expressed as the mean of three triplicates. Different

concentrations of the extract and extract generated AgNPs differ-
ences were analyzed with one-way analysis of variance (ANOVA)
using SPSS (version 17). Differences of p ≤ .05 were considered to
be statistically significant.

4 | R E S U LT S A N D D I S CU S S I O N

4.1 | Characterizations and sample analysis

Diluted honey samples were mixed with silver nitrate to synthe-

size AgNPs. The change in color of the mixture was an indication
of AgNPs synthesis where the color of the solution changed from
pale yellow to brown and continued to dark brown (Figure 1i–vi). The
degree of color change was pH-dependent that enabled the visual
monitoring by observation. The pH of the samples was 3.52, 3.59,
and 3.25 for H1, H2, and H3, respectively. The color change was
complete when the pH reached 9.
Both diluted honey (20%) before adding AgNO3 and honey after
the complete color change to brown were scanned spectrophoto-
metrically. Results indicated the formation of silver nanoparticles
after treated with AgNO3. H1 showed a peak at 410 nm (Figure 1B),
H2 at 405 nm (Figure 1D), and H3 at 395 nm (Figure 1F).
Honey is an extremely complex food product that has been re-
ported to contain at least 181 different substances including pro-
teins, enzymes, amino acids, minerals, vitamins, and polyphenols
(Balasooriya et al., 2017; Philip, 2009). There is a possibility that su-
crose, glucose, and proteins/enzymes play a part in the reduction
process. The addition of NaOH, which consequently increased the F I G U R E 2 The SEM images showing the spherical silver
pH of the solution, has an effect on the size of the nanoparticles pro- nanoparticles produced by H1 (a), H2 (b), and H3 (c)
duced. This probably due to the increased formation of gluconic acid
from glucose as pH increased. Based on a number of literature stud- Scanning electron micrographs showed that the H1-, H2- and
ies, gluconic acid is formed from glucose because the base drives H3-synthesized AgNPs are spheres and of a size about 70–80 nm
the opening of the glucose ring by abstraction of the α-proton of the (Figure 2a), 80–90 nm (Figure 2b), and 50–60 nm (Figure 2c),
sugar ring oxygen. The Ag ions then oxidized glucose to gluconic acid respectively.
and itself reduced to metallic Ag. However, the actual ingredients Mock, Barbic, Smith, Schultz, and Schultz (2002) using
which are responsible for the reduction of the Ag ions still remain high-resolution TEM images of silver nanoparticles with differ-
unknown and need further study. ent sizes and the geometrical shape showed that at the surface
GHRAMH et al.
plasmon resonance (SPR) peak range 410–500 nm the shape of the
particles is spherical, whereas pentagons and triangular shapes are
mostly formed at wavelengths from 500 to 700 nm. This obser-
vation strongly suggests that the Ag nanoparticles formed here
were spherical.
4.2 | Functional groups characterization
FTIR spectroscopy is useful in probing the chemical composition of
the surface of the silver nanoparticles and the local molecular envi-
ronment of the capping agents on the nanoparticles. Figure 3 shows
the FTIR spectra of honey and honey containing silver nanoparticles
obtained in this study.
The bioactive compounds of honey 1 (H1) and the biosynthe-
sized silver nanoparticles (AgNPs) were traced by FTIR spectropho-
tometer shown in Figure 3 (Figure 3,4-H1 and Figure 3,4-H1-Ag).
Totally, 7 peaks were obtained in the case of honey. The broad-
−1
band appearing at 3,278 cm is assigned for O–H stretching vi-
bration indicating the presence of alcohol or phenol as a reducing
agent. Weak bands at 2,987, 1,711, and 1,511 cm−1 are assigned to
C-H, C=O, and N-O stretching vibration indicating the presence
of alkane, aliphatic ketone, and nitro compound, respectively. The
−1
strong, intense peaks at 1,022, 711, and 578 cm correspond to
C-O, C=C, and C-Br stretching vibrations indicating the presence of
vinyl ether, alkene, and bromocompounds. The result of this FTIR
spectroscopic study confirmed that the red apple fruit extract has
F I G U R E 3 FTIR spectra of H1, H2,
and H3. Where H1, H2, and H3 before
and H1-Ag, H2-Ag, and H3-Ag after the
addition of AgNO3
| 449
the ability to perform dual functions of reduction and stabilization
of silver nanoparticles. FTIR of AgNPs showed 6 peaks which are
merely similar to that obtained by honey indicating the presence
of alcohol, alkane, aliphatic ketone, nitro compound vinyl ether,
alkene, and bromo compounds. The noticed difference is that
peaks indicating alcohol, nitro compound, and alkene decreased
in intensity indicating the exploitation of these compounds in the
reduction and capping of silver nanoparticles.
The FTIR spectrum was documented for both honey (H2)
and the biosynthesized silver nanoparticles (AgNPs) as shown in
Figure 3 (Figure 3,4-H2 and Figure 3,4-H2-Ag). The FTIR spec-
tra of honey (H2) showed the characteristic 7 peaks of bioactive
compounds. The broadband appearing at 3,278 cm−1 is assigned
for O–H stretching vibration indicating the presence of alcohol
as a reducing agent. Weak bands at 2,956, 1,744, and 1,511 cm−1
are assigned to C-H, C=O, and N-O stretching vibration indicat-
ing the presence of alkane, cyclopentanone, and nitro compound,
respectively. The strong peaks at 1,022, 711, and 578 cm−1 cor-
respond to C-O, C=C, and C-Br stretching vibrations indicating
the presence of vinyl ether, alkene, and bromo compounds. On
the other hand, FTIR of AgNPs showed 7 peaks, some of them
similar to that obtained in honey spectrum meanwhile some new
peaks arisen, such as weak band at 2,140 cm−1 may correspond
to nitrile CΞN stretch or alkynyl CΞC stretch. Medium peak at
1,578 cm−1 assigned to C=C stretching of cyclic alkene. Another
peak observed at 1,578 cm−1 assigned to C=O stretching vibra-
tions of amide. Medium peak at 1,378 cm−1 may be attributed to
450 | GHRAMH et al.
C-H bending due to alkane. So, these compounds produced as a
result of the reduction of silver nitrate to silver nanoparticles. On
the other hand, some peaks disappeared such as that correspond-
ing to cyclopentanone, alkene, and bromo compounds, indicating
that they are used in the reduction and stabilization process of
silver nanoparticles.
The functional groups involved in honey 3 and the forma-
tion of AgNPs using FTIR spectroscopy were shown in Figure 3
(Figure 3,4-H3 and Figure 3,4-H3-Ag). Representative spectra of
both H3 and H3 containing AgNPs manifest absorption peaks in
the region 3,500–500 cm−1. The broad peak around 3,289 cm−1
in the spectra indicates the existence of O-H group of alcohol.
Weak bands at 2,967, 1,733, 1,522, 1,033, 722, and 600 cm−1 are
associated with stretch vibration of C-H, C=O, C=C, N-O, C-O, and
C-Cl that correspond to alkane, ketone, alkene, nitro compounds,
vinyl ether, and alkyl halide, respectively. After nanoparticle syn-
thesis, the bands shifted to 3,200, 2,967, 1,758, 1,000, 711, and
−1
600 cm bands with lower intensity which could be assumed that
they were used in the reduction and capping of silver nanoparti-
cles. Appearance of a new strong peak around 1,333 cm−1 corre-
sponding to C-N stretching of aromatic amine as a byproduct of
reduction process. On the other hand, disappearance of the band
at 1,522 cm−1 means that alkene is exhausted in the reduction and
stabilization of nanoparticles.
4.3 | Antimicrobial susceptibility testing (AST)
The results of antibacterial activity of Sidr honey against gram-posi-
tive bacteria (B. subtilis), gram-negative bacteria (E. coli and P. aerugi-
nosa), and the fungal strain C. albicans are shown in Table 2.
Regarding the results of antimicrobial activity of different honey
types against B. subtilis, P. aeruginosa, and E. coli and the fungal strain
C. albicans, it was clear that all honey alone at the 20% concentration
showed inhibition of bacterial growth. H1, but not H2 and H3, showed
bacterial growth inhibition effect more than that of the positive con-
trol (Penicillin 10 µg). Notably, addition of AgNPs to both H1 and H3
clearly increased their growth inhibition effect but did not regarding
to H2. The inhibition of bacterial growth may be due to many factors
as the osmotic effect of honey (Kwakman et al., 2010; Kwakman &
TA B L E 2 Antimicrobial potentials of honey types alone and containing AgNPs
Inhibition zone (mm)
Escherichia coli Pseudomonas aeruginosa
Honey 1 13.50 ± 0.20 11.90 ± 0.19
Honey 1 + AgNPs 20.12 ± 0.29 12.65 ± 0.16
Honey 2 10.9 ± 0.35 10.9 ± 0.35
Honey 2 + AgNPs 18.10 ± 0.39 11.26 ± 0.12
Honey 3 7.8 ± 0.22 7.8 ± 0.22
Honey 3 + AgNPs 15.10 ± 0.18 11.80 ± 0.14
Penicillin (10 µg) 12.60 ± 0.02 9.80 ± 0.09
Zaat, 2012; Voidarou et al., 2011), the presence of hydrogen perox-
ide (Nassar et al., 2012), nonperoxide substances (Mandal & Mandal,
2011), and volatile antibacterial substances (Boateng & Diunase, 2015;
Olaitan et al., 2007). Also, Jeddar et al. (1985) evaluated the growth of
various gram-positive and gram-negative bacteria in media containing
various concentrations of honey, and they found that most pathogenic
bacteria failed to grow in honey at a concentration of 40% or above.
The pH of honey being between 3.2 and 4.5 is low enough to inhibit
pathogen growth. But if this honey is diluted with other fluids, for ex-
ample by body fluids, the pH will raise and would not lower that effec-
tively can inhibit bacterial growth (Molan, 1992, 2002, 2015).
The enzyme glucose oxidase (bee-origin) and the enzyme cata-
lase (floral origin) play an important role in the biological activities
of honey (White et al., 1963). Regarding glucose oxidase enzyme, as
this enzyme concentration increases in honey, the ability to hydro-
lyze glucose producing hydrogen peroxide (H2O2) increases, result-
ing in higher oxidative stress on microbial growth. In contrary, the
increase in catalase enzyme concentration, which destroy H2O2, will
determine the final antimicrobial effects. The balance of these two
enzymes determines, at least in part, the antimicrobial activity of the
honey (Zainol et al., 2013).
Some researcher demonstrated that undiluted honey has inactive
glucose oxidase (White et al., 1963), meaning that the action of H2O2 is
minimal and the antimicrobial effects of honey depend mainly on the
very high osmotic pressures coupled with the high acidity are the two
main factors contributing to the antimicrobial properties (Kwakman &
Zaat 2012; Zainol et al., 2013). But, if honey is diluted, glucose oxidase
will get activated and utilize glucose to produce H2O2. In the current
study, all honeys were diluted using sterile distilled water to get the
glucose oxidase activated. In diluted honey, if the osmotic pressure is
decreased as a result of dilution, the antimicrobial potentials will be
referred to the pH value and peroxide activity. In addition, some other
components in the diluted honey can contribute to its antimicrobial
activities that may include phenolic compounds, flavonoids, antibac-
terial peptides, methylglyoxal, methyl syringate, antibiotic-like deriva-
tives, and other components present in trace amounts (Jaganathan &
Mandal, 2009; Mandal & Mandal, 2011). Others (AL-Waili et al., 2013)
concluded that, regarding that geographical areas and plant origins, all
honey may show antimicrobial activities despite considerable variation
in their composition.
Bacillus subtilis Candida albicans
12.43 ± 0.21 12.5 ± 0.80
11.82 ± 0.12 15.20 ± 0.29
10.9 ± 0.35 10.9 ± 0.35
10.22 ± 0.11 10.10 ± 0.13
7.8 ± 0.22 7.8 ± 0.22
11.60 ± 0.17 9.70 ± 0.09
10.70 ± 0.03 10.40 ± 0.17
GHRAMH et al.
4.4 | Effects of different honey samples on normal
rat splenic cell proliferation
Until now, the immunomodulatory effects of Sidr honey are rela-
tively unknown. Therefore, in this study, we investigated the effects
of Sidr honey collected from three different geographical locations
on immune function and antitumor activity in vitro.
The proliferative/antiproliferative potentials in the tested
honeys were examined using normal rat splenic cells (Figure 4a).
Treatment with 20% H1 leads to growth stimulation of normal
splenic cells. This growth stimulatory effect significantly (p < .001)
increased when cells are treated with H1 containing AgNPs. In con-
trary, H2 and H3 inhibited the cell's growth. H2 nonsignificantly
F I G U R E 4 Effects of different honey (H1-H3) treatments, alone
or containing AgNPs, on normal rat splenic cells and cancer cell
lines
| 451
inhibited cell's growth more than H3. The degree of inhibition of
H2 was nonsignificantly lowered when H2 contained AgNPs. H3
inhibited splenic cell's growth nearly the same as H3 containing
AgNPs.
Some researchers (Tonks et al., 2003; Tonks, Cooper, Price,
Molan, & Jones, 2001) reported that honey (Manuka) increased fac-
tors that decrease cell growth like IL-1β, IL-6, and TNF-α production
by monocytes through a 5.8 kDa protein. The expected mechanism
by which the increase in these cytokines production is via TLR4
(Tonks et al., 2007). Others (Al-Waili & Haq, 2004) reported that in-
take of honey (oral) augmented the production of antibodies in pri-
mary and secondary immune responses.
One of the explanations that honey lowers the cell growth
is by arresting cell cycle (Tomasin & Cintra Gomes-Marcondes,
2011). The components contained by honey (e.g., flavonoids and
phenolics) are reported to block the cell cycle of many cell types
(Jaganathan & Mandal, 2009; Lee et al., 2003; Pichichero, Cicconi,
Mattei, Muzi, & Canini, 2010) in G0/G1 phase. This inhibitory
effect exerted on the proliferation of cells directly follows the
downregulation of several cellular pathways through tyrosine cy-
clooxygenase, ornithine decarboxylase, and kinase (Jaganathan &
Mandal, 2009; Oršolić et al., 2010; Pichichero et al., 2010). Others
showed that 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) assay method confirmed that antiproliferative ef-
fect of honey is in a dose- and time-dependent manner (Pichichero
et al., 2010). Honey or its components mediate inhibition of cell
growth due to its perturbation of the cell cycle (Oršolić et al.,
2010; Pichichero et al., 2010).
Another explanation was shown by some workers (Duddukuri, Rao,
& Athota, 2008) where they suggested that the inhibitory potential of
honey may be due to the direct suppressive effect of honey on T-cell
proliferation. When adding silver nitrate to the honey, some biomole-
cules are consumed to produce silver nanoparticles as indicated ear-
lier using FTIR analysis in this work. The inhibitory effects of honey
nonsignificantly decreased when contained AgNPS. This is may be due
to the use of some inhibitory biomolecules in nanoparticle formation.
The consumption of these expected biomolecules gave the chance to
stimulatory biomolecules to dominate in the medium, explaining the
stimulatory behaviors of the honey when mixed with AgNPs.
4.5 | Anticancer activity
The potentials of the three Sidr honeys to kill or stop cancer cell
proliferation were tested using two cell lines (Figure 4b). All honeys
did not show any anticancer activities against the Hela cell line. But,
when honey contained AgNPs, anticancer activities were shown by
all honeys. The anticancer activity against the Hela cell line of H1,
H2 and H3 containing AgNPs was shown to be not significantly dif-
ferent. In contrary to the anticancer activity against Hela cells, H1,
H2 and H3 showed anticancer potentials against the HepG2 cell line.
The anticancer activity exerted by H1, H2, and H3 was not signifi-
cantly different. This anticancer activity of H, H2, and H3 increased
452 | GHRAMH et al.
significantly (0.02, 0.03, and 0.02, respectively) with the presence of
AgNPs. H1 showed anticancer.
Honey as a traditional medicine and dietary natural product has
recently become the focus of attention in the treatment of certain
diseases as well as promoting overall health and well-being. There
is strong evidence supporting the positive role of natural food and
food product on the induction of apoptosis in different tumor cells
(Samarghandian et al. 2011). In this regard, we investigated the an-
tiproliferative honey kinds on Hela and HepG2 cell lines. We found
that honey was cytotoxic toward the cancer cells. Some worker
(Sadeghi-Aliabadi et al., 2015) showed the same result when tested
on HepG2 cell line as the IC50 was 3.12% and this effect was dose
dependent. In the oral health setting, honey has been found to be ef-
fective for the treatment of radiation-induced oral mucositis (Biswal,
Zakaria, & Ahmad, 2003) and is also found to be anticarcinogenic
(Sela, Maroz, & Gedalia, 2000) and antiproliferative and induces
apoptosis in prostate cancer cells (Samarghandian et al., 2011). This
is consistent with results being presented in this study. The honey
tested in this study has been originally produced in Saudi Arabia
(Rijal Ulma, Aseer, Saudi Arabia) and Pakistan, and sold as Sidr honey
in the local markets. According to our best knowledge, Sidr honey
has not been reported to be tested against Hela and HepG2 cancer
cells. Some researchers (Attia, Gabry, El-Shaikh, & Othman, 2008;
Gribel' & Pashinskiĭ, 1990) reported that honey revealed moderate
antitumor and pronounced antimetastatic effects. Their results also
showed that the antitumor activity of 5-fluorouracil and cyclophos-
phamide has been increased in combination with honey. Honey in-
hibited the growth of bladder cancer cell lines in vitro, and bladder
cancer antiproliferative activity of honey may relate to its low pH
(3.2–4.6). It was suggested that the polyphenols found in honey, in-
cluding caffeic acid, and its phenyl esters, present in natural honey
at the levels of 20%–25%, to be promising pharmacological agents
in the treatment of cancer by reviewing their antiproliferative and
molecular mechanisms (Jaganathan & Mandal, 2009). These com-
pounds are thought to exhibit a broad spectrum of activity including
tumor inhibition (Rao et al., 1993) by downregulation of many cellu-
lar enzymatic pathway including protein tyrosine kinas cyclooxygen-
ase and ornithine decarboxylase pathways (Rao et al., 1993). Jungle
honey obtained from the tropical forest of Nigeria showed chemo-
tactic activity for neutrophils, which were found to possess potent
antitumor activity (Fukuda et al., 2011). Moreover, the expression of
various proapoptotic and antiapoptotic proteins was found to be al-
tered during apoptosis (Ghashm et al., 2010). Unfractionated honey
induces cell-growth arrest, resulting in cell cycle blockage at the sub-
G1 phase (Jaganathan & Mandal, 2010).
Studies have shown several possible mechanisms mediated the
antiproliferative effect of honey toward cancer cells such as involve-
ment in inducing antioxidant effects (Antony, Han, Rieck, & Dawson,
2000), stimulation of TNF-α, involvement in the inhibition of lipopro-
tein oxidation (Swellam et al., 2003), and induction of apoptosis and
cell cycle inhibition (Dai et al., 2013).
Regarding the induction of mild growth of Hela cells by honey,
some authors considered honey as mitogenic agent (Tonks et al.,
2001) where it enhances cell proliferation (Aljady, Kamaruddin,
Jamal, & Yassim, 2000). Enhanced proliferation induced by honey
was suggested to be a nutritional effect caused by the carbohydrate
of honey which provides substrates for glycolysis. This does not con-
tradict with its other antitumor effect (El-Sayed et al., 2010). This
idea was proved by extracting the sugar from honey (Aziz, Rady,
Amer, & Kiwan, 2009).
5 | CO N C LU S I O N S
Sidr (Ziziphus spina-christi) honey was collected from three different ge-
ographical locations, two from KSA and one from Pakistan. All collected
honeys could produce AgNPs from silver nitrate solution in a spherical
shape. Sidr honey collected from KSA and produced by Apis mellifera
jemenitica, alone or containing AgNPs, stimulated the growth of nor-
mal rat splenic cells while both Sidr honey collected from Pakistan and
KSA and produced by Apis florea inhibited the splenic cells' growth. All
honeys alone stimulated Hela cancer cell line, but inhibited its growth
when contained AgNPs. All honeys, alone or containing AgNPS, inhib-
ited HepG2 cancer cell line proliferation. All honeys showed antimi-
crobial activities, these activities increased when honeys contained
AgNPs. Sidr honey can be used for antimicrobial agent, but can be used
as anticancer agent with care as it stimulated cell growth of some lines
(e.g., Hala) and inhibited another (e.g., HepG2).
AC K N OW L E D G M E N T S
The authors extend their appreciation to the Deanship of Scientific
Research at King Khalid University for funding this work through the
grant number: RCAMS/KKU/011-19.
C O N FL I C T O F I N T E R E S T
All authors state that they have not any financial/commercial con-
flict of interest regarding this work.
E T H I C A L A P P R OVA L
This study was approved by the Ethical Committee of King Khalid
University.
ORCID
Hamed A. Ghramh https://orcid.org/0000-0001-7995-0663
Essam H. Ibrahim https://orcid.org/0000-0003-0130-2257
Mona Kilany https://orcid.org/0000-0002-9538-6799
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 Biomedicine & Pharmacotherapy 84 (2016) 158–165

Available online at

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Original article

Anticancer activity of silver nanoparticles from Panax ginseng fresh

leaves in human cancer cells
Verónica Castro-Aceitunoa , Sungeun Ahna , Shakina Yesmin Simub , Priyanka Singhb ,
Ramya Mathiyalaganb , Hyun A. Leea , Deok Chun Yanga,b,*
a
Department of Oriental Medicinal Biotechnology, College of Life Sciences, Kyung Hee University, Yongin, 446-701, Republic of Korea
b
Graduate School of Biotechnology and Ginseng Bank, College of Life Sciences, Kyung Hee University, Yongin, 446-701, Republic of Korea

A R T I C L E I N F O A B S T R A C T

Article history:
Received 30 March 2016 The pharmaceutical role of silver nanoparticles has been increased over the last decades, especially those
Received in revised form 29 August 2016 synthesized through herbal medicinal plants, due to their variety of pharmacological importance. Panax
Accepted 6 September 2016 ginseng Meyer (P. ginseng) has been widely used as a therapeutic herbal medicine for a long time in cancer
treatment. In this study, the cytotoxic and oxidative effect of a novel silver nanoparticles synthesized
Keywords: from P. ginseng fresh leaves (P.g AgNPs) were evaluated in different human cancer cell lines. In addition,
Panax ginseng the effect of P.g AgNPs on cell migration, apoptosis and the determination of the mechanism involve was
Silver nanoparticles determinate by the use of A549 lung cancer cell line. It was found that P.g AgNPs treatment inhibited cell
Apoptosis
viability and induced oxidative stress in A549, MCF7 and HepG2 cancer cell lines. Likewise, P.g AgNPs
Cell migration
treatment inhibited the epidermal growth factor (EGF)-enhanced migration, as well as decreased the
p38 MAPK
mRNA levels and phosphorylation of EGF receptors in A549 cells. Moreover, P.g AgNPs modified the
morphology of the cell nucleus and increase apoptosis percentage; this effect was linked to the
stimulation of p38 MAPK/p53 pathway. Taken together, our results showed that P.g AgNPs exhibited anti-
cancer activity in A549 and the regulation of EGFR/p38 MAPK/p53 pathway might be the possible
mechanism of its anti-activity. Further experiments are suggested to determinate the mechanism by
which P.g AgNPs induce cytotoxicity and ROS generation in MCF-7 and HepG2 cells.
ã 2016 Elsevier Masson SAS. All rights reserved.

1. Introduction
Cancer is one of the first causes of mortality worldwide [1].
Some cancer types are well known to undergo mutations. This
mutation in cancer cells gives new properties that allow to the
cancer to persist, and in some cases, to spread to other organs.
Unfortunately, the current therapies which exhibits properties to
control and/or reduce the growth of cancer cell or tumors, also can
induce severe side effect in the patient decreasing its life quality
[2]. Over the years, the seek of therapies that can be used against
different cancers, as well as decrease the side effect of current
* Corresponding author at: Department of Oriental Medicinal Biotechnology,
College of Life Sciences, Kyung Hee University, Yongin, 446701, Republic of Korea.
E-mail addresses: ca.veronica@khu.ac.kr (V. Castro-Aceituno),
se8688@gmail.com (S. Ahn), simu.sha2@gmail.com (S.Y. Simu),
prnksingh254@gmail.com (P. Singh), ramyabinfo@gmail.com (R. Mathiyalagan),
Khuhanbang@gmail.com (H.A. Lee), dcyang@khu.ac.kr (D.C. Yang).
therapies have been increased [3]. Unfortunately, a drug with such
characteristics has not been reported so far.
Nanotechnology has gained interest in the range of biomedical
applications to its application in the development of agent with
different properties [4]. Silver nanoparticles (AgNPs) had been
used as an anti-microbial agent due to their ability to link with the
microbial membrane and to induce the production of the reactive
oxygen species (ROS), and finally induce apoptosis [5,6]. The effect
of AgNPs to induce oxidative stress in mammalian cells has been
previously evaluated. Similarly, it has been reported that AgNPs
were able to induce toxicity by trigger ROS production in cancer
cells [7,8]. Also, an AgNPs synthesized from a natural plant extract
had exhibited anti-cancer activity in lung cancer cells [9]. These
findings suggest that AgNPs synthesized from natural sources
might be used as an anti-cancer agent.
Panax ginseng Meyer (P. ginseng) have been used for the
treatment of several diseases [10–12]. Previously, the synthesis of
silver nanoparticle from P. ginseng fresh leaves, which exhibited
anti-bacterial and anti-coagulant effect have been reported [6].

http://dx.doi.org/10.1016/j.biopha.2016.09.016
0753-3322/ã 2016 Elsevier Masson SAS. All rights reserved.
 V. Castro-Aceituno et al. / Biomedicine & Pharmacotherapy 84 (2016) 158–165
Based on the premise that induction of oxidative stress by P.g
AgNPs is the responsible for the anti-microbial effect, and the
relation between ROS generation and apoptosis in cancer cell lines,
we envisage a possible anti-cancer effect from P.g AgNPs. In this
study, we focus in elucidate the cytotoxicity and the ROS
generation response of different cancer cell lines by P.g AgNPs
treatment. A549 non-small cell lung cancer line used for the
evaluation of the anti-migration and apoptotic effect of several
compounds in the presence of EGF external stimulation in previous
studies [13–16], had been selected to evaluate the anti-cancer
activity of P.g AgNPs. Furthermore, in order to underline the
possible mechanism involved in the anti-cancer activity of P.g
AgNPs, we determinate the mRNA and protein expression of
markers related to the EGFR/p38/p53 MAPK pathway.
2. Material and methods
2.1. AgNPs synthesis
Panax ginseng (P. ginseng) fresh leaves were used for the
synthesis of silver nanoparticles (AgNPs) as reported previously
[6]. Briefly, 15 g of P. ginseng fresh leaves were cut and boiled for
20 min in 100 mL of sterile water. Five milliliters of the total
volume of the filtrated solution were mixed with 25 mL of sterile
water. The AgNO3 solution was added to a final concentration of
one micromolar in the mixture. The reaction mixture was then
kept at 80
C for reduction of Ag+ to Ag atoms. After the
synthesis, indicated by a color changed, the nanoparticles were
collected, washed, dried overnight and stored at room tempera-
ture.
2.2. Cell culture
The lung cancer cell line A549, breast cancer cell line MCF7 and
hepatocellular carcinoma cell line HepG2 were purchased from the
Korean Cell Line Bank (Seoul, South Korea). The A549 cells and
MCF7 were grown in RPMI 1640 culture medium (GenDEPOT Inc.,
Barker, TX) and HepG2 cells were grown in DMEM media
(Welgene, Gyeongsangbuk-do, South Korea), supplemented with
10% fetal bovine serum (FBS), 1% penicillin G and streptomycin
(Gibco-BRL, Gaithersburg, MD), at a temperature of 37
C in a
humidified incubator with a 5% CO2 atmosphere.
2.3. Cell viability assay
MTT ((3, 4, 5-dimethylthiazol-2-yl)-2-5-diphenyletrazolium
bromide) reagent was assessed to evaluate the toxicity of P.g
AgNPs in A549, MCF7 and HepG2 cell lines. Cells were exposed to P.
g AgNPs at different dose (1–20 mg/mL) for a period of 48 h. Next,
20 mL of MTT solution (5 mg/mL) was added to each well and
incubated for 3 h. Old media were replaced with 100 mL of DMSO
and incubated for 20 min. The total amount of formazan formed by
viable cells was measured using multi-model plate reader (BioTek
Instrument, Winooski, VT) at a test wavelength of 570 nm with a
reference wavelength of 630 nm [17].
2.4. Determination of ROS
The reactive oxygen species generation was determinate by
20 , 70 -dichlorofluorescein (DCF-DA). A549, MCF7 and HepG2 cells
were seeded at 1.0–1.2  104 cells per well and incubated
overnight. Cell were treated at different concentration of P.g
AgNPs for 48 h. The addition of 25 mM of the DCF-DA solutions was
done 30 min prior treatment time finished. A multi-model plate
reader was used to measure the fluorescence by an excitation
wavelength of 485 and emission of 495 nm [18].
159
2.5. Cell proliferation
A549 cells were seeded in 96 well plate at 1 105 cells/mL and
incubated during 24 h at 37
C in a humidified chamber with a 5%
CO2 atmosphere. Then, the cells were exposed to P.g AgNPs at
different concentration for 48 h. The effect of P.g AgNPs on cell
proliferation were determined by the BrdU Cell Proliferation Assay
(Calbiochem1, La Jolla, CA) according to the manufacturer’s
protocol. Briefly, BrdU was added to the medium 18 h before
termination of the experiment. The BrdU incorporated into the
cells were determined by anti-BrdU antibody. The absorbance was
measured at dual wavelengths of 450/540 nm with a multi-model
plate reader [19].
2.6. Nuclear staining assay
To evaluate the effect of P.g AgNPs on the nucleus morphology of
A549 cells a Hoechst 33258 dye was used. After 48 h of treatment,
the medium was removed and the cells were washed twice with 1X
PBS and fixed with 3.7% (v/v) formaldehyde for 5 min at room
temperature. Then, the nucleus of the permeabilized cells was
stained with Hoechst 33258 (2 mg/mL) for 30 min in dark condition
at room temperature. Nuclear morphologies of the Hoechst-
positive cells were observed and photographed under a fluores-
cence microscope (400, Optinity, Korean Labtech, South Korea)
for further analysis. Scale bar was added by the Image J software
[20].
2.7. Apoptosis quantitation or quantitation of apoptosis
A549 cells were plated and exposed to P.g AgNPs for 48 h. The
quantification of DNA damage was evaluated by HT TiterTACSTM (R
& D System Minneapolis, MN) following manufacturer’s instruc-
tions. Briefly, the cells were fixed and labelled prior colorimetric
analysis. Cells were incubated with TACS-SapphireTM substrate and
then, the colorimetric reaction was stopped with 5% phosphoric
acid after 1 h. The absorbance was measured at 450 nm using a
multi-model plate reader.
2.8. Cell migration assay
Twelve-well plate was used to grow A549 cells. The cells were
incubated at 5% CO2 and 37
C humidified atmosphere, until reach
complete confluence. Next, A549 cells were subject to serum
starvation by replacing the old medium for serum-free growth
media during a period of 24 h. Prior treatment, a scratch was made
to each well by using 10 mL plastic pipette tip, and washed twice
with 1X PBS to remove floating cells. Then, cells were exposed to P.g
AgNPs at 10 mg/mL in the presence or absence of EGF (epithelial
growth factor) stimulation. Two randomly areas were photo-
graphed at the moment of the treatment and after 24 h under an
optical microscope Eclipse ME600L (Nikon Instruments, Melville,
NJ). The images were analyzed by T-scratch program [21].
2.9. Reverse transcription polymerase chain reaction analysis
The A549 cells (1 105 cells in a 6 well-plat) were exposed to P.g
AgNPs (10 mg/mL) with or without EGF (20 ng/mL) for 48 h at 37
C
prior total RNA being extracted using TRIzol reagent (Sigma, St.
Louis, MO) as we did previously [22,23]. cDNAs were synthesized
using a cDNA synthesis kit (Thermo Scientific, Lithuania, EU)
according to the manufacturer’s instructions. Quantitative real-
time polymerase chain reaction (qPCR) amplification was per-
formed using an R-Corbett Rotor-Gene Model 6000 (Corbett
Research, Sidney, Australia), with a SYBR Green qPCR Super Mix
UDG kit (Invitrogen, Carlsbad, CA, USA). qPCR was carried out using
160 V. Castro-Aceituno et al. / Biomedicine & Pharmacotherapy 84 (2016) 158–165

Fig. 1. P.g AgNPs reduce cell viability in A549, MCF7 and HepG2 cell lines. (a) Cell viability was measured after 48 h of treatment by MTT assay. Data are representative of three
independent experiments. *p < 0.05 and ***p < 0.001 vs Control. P.g AgNPs, Silver Nanoparticles from Panax ginseng fresh leaves.

Fig. 2. P.g AgNPs induce ROS generation in A549, MCF7 and HepG2 cell lines. (a) ROS generations was determinate by DCFH-DA fluorescence assay. Data are representative of
three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 vs Control. P.g AgNPs, Silver Nanoparticles from Panax ginseng fresh leaves. ROS, reactive oxygen species.

the following primers: EGFR 50 -CCA ACC AAG CTC TCT TGA GG-30
(forward) and 50 -GCTTTCGGAGATGTTGCTTC-30 (reverse); ELK1
50 -TGA GCT GTA GGG AAA CGC AG-30 (forward) and 50 -CAG GGG
TAC CTG TGT GTA GC-30 (reverse); p38 MAPK, 50 - CGA CTT GCT GGA
GAA GAT GC-30 (forward) and 50 - TCC ATC TCT TCT TGG TCA AGG-30
(reverse); JNK, 50 - CGG TGA GGA ACT ACG TGG AG-30 (forward) and
50 - ACC ATC GCT CTC AAC CCT TG-30 (reverse); ERK, 50 - CCT AAG
GAA AAG CTC AAA GA-30 (forward) and 50 - AAA GTG GAT AAG CCA
AGA C-30 (reverse); p53, 50 - TCT TGG GCC TGT GTT ATC TCC’
(forward) and 50 - CGC CCA TGC AGG AAC TGT TA-30 (reverse); p21,
50 -AGA AGA GGC TGG TGG CTA TT’ (forward) and 50 - TCG AAG TTC
CAT CGC TCA CG-30 (reverse); BcL-2, 50 -AAT GGG CAG CCG TTA GGA
AA-30 (forward) and 50 -GCG CCC AAT ACG ACC AAA TC-30 (reverse);
Bax, 50 -GGC CCT TTT GCT TCA GGG TT-30 (forward) and 50 -GGA AAA
AGA CCT CTC GGG GG-30 (reverse); EGFR 50 -CCA ACC AAG CTC TCT
TGA GG-30 (forward) and 50 -GCTTTCGGAGATGTTGCTTC-30
(reverse); Caspase-3, 50 -TTT GTT TGT GTG CTT CTG AGC C-30
(forward) and 50 -ATT CTG TTG CCA CCT TTC GG-30 (reverse);
GAPDH, 50 -AAT GGG CAG CCG TTA GGA AA-30 (forward) and 50 -GCG
CCC AAT ACG ACC AAA TC-30 (reverse). Amplifications were
performed at an initial temperature of 95
C for 10 min, followed
for 40 cycles at 95
C for 10 s, 60
C 15 s, and 72
C for 20 s. The
relative gene expression levels were normalized to the amount of
GADPH mRNA using delta cycle threshold (Ct) method [24].
visualize the bands by the use of a chemiluminescence detection
reagent.
2.11. Statistical analysis
Statistical analysis and data visualization were performed using
Graph Pad 6.04 software (La Jolla, CA 92037, USA). The statistical
significance of differences between values was evaluated by one-
way ANOVA. Results are expressed as mean  SEM.
3. Results and discussion
3.1. AgNPs induce cytotoxicity and ROS production in cancer cells
Panax ginseng Mayer (P. ginseng) has been used widely in the
treatment of different diseases [10]. Compounds derived from
leaves of P. ginseng such as ginsenosides, polysaccharides,
triterpenoids and flavonoids have been found to possess a lot of
pharmacological activities including anti-cancer activity [26–29].
Among these compounds, ginsenoside Re, Rd and Rg1 are highly

2.10. Immunoblotting

Cell were lysed and protein was extracted using 2X sodium

dodecyl sulfate (SDS) loading buffer (100 mM Tris-Cl (pH 6.8), 4%
(w/v) SDS, 0.2% (w/v) bromophenol blue, 20% glycerol and 200 mM
b-mercaptoethanol) for 5 min at room temperature [25]. Aliquots
of protein lysates were separated on 10% SDS PAGE gel and
transferred to PVDF membrane which was blocked with 5% skim-
milk in 1% TBS. The following antibodies were used for
immunoblotting: phospho-EGF Receptor (Tyr1068), EGF receptor, Fig. 3. P.g AgNPs decrease cell proliferation in A549 lung cancer cells. DNA synthesis
phosphor-p38 MAPK, p38 MAPK, p53, Nf-Kb, cleaved caspase-3 was examined by BrdU cell proliferation enzyme-linked immunosorbent assay.
These cells were exposed to various concentrations of P.g AgNPs for 48 h followed by
and GAPDH. The resultant protein bands were visualized after
BrdU incorporation assay. Data are representative of three independent experi-
incubation with goat anti-mouse or anti-rabbit IgG secondary ments. *p < 0.05 and **p < 0.01 vs Control. P.g AgNPs, Silver Nanoparticles from
antibody. The membranes were exposed to an X-ray film to Panax ginseng fresh leaves.
 V. Castro-Aceituno et al. / Biomedicine & Pharmacotherapy 84 (2016) 158–165 161

present in the leaves of P. ginseng [30,31]. Previous study reported
the anti-cancer effect of ginsenoside Rd in cancer cells [32]. In
addition, silver nanoparticles (AgNPs), synthesized from different
source by different methods, had been shown anti-cancer effect in
various cancer cell lines by the reduction of cell viability through
different mechanisms [9,33–35]. The effect of P.g AgNPs (silver
nanoparticle synthesized from Panax ginseng fresh leaves) has not
been studied so far. Thus, the aim of this study, was to determinate
its effect on cancer cells.
First, the cytotoxic effect of P.g AgNPs in various cancer cell lines
was assessed. By following the premise that P.g AgNPs did not
modify cell viability of the murine macrophage cell line RAW 264.7
up to 20 mg/mL (data not showed), the cytotoxicity and reactive
oxygen species (ROS) generation was evaluated up to 40 mg/mL
and 20 mg/mL, respectively. As a result, we found that P.g AgNPs at
2.5 mg/mL exhibited higher toxicity in MCF7 cell line than A549
and HepG2 cell lines, which toxicity was significantly reduced at
5 mg/mL and 10 mg/mL, respectively (Fig. 1). On the other hand, the
ROS generation was higher in HepG2 cells than A549 cells and
MCF7 cells (Fig. 2). These results suggest that the reduction on cell
viability of A549 and HepG2 cells induce by P.g AgNPs treatment
might be a consequence of the induction of ROS. However, the

Fig. 4. P.g AgNPs reduces EGF-enhanced migration in A549 lung cancer cells. (a) Wound healing assay was used to determinate the effect of P.g AgNPs. (b) The analysis of the
migration ratio (%) within 24 h measure by wound healing assay. Scale bars: 50 mm. Data are shown as mean  SEM (n = 4). ##p < 0.01, vs. Control; **p < 0.01 versus EGF-
Control. EGF, epidermal growth factor. P.g AgNPs, Silver Nanoparticles from Panax ginseng fresh leaves.
162 V. Castro-Aceituno et al. / Biomedicine & Pharmacotherapy 84 (2016) 158–165

induction of cell toxicity might not be correlated with the
induction of oxidative stress in MCF7 cell line. The result of cell
toxicity and ROS generation in A549 cells were similar to the
results reported in previous studies [7,36]. Thus, A549 cell line was
used for further evaluation of the anti-cancer effect of P.g AgNPs
3.2. AgNPs reduce cell proliferation in A549 lung cancer cells
Evaluation of cell cycle progression by the measurement of [3H]
thymidine incorporation as cells enter S phase has long been the
traditional method for the detection of cell proliferation [37]. Thus,
in this study, bromodeoxyuridine (BrdU), a thymidine analog
replaces [3H] thymidine, was used to evaluate the newly
synthesized DNA strands of actively proliferating A549 cells in
the presence or absence of P.g AgNPs treatment.
Inhibition of cell proliferation by compounds isolated from
ginseng such as ginsenoside Rg1, had been previously reported
[38]. In addition, AgNPs derived from Podophyllum hexandrum
were reported to decrease cell proliferation in cervical cancer cells
[34]. Among this, we hypothesize that the AgNPs synthesized from
Panax ginseng fresh leaves (P.g AgNPs) extract might exhibit an
anti-proliferative effect. We found that the DNA synthesis of A549
cells, detected by BrdU incorporation assay, was significantly
inhibited by P.g AgNPs by 75% and 60% at 5 and 10 mg/mL
respectively (Fig. 3). Interestingly, duplication of the dose of P.g
AgNPs treatment (20 mg/mL) does not significantly decreased the
DNA synthesis of A549 in comparison with the cells treated at
10 mg/mL. Therefore, this data along with the cytotoxic and ROS
generation results suggest that P.g AgNPs at 10 mg/mL can be used
as an optimal concentration for further experiment to evaluate its
anti-cancer effect in A549 lung cancer cells.
3.3. AgNPs reduce cell migration and EGFR phosphorylation in EGF-
enhanced A549 cells
Previously, overexpression of epithelial growth factor receptors
(EGFR) in cancer cells, was related to cell survives, drug resistance
and cell migration [39]. Thus, in order to determinate the effect on
cell migration of P.g AgNPs we carry out a wound-healing assay.
EGFR overexpression in A549 was already reported to be done by
external stimulation with EGF at 20 mg/mL [15]. However, the
effect on cell migration by silver nanoparticle in EGF-induced
migration had not been reported so far. Thus, we used EGF
stimulation at 20 ng/mL in this study to determinate the effect of
silver nanoparticles in EGF-induced migration. P.g AgNPs showed
to decrease cell migration at 10 mg/mL in EGF-enhanced A549 cells
after 24 h of treatment (Fig. 4). Similarly, was reported the effect of
silver nanoparticles to decrease cell migration on VEGF-induced
migration cells [40]. Additionally, the effect on mRNA and protein
levels of EGFR was determinate by PCR and Western blot
respectively. EGFR and ELK1 mRNA levels decreased after 48 h of
treatment with P.g AgNPs (Fig. 5a,b). Also, P.g AgNPs reduce the
phosphorylation of EGFR but not modified the expression of EGFR
(Fig. 5c,d). Taken together, these findings propose to EGFR as a
possible mechanism by which P.g AgNPs reduce cell migration in
A549 cells.

Fig. 5. P.g AgNPs decreased EGFR-induced expression in A549 lung cancer cells. (a) Expression levels were evaluated by RT-PCR and visualized via gel. (b) mRNA expression
levels were quantified using qRT-PCR normalized to GAPDH. (c) Protein expression levels of phospho-EGFR and EGFR by Western Blot. (d) Analysis of band density of Western
Blot results was done by Image J software. The figure is from a representative result of three experiments. #p < 0.05 and ##p < 0.01 vs control. *p < 0.05 and **p < 0.01 vs EGF-
Control. EGF, epithelial growth factor. EGFR, epithelial growth factor receptor. P.g AgNPs, Silver Nanoparticles from Panax ginseng fresh leaves. GAPDH, glyceraldehyde
3-phosphate dehydrogenase.
 V. Castro-Aceituno et al. / Biomedicine & Pharmacotherapy 84 (2016) 158–165 163

Fig. 6. P.g AgNPs induce apoptosis in A549 lung cancer cells. (a) The Hoechst 33258 staining result in A549 cells. Apoptotic cells are indicated with arrows. Scale bar, 10 mm. (b)
The percentage of apoptotic cells was determinate by HT TiterTACSTM assay. (c) Gene expression levels were evaluated by RT-PCR and visualized via gel. (d) mRNA expression
levels were quantified using qRT-PCR and normalized to GAPDH. (e) Protein expression levels of phospho-p38 MAPK and p38 MAPK by Western Blot. (f) Analysis of band
density of Western Blot results was done by Image J software. Data are representative of three independent experiments. *p < 0.05 and **p < 0.01 vs Control. EGF, epithelial
growth factor. P.g AgNPs, Silver Nanoparticles from Panax ginseng leaves.

3.4. AgNPs induces cell apoptosis through p38 MAPK/p53 pathway
The apoptotic effect is currently evaluated to determinate new
anti-cancer therapies among of the ability of cancer cells to
decrease the expression or lead mutation of pro-apoptotic
markers. Previous studies suggest, that the increased of oxidative
stress is related to the apoptotic response induced by several
anticancer agents [41]. In this study was found an induction of ROS
generation in A549 trigger by P.g AgNPs treatment. In order to
determinate the effect of P.g AgNPs on the nucleus morphology of
A549 cells as well as quantified the percentage of apoptosis rate
after treatment. We found that P.g AgNPs induced morphological
changes in the nucleus of A549 cells, such as chromatin
condensation and DNA fragmentation (Fig. 6a), as well as increased
the percentage of apoptotic cells (Fig. 6b). In additional, the mRNA
expression of p53 and p21 genes and protein expression of p53 and
Nf-kB were evaluated among that the induction of apoptosis and
senescence is thought to be the major biological outputs of the p53
pathway in response to various types of cell-physiologic stresses
[42]. Our gene expression studies shown an increase in the mRNA
levels of p53 and p21 genes by P.g AgNPs treatment (Fig. 6c,d).
Likewise, p53 protein expression was enhanced after treatment
(Fig. 6e,f). On the other hand, we found that the protein expression
of Nf-kB was not significantly modified. In previous studies have
been reported that p53 induces apoptosis by stimulate mitochon-
drial markers [43,44]. Likewise, further analysis shown that P.g
AgNPs lead to the cleaved of caspase-3 in A549 cells (Fig. 7a,b). As
well as a decrease in the mRNA levels of Bcl-2 and an increase in

Fig. 7. Cleaved caspase-3 and mRNA levels of Bax, Bcl-2, caspase 3 of A549 lung cancer cells treated with P.g AgNPs. (a) Protein expression levels of cleaved caspase-3 by
Western Blot. (b) Analysis of band density of Western Blot results was done by Image J software. (c) mRNA expression levels were quantified using qRT-PCR and normalized to
GAPDH. Data are representative of three independent experiments. *p < 0.05 and **p < 0.01 vs control. P.g AgNPs, Silver Nanoparticles from Panax ginseng fresh leaves. GAPDH,
glyceraldehyde 3-phosphate dehydrogenase.
164 V. Castro-Aceituno et al. / Biomedicine & Pharmacotherapy 84 (2016) 158–165
Fig. 8. P.g AgNPs up-regulated p38 MAPK pathway in A549 lung cancer cells. (a) Gene expression levels were evaluated by RT-PCR and visualized via gel. (b) mRNA expression
levels were quantified using qRT-PCR and normalized to GAPDH. Data are representative of three independent experiments. #p < 0.05 and ##p < 0.01 vs control. *p < 0.05
**p < 0.01 and ***p < 0.001 vs EGF-Control. EGF, epithelial growth factor. P.g AgNPs, Silver Nanoparticles from Panax ginseng fresh leaves. GAPDH, glyceraldehyde 3-phosphate
dehydrogenase.
mRNA levels of Caspase-3 and Bax occurs in the presence of P.g
AgNPs treatment (Fig. 7c). These findings suggest a relation
between p53 activation and mitochondrial-caspase-3 activation in
the presence of P.g AgNPs treatment.
The relation between ROS generation and apoptosis has been
suggested to be linked to the activation of p38 MAPK and c-Jun N-
terminal kinase (JNK) in previous studies [45–47]. Thus, the effect
of P.g AgNPs on the mRNA levels of p38 MAPK, JNK and ERK genes
and protein levels of p38 MAPK was evaluated. In this study, P.g
AgNPs showed to increase the mRNA levels of p38 MAPK, JNK and
ERK after treatment (Fig. 8a,b), as well as the phosphorylation of
p38 MAPK (Fig. 8c,d). Similarly, it has been reported that the effect
on the activation of p38 MAPK pathway to induce apoptosis [48]. In
addition, it has been reported that silver nanoparticle increased the
expression of JNK and p53 protein in A549 cells [49]. Beside, cell
apoptosis have been considered one of the major mechanisms for
p53’s anti-tumor effect [50]. Taken together, our finding suggests
that the apoptotic effect of P.g AgNPs in A549 lung cancer cells was
related to the activation of p38 MAPK-p53- mitochondria-caspase-
3 apoptotic cascades.
4. Conclusion
In this study, for the first time, we showed the anti-cancer effect
of the novel silver nanoparticle synthesized from Panax ginseng
Meyer fresh leaves (P.g AgNPs). P.g AgNPs decreased the cell
viability and induced reactive oxygen species production in A549,
MCF7 and HepG2 cell lines. Moreover, P.g AgNPs decrease cell
migration and reduce mRNA and phosphorylation of EGFR of A549
cells. In addition, P.g AgNPs induces cell apoptosis and up-
regulated the p38 MAPK/p53-mitochondria caspase-3 pathway in
A549 cells. Further experiments are advised in order to elucidate
the mechanism related to the cytotoxic effect of P.g AgNPs in MCF7
and HepG2 cancer lines.
Conflicts of interests
The authors declare that they have no conflict of interest.
Acknowledgements
This work was supported by the Korea Institute of Planning and
Evaluation for Technology in Food, Agriculture, Forestry and
Fisheries, Republic of Korea (313038-03-2-SB010).
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 Online - 2455-3891
Vol 10, Issue 3, 2017 Print - 0974-2441
Review Article

ANTIMICROBIAL AND ANTICANCER ACTIVITY OF SILVER NANOPARTICLES FROM EDIBLE

MUSHROOM: A REVIEW

PRIYADARSHNI KC*, MAHALINGAM PU

Department of Biology, The Gandhigram Rural Institute- Deemed University, Gandhigram, Dindigul, Tamil Nadu, India.
Email: priyakcdarshu@gmail.com
Received: 04 November 2016, Revised and Accepted: 08 December 2016

ABSTRACT

Biologically inspired nanoparticle synthesis is currently a rapid expanding area of research in nanotechnology. Nanoparticle synthesis utilizing the
bioresources such as plants and microbes appears to be a viable, low-cost, and eco-friendly approach. Especially mushrooms can be used for large-
scale synthesis of silver nanoparticles as mushroom produces many proteins that reduce the silver nitrate during the biosynthesis. Silver nanoparticles
can be characterized using ultraviolet-visible (UV-VIS) spectroscopy, fourier transform infrared spectroscopy, X-ray diffraction, scanning electron
microscopy, energy dispersive X-ray, and transmission electron microscope. Silver nanoparticles possess high antibacterial activity since silver in
different forms has been extensively used as a medicine for curing diseases and promote wound healing. Silver nanoparticles have high surface
specific area, which will lead to excellent antimicrobial activity as compared with bulk metallic silver. Further, the silver nanoparticles show anticancer
activity against various cell lines such as human epidermoid larynx carcinoma (HEP-2), colon adenocarcinoma (HCT-116), breast adenocarcinoma
(MCF-7), liver carcinoma (Hep-G2), and intestinal adenocarcinoma (Caco2) were well documented. This review intends to present green synthesis of
silver nanoparticles and their application as antimicrobial and anticancer agents.

Keywords: Silver nanoparticles, Bioresources, Mushroom, Antimicrobial activity, Anticancer property.

© 2017 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.
org/licenses/by/4. 0/) DOI: http://dx.doi.org/10.22159/ajpcr.2017.v10i3.16027

INTRODUCTION
Nanoparticles are the basic essential elements of nanotechnology
and it exhibits fabulous advanced characteristic features based on
their properties such as size, morphology, and other size-dependent
properties [1]. These unique features of nanoparticles may lead to play a
crucial role in a variety of surprising and interesting uses in biomedicine,
energy science, optics, and other health-care applications [2,3]. Among
the nanoparticles, nanosilver has potential applications in the field of
biomedicine since silver have a disinfecting effect and has found uses
in traditional medicines for a long time. Silver nanoparticles have high
surface specific area, which will lead to excellent antimicrobial activity
as compared with bulk metallic silver.
Originally, silver nanoparticles can be synthesized using various chemical
and physical methods, but these approaches are not environmentally
benign [4]. Each method has advantages and disadvantages with
common problems being cost, scalability, wide size distribution [5]
and decrease in the stability of the nanoparticles on application can
be problematic since aggregation decreases the specific surface area
lowering the antimicrobial and catalytic activity [6]. Furthermore, the
presence of some toxic metals in the synthesis process may create
some adverse effects in biomedical applications [7]. Thus, biosynthesis
of nanoparticles emerged as an effective alternative to overcome the
disadvantages of the classical methods.
The bio-route attracts a considerable interest because of its eco-
friendliness and biocompatibility. A great deal of effort has been put
into the biosynthesis of metal nanoparticles, using microorganisms
such as bacteria [8,9], fungus [10,11], algae [12], and plants [13]. The
use of macro-fungi especially mushroom in the nanoparticle synthesis
is a new addition and holds a promising role in large-scale nanoparticle
production in lesser time period. In fact, the mushroom fungi produce
many proteins and extracellular enzymes involved in the reduction of
silver nitrate during the synthesis of silver nanoparticles and are very
simple to grow both in lab scale and industrial scale and also the yield
is high.
This review intends to present a brief note on the biosynthesis of silver
nanoparticles from edible mushroom extract, their characterization
using various techniques, antimicrobial and anticancer activity.
BIOSYNTHESIS OF SILVER NANOPARTICLES FROM EDIBLE
MUSHROOM EXTRACT
Mushroom extract was prepared by boiling cut pieces of edible
mushroom (Pleurotus sp.) with sterile distilled water for 10 minutes
and filtered. The extract was mixed with 1mM AgNO3 (Fig. 1). The
mixture turned brown color which indicates the formation of silver
nanoparticles. The nanoparticles are then pelleted and dried.
CHARACTERIZATION OF SILVER NANOPARTICLES
UV-VIS spectroscopy
Metal nanoparticles exhibit strong absorption of electromagnetic
waves due to surface plasmon resonance in the visible range [14]. Thus,
UV-VIS spectroscopy is used to investigate the formation and stability
of metal nanoparticles in solution. Dark brown color indicates the
production of silver nanoparticles in solution [15].
Fourier transform infrared spectroscopy (FTIR)
When infrared radiation is passed through a sample, specific wavelengths
are absorbed which causes the chemical bonds present in the material to
undergo vibrations such as stretching, contracting and bending. Thereby,
FTIR can be used to determine the functional groups of the biomolecules
in the extract which are responsible for the reduction of the silver ions.
X-ray diffraction (XRD)
XRD is a conventional technique used to establish the crystalline nature,
domain size, and structure of the nanoparticles [16]. It can be used to
look at a single crystal or polycrystalline materials.
 Priyadarshni and Mahalingam
Fig. 1: Schematic representation of silver nanoparticle synthesis
Scanning electron microscopy (SEM)
SEM is a direct visualization technique used for the morphological
examination of nanoparticles. Surface morphology and size of the
nanoparticles can be examined, however; this technique provides
limited information about the size distribution.
Energy dispersive X-ray spectroscopy (EDX)
EDX spectroscopy is used for the elemental analysis or chemical
characterization of nanoparticle samples. EDX confirms the formation
of silver nanoparticles by recording the strong signal of elemental
silver.
Transmission electron microscope (TEM)
TEM is used to study the size and shape of the silver nanoparticles. It is
a direct imaging technique, provides higher resolution than SEM. The
synthesized silver nanoparticle solution is dropped on carbon-coated
TEM grids, dried and viewed for determining the particle size [16]. The
sample preparation for TEM is complex and time-consuming because
of its requirement to be ultra thin for the electron transmittance under
high vacuum.
ANTIMICROBIAL ACTIVITY OF SILVER NANOPARTICLES
Since several years silver has been known as a strong disinfecting agent
and has found many uses in traditional medicinal practices. Several
silver-based compounds have been utilized effectively as antimicrobial
specialists [17]. Compounds of silver are also used in the medical field to
treat burnt wounds and various other types of infections. Nanoparticles
of silver have aptly been investigated for their antibacterial property
because the silver nanoparticles have high specific area than their
volume, which will lead to excellent antimicrobial activity as compared
with bulk silver metal [18-21].
Silver nanoparticles showed potential antibacterial activity against
Gram-positive bacteria such as Staphylococcus aureus and Bacillus
subtilis [22] and Gram-negative organisms such as Klebsiella
pneumonia [22] and Salmonella typhus [23]. Praiseworthy endeavors
have been made to investigate this property utilizing electron
microscopy, which has uncovered size-dependent interaction of silver
nanoparticles with microorganisms [19]. However, the inhibitory
mechanism of silver nanoparticles is only partially understood. There
are different speculations on the activity of silver nanoparticles on
microorganisms to bring about the microbicidal impact.
Silver nanoparticles attach with the microbial cell wall membrane by
electrostatic attraction [24,25] and subsequently penetrate it, thereby
changing the permeability of the cell membrane and causes cell death.
Sondi and Salopek-Sondi [26]. stated that the nanoparticles associated
with the bacterial cell wall form “pits,” thereby affects the permeability
and causes cell death.
Electron spin resonance spectroscopy studies proposed that there is
the development of free radicals by the silver nanoparticles that might
be thought to be another mechanism by which the cells die. At the
point when the free radicals formed by the silver nanoparticles are in
Asian J Pharm Clin Res, Vol 10, Issue 3, 2017, 37-40
close contact with the bacteria, they can harm the cell membrane and
make it permeable which can at last prompt to cell death [27,28]. It was
suggested that there can be release of silver ions by the nanoparticles,
which acts on the thiol groups of many crucial enzymes and make
them inactive, thereby repress several cellular activities and harm the
cells [29].
Another reality is that the DNA has sulfur and phosphorus as its
important constituents which are soft bases. Silver being a soft acid can
act on these soft bases and destroy the DNA which would definitely lead
to cell death [19]. The interaction of the silver nanomaterials with the
sulfur and phosphorus of the DNA can prompt to issues in the bacterial
DNA replication and thus terminate the microscopic organisms (Fig. 2).
However, further research is required to thoroughly establish the
bactericidal mechanism.
ANTICANCER ACTIVITY OF SILVER NANOPARTICLES
Nanotechnology plays a tremendous role in overcoming many of the
problems that conventional methods face in the treatment, diagnosis,
and detection of cancer [31]. The primary feature of these nanoparticles
is that their surfaces can be functionalized, exploiting reactive terminal
groups, with particular proteins, peptides or monoclonal antibodies
that are capable specifically to bind at a site of action or a specific
targeted tissue, without interacting with other cells [32]. Therefore, the
use of nanotechnology in cancer treatment offers exciting possibility
of destroying cancer cells with minimal damage to healthy tissues and
organs.
Nanoparticles can be used in the detection and elimination of cancer
cells before they form tumors. Nanoparticles can also be designed to
carry drugs and release them at the targeted site. These particles can
be employed as contrast agents in magnetic resonance imaging for
diagnostic purposes and treatment monitoring. In particular, silver
nanoparticles secure much enthusiasm among the rising nanoproducts
in the field of nanomedicine because of their exceptional properties and
clear therapeutic potential in treating a variety of diseases.
Nanoparticles will have a lethal effect on the cell wall of the cancer
cells [33]. Water soluble organic moieties in the nanoparticles induce
a synergistic antiproliferative effect in various cancer cell lines, thus
prove to be useful in various types of cancer control system (Fig. 3).
Silver nanoparticles have been found to induce the apoptotic pathway
in vitro through free oxygen radical generation, which showed
antitumor, antiproliferative and antiangiogenic effects in-vitro [34].
Compounds having antiangiogenic properties are known for their
potential capacity to hinder the action of abnormally expressed
signaling proteins, for example, Ras and Akt, subsequently, exhibit a
reliable antitumor effect [35].
Cytotoxic activities of silver nanoparticles are documented in the
following research works. Asharani et al. [36] reported that silver
nanoparticles exhibit antiproliferative effect on human glioblastoma
cells. Franco-Molina et al. [37] evaluated the effects of colloidal silver on
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Asian J Pharm Clin Res, Vol 10, Issue 3, 2017, 37-40

Fig. 2: Various mechanisms of bactericidal action of silver nanoparticles [30]

Fig. 3: Antiproliferative activity of nanoparticles [33]

MCF-7 human breast cancer cells. Sanpui et al. [38] demonstrated that
nanosilver not only disrupted normal cellular function but also affected
membrane integrity, inducing various apoptotic signaling genes of
mammalian cells, leading to programmed cell death. Hsin et al. [39]
reported that nanosilver induced apoptosis in murine embryonic
fibroblast cells (NIH 3T3) by increasing the generation of reactive oxygen
species and activating the c-Jun N-terminal kinase pathway, leading
to mitochondria-dependent apoptosis. Silver oxide nanoparticles
displayed antitumor properties in transplanted Pliss lymphosarcoma
tumor models when given through intravenous injection in aqueous
form [40]. Silver nanoparticles have shown anticancer activity against
some of the human cancer cell lines such as colon carcinoma (HCT-116),
breast carcinoma (MCF-7), liver carcinoma (Hep-G2), and intestinal
carcinoma (Caco2) as stated by Shawkey et al. [41]. The cytotoxicity of
nanosilver is the consequence of dynamic physicochemical interaction
of silver particles with the functional groups of intracellular proteins,
as well as with the nitrogen bases and phosphate groups of DNA [42].
Silver nanoparticles serve as antitumor agents by decreasing
progressive development of tumor cells. This might be because of
their inhibitory actions in several signaling cascades liable for the
development and pathogenesis of cancer. Taken together, these
informations recommend that silver nanoparticles can actuate
cytotoxicity on cancer cells and hindering tumor progression without
lethality to normal cells.
CONCLUSION
Biologically prepared silver nanoparticles tend to be biocompatible and
cost-effective. Thus, they are handy in wide variety of nanobiotechnology
applications. Silver nanoparticle is a good alternative in antibacterial
treatment with less side effect and potential action. More research is
needed to study the anticancer activity of silver nanoparticles in detail.
Under this backdrop, the edible mushroom will be a suitable source
for green synthesis of silver nanoparticles having potential anticancer
properties. Care has to be taken to utilize this marvel well and in a good,
effective and efficient way for human betterment.
ACKNOWLEDGMENTS
The authors would wish to acknowledge the Head of the Department of
Biology, Gandhigram Rural Institute, Deemed University, for providing
research facilities and encouragement.

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 Priyadarshni and Mahalingam
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On the anti-cancer activities of silver nanoparticles

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DOI: 10.15406/jabb.2018.05.00116

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 Journal of Applied Biotechnology & Bioengineering
On the Anti-Cancer Activities of Silver Nanoparticles
Abstract
In the present mini-review silver nanoparticles (AgNPs) due to their superior
physicochemical, and biological properties are intensively dealt with. The proper
knowledge of these characteristics is essential to maximize their potential
applications in many areas while minimizing their hazards to humans and the
environment. This manuscript aims to critically review AgNPs synthesized via
different approaches, its utilization in cancer treatment and future challenges.
Keywords: Anti-cancer activities; AgNPs; IC50; Plasmon; HepG2; MCF-7;
MDAMB231; SKBR3; Rosa indica
Introduction
Silver nanoparticles (AgNPs) constitute a class of materials
with sizes in the range 1-100 nm. The interest in the study
of AgNPs concerning their various behaviours has recently
increased because of their unique and attractive physical,
chemical, and biological properties [1-7]. AgNPs are also known
to have particular functions regarding toxicity, surface plasmon
resonance, and electrical resistance. Based on these, intensive
research works have been conducted to investigate their
properties and potential applications for several purposes such
as antimicrobial agents in wound dressings, anticancer agents,
electronic devices, and water treatment as well [8-15].
Cancer is a group of diseases, generating various pathological
and metabolic changes in cellular environments. It is developed
through diverse signaling mechanisms including cell proliferation,
angiogenesis, and metastasis [16,17]. Cancer cells have abnormal
metabolic activities in aerobic glycolysis, mitochondrial DNA
depletion, and alterations in respiratory chains and genomic
expressions. The physical and chemical treatments of cancer are
Figure 1: Mechanism of action of silver nanoparticles against cancer cells.
There is several review papers published to address the issues
associated with AgNPs regarding their toxicology properties
during their use as antimicrobial agents for textiles, dental
biomaterials, and bio-detectors, as well as during their syntheses
[26-34]. For instance, toxicity properties including cytotoxicity and
Submit Manuscript | http://medcraveonline.com
Mini Review
Volume 5 Issue 2 - 2018
Department of Refractories, Ceramics and Building Materials,
National Research Centre, Egypt
*Corresponding author: Wafa I Abdel-Fattah, Refractories,
Ceramics and Building Materials Dept. Biomaterials Group,
Inorganic Chemical Industry Division, National Research
Centre, Egypt,
Email:
Received: December 20, 2017 | Published: February 09,
2018
limited at different stages. However, currently available therapies
have an adverse effect and affect normal cell functions while
giving excess drug and radiation exposures [18,19].
A marginal increase in cancer cases within the last few years
ends up mostly, with death [20]. In several cancer types, we have
to manipulate satisfactory medicine carriers similar to drug
delivery to be applied as adequate chemotherapeutic agents
[21,22]. Recently, AgNPs are reported to modulate the Pgp activity
and therefore enhance the chemotherapeutic efficacy against
multi-drug resistant cancer cells, thus, further emphasizing their
excellent potential as combinational partners [23]. Moreover, the
genotoxicity of AgNPs is supported by the generation of double-
stranded DNA breaks along with chromosomal instability that
drives the initiation of apoptotic execution [24,25]. This acting
mechanism implies that AgNPs can be mutually associated with a
great many DNA-targeting anticancer drugs (Figure 1).
genotoxicity of capped or uncapped AgNPs have been reviewed in
detail [35]. Their toxicity mechanisms after oral exposure were
also thoroughly discussed [36]. Also, a recent review of AgNPs had
focused on their synthesis using plant extracts for antimicrobial
applications [37]. Most of the above studies concentrate chiefly on
J Appl Biotechnol Bioeng 2018, 5(2): 00116
 Copyright:
On the Anti-Cancer Activities of Silver Nanoparticles ©2018 Abdel-Fattah et al. 2/4

the different synthesis methods and their bactericidal activities.
The reviews on the anticancer activity are somewhat seldom.
Therefore, this review aims to present the anticancer activities for
silver nano-particles synthesized from different sources.
Anticancer activity of silver nanoparticles
The Metallo-pharmaceuticals were included within the research
field that was previously dominated by organic compounds and
natural products. Many platinum and platinum-based compounds
including carboplatin and oxaliplatin were approved as antitumor
agents [38]. However, numerous drawbacks of platinum-based
Table 1: Silver nanoparticles from different sources against several cancer cells.
pharmaceuticals were reported proving, therefore, their curative
effects.
Many cancer types are not susceptible to platinum drugs, and
there are many toxic side effects, including gastrointestinal and
haematological toxicity [39]. Moreover, several cancer cells have
either intrinsic or acquired resistance to other platinating agents
and cisplatin [40]. Consequently, current anticancer research
has been devoted to the discovery of novel transition metal
compounds. While silver was initially investigated because of
its advantageous antimicrobial activity, there has been a recent
interest in its anticancer functions (Table 1).

AgNPs Synthesis Route Tested Cancer Cell Reference

plant dandelion- Taraxacum officinale human liver cancer cells (HepG2) [41]

Plant Extract-Commelina nudiflora L HCT- 116 colon cancer cells [42]

human colorectal adenocarcinoma, the human kidney, human chronic myelogenous,

plant extracts of guava and clove [43]
leukaemia, bone marrow, and human cervix

Plant Extract- Nostoc linckia MCF-7 [31]

A549 (Human lung carcinoma), HeLa (Human cervical adenocarcinoma), MCF7 (Human
Chemical synthesis breast adenocarcinoma), MDAMB231 (Human breast adenocarcinoma), and SKBR3 (Human [44]
breast adenocarcinoma) cells

Plant Egtract-ethanolic extract of rose

human colon adenocarcinoma cancer cell line HCT 15 [42]
(Rosa indica) petals

Saratale et al. [41] developed AgNPs from common medicinal
plant dandelion, Taraxacum officinale and showed their high
cytotoxic effect against human liver cancer cells (HepG2)
[41]. The AgNPs Synthesized by Kuppusamy et al. [42] Using
Commelina nudiflora L [42] aqueous extract showed a reduced
cell viability and increased cytotoxicity against HCT-116 colon
cancer cells. Biofunctionalized silver nanoparticles synthesized
within different plant extracts of guava and clove showed the
satisfactory anti-cancer effect against four different cancer cell
lines; human colorectal adenocarcinoma, the human kidney,
human chronic myelogenous, leukaemia, bone marrow, and
human cervix [43]. The developed silver nanoparticles using a
proteinaceous pigment phycocyanin extracted from Nostoc linckia
as reducing agent exhibited effective cytotoxic activity against
MCF-7. The inhibitory concentration (IC50) was 27.79± 2.3μg/
mL [31]. Moreover, the chemically synthesized AgNPs composites
possessed promising anticancer activity against the A549 (Human
lung carcinoma), HeLa (Human cervical adenocarcinoma), MCF7
(Human breast adenocarcinoma), MDAMB231 (Human breast
adenocarcinoma), and SKBR3 (Human breast adenocarcinoma)
cells [44]. Kuppusamy et al. [42] successfully bio-synthesized
silver nanoparticles using the ethanolic extract of rose (Rosa
indica) petals. The Ag functionalized extract proved potential
anticancer activity against human colon adenocarcinoma cancer
cell line HCT 15 [42].
Effect of silver nanoparticles size on their anticancer
activity
The biological effects of various metal nanoparticles in p53-
deficient tumor cells as well as in vitro tumor stroma and in vivo
metastasis models were investigated [45]. A higher cytotoxicity
was recorded for the smaller, 5 nm sized silver nanoparticles
compared to their larger counterparts. Additionally, it was
concluded that silver nanoparticles could induce apoptosis-
dependent programmed cell death in the absence of the tumor
suppressor p53. Conventional cancer therapy often fails to
cause cell death in p53-deficient cancer cells. The unique
chemotherapeutic potential of such developed AgNPs was proved.
Moreover, it was concluded that nanoparticles of size 5-35 nm
primarily induced cell death through the mitochondrial structure
and function targeting. Although the smaller Ag nanoparticles are
more cytotoxic, the apoptotic action mechanism of both 5 and
35 nm was identical [46]. Interestingly, the cytotoxic features of
silver and silver hybrid nanoparticles are cell-type dependent. In
this domain, a higher cytotoxicity was recorded against cancer
cells compared to non-cancerous fibroblasts. Conclusively, the
stimulation of tumor-associated fibroblast cells with metal
nanoparticles represents a typical therapeutic strategy. Since the
treatment by Ag and Ag hybrids suppress the cancer cell promoting
the activity of a tumor associated fibroblasts. Additionally, the in
vivo results proved the ability of Ag/hybrids to inhibit the 4T1
tumor metastatic spreading in mice. Impressively, Ag hybrids
can enhance the therapeutic efficacy of intravenous doxorubicin
treatment [47].
Conclusion
The silver nanoparticles proved unique anticancer activity
against different types of cancer cells. The several syntheses
approaches significantly affect the cytotoxic activity of the
achieved Ag nanoparticles. Future challenges on AgNPs synthesis

Citation: Abdel-Fattah WI, Ali GW (2018) On the Anti-Cancer Activities of Silver Nanoparticles. J Appl Biotechnol Bioeng 5(2): 00116.
DOI: 10.15406/jabb.2018.05.00116

On the Anti-Cancer Activities of Silver Nanoparticles
and their release into the environment other than scaling up
production, assess several potential avenues for future works
are to promote a safer and more efficient utilization of these
nanoparticles.
Acknowledgement
None.
Conflict of Interest
None.
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Citation: Abdel-Fattah WI, Ali GW (2018) On the Anti-Cancer Activities of Silver Nanoparticles. J Appl Biotechnol Bioeng 5(2): 00116.
DOI: 10.15406/jabb.2018.05.00116
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 Academic Sciences International Journal of Pharmacy and Pharmaceutical Sciences

ISSN- 0975-1491 Vol 4, Suppl 4, 2012

Research Article

INVITRO ANTICANCER ACTIVITY OF SILVER NANOPARTICLES SYNTHESIZED USING THE

EXTRACT OF GELIDIELLA Sp.

J. SARANIYA DEVI1,2, B. VALENTIN BHIMBA2, KRUPA RATNAM3

1Department of Microbiology, Asan Memorial College of Arts and Science, Chennai-100, 2Department of Biotechnology, Sathyabama
University, Chennai, 3Marine Biotechnology, National Institute of Technology, Chennai. Email: bvbhimba@yahoo.co.in
Received: 18 Apr 2012, Revised and Accepted: 28 May 2012
ABSTRACT
The eco-friendly synthesis of nanoparticles through various biological means helps us to explore various seaweeds for their ability to synthesize
silver nanoparticles (AgNPs). It was found that aqueous silver ions when exposed to aqueous extract of Gelidiella sp. are reduced in solution, thereby
leading to the formation of the AgNPs within 10 min. at 121˚C. These AgNPs were characterized by means of several techniques. The nanoparticles
show absorbance at 435nm on UV-Vis spectroscopy. The presence of proteins was identified by Fourier transform-infra red spectroscopy (FT-IR).
The presence of elemental silver was characterized by Energy dispersed spectroscopy (EDS). The morphology of AgNPs was characterized by
Scanning electron microscopy (SEM). The synthesized AgNPs are very stable in aqueous solution for about 1 month without any sign of
precipitation. The nanoparticles were assessed for their cytotoxic activity on Hep-2 (Humman laryngeal) cell lines.
Keywords: Ecofriendly, Silver nanoparticles, Gelidiella sp., UV-Vis, FT-IR, EDS, SEM, Cytotoxic activity, Hep-2 cell lines.

INTRODUCTION fashion, leading to a progressive increase in the number of dividing

Nanotechnology deals with processes that take place on the
nanometer scale, that is, from approximately 1 to 100 nm.
Nanoparticles with controlled size are of fundamental and
technological interest as they provide solutions to technological and
environmental challenges in the areas of medicine, solar energy
conversion, catalysis and water treatment. Thus, production and
application of nanomaterials from 1 to 100 nanometers (nm) is an
emerging field of research (1). Nanoparticles are collection in
aggregate of atoms in the range of 1-100 nm with unique structure
and properties, which are widely used in an increase amount of
applications. Silver nanoparticles (Ag-NPs) in particular, provide
effective growth inhibition of various microorganisms in suspension
and on solid medium.
Although chemical and physical methods may successfully produce
pure, well-defined nanoparticles, these methods are quite expensive
and potentially dangerous to the environment. Use of biological
organisms such as microorganisms, plant extract or plant biomass
could be an alternative to chemical and physical methods for the
production of nanoparticles in an eco-friendly manner (2). In recent
years, plant-mediated biological synthesis of nanoparticles is gaining
importance due to its simplicity and eco-friendliness. Green
synthesis of nanoparticles is an easy, efficient and eco friendly
approach, where most researchers are looking at the eco-friendly
and green synthesis of nanoparticles paves the way for the
researchers around the world to explore the ability of various herbs
in synthesizing nanoparticles. Nanoparticles synthesized by
chemical method are not eco-friendly (3).
AgNPs have several characteristics that make it currently among the
most widely used nanoparticle in science. One highly useful
characteristic is its antimicrobial property (4). Silver in its pure form
was known as a great material to keep microbes at bay. If silver is
transformed into a nanoparticle, this anti-microbial property is
intensified, making it useful in effectively eliminating fungus,
bacteria, and viruses. As a natural material, silver is known to be safe
to man and produce little to no allergic reactions when tested for
curing various diseases.
Silver is an effective antibacterial agent with low toxicity which is
important especially in the treatment of burn wounds. Given its
broad spectrum activity, AgNPs have been the focus of increasing
interest and are being used as an excellent candidate for therapeutic
purposes (5).
Cancer is an abnormal type of tissue growth in which the cells
exhibit an uncontrolled division, relatively in an autonomous
cell (6). There is increasing demands for anticancer therapy (7).
Invitro cytotoxicity testing procedures reduces the use of laboratory
animals (8) and hence use of cultured tissues and cells have
increased (9).
The discovery and identification of new antitumor drug with low
side effects on immune system has become an essential goal in many
studies of immuno-pharmacology (10). With this aim, many
attentions have been paid to natural compounds in plants, marine
organism and microorganisms.
Many medically relevant nanoparticles such as AgNPs were
investigated for their cytotoxicity aspect. AgNPs showed different
degrees of in vitro cytotoxicity (11). The Apoptotic Effect of
Nanosilver is mediated by a ROS- and JNK-Dependent Mechanism
Involving the Mitochondrial Pathway in NIH3T3 Cells (12).
The present study was carried out to verify the possible cytotoxic
action of AgNPs synthesized using Gelidiella sp. on HEp2 cells,
evaluating morphology and the number of viable cells after
incubation with the plant extract in different concentrations and
duration of exposure.
MATERIALS AND METHODS
Collection of seaweed samples
Seaweeds were collected from the intertidal regions of the
Mandapam coastal regions (Lat.09˚ 17.417 N; Long.079˚ 08.558 E) of
Gulf of Mannar. Samples were washed with freshwater to remove
adhering debris and associated biota and identified as Gelidiella sp.
by CAS Botany, Madras University. Collected samples were
transferred to the lab in a cool bag, lyophilized, powdered and
stored in -4˚C.
Seaweed extraction
About 5gm of the powdered material were extracted with 1000ml of
sterilized double distilled water. Then the crude extract was blended
thoroughly and filtered through Whatmann filter paper No.1 (42
µm) and the residue was again blended with water and filtered. The
filtrate was stored and used for further analysis.
Synthesis of silver nanoparticles
900ml filtrate was treated with 100ml of aqueous 1mM silver nitrate
solution in an Erlenmeyer flask slowly under the magnetic stirring
conditions. The bio reduction of silver nitrate occurred within 10
min. at 121˚C. The color change to dark brown of the medium was
noted by visual observation indicating the formation of AgNPs,
 Bhimba et al.
which remain stable for more than 3 months without any changes in
the absorption spectrum. The reduction of pure Ag+ ions was
monitored by measuring the UV-Vis spectrum of the reaction
medium after diluting a small aliquot of the sample into distilled
water. Aliquots of the reaction solution were measured using a UV-
1601 Schimadzu spectrophotometer operated at a resolution of
1nm.
Fourier transform infrared spectroscopy
The interaction between protein-silver nanoparticles were analyzed
by Fourier transform infrared spectroscopy (FTIR) in the diffuse
refluctance mode at a resolution of 4cm-1 in the KBr pellets and the
spectra were recorded in the wavelength interval of 4000 to 400nm-
1. FTIR measurements were carried out to identify the possible
biomolecules responsible for the reduction of the Ag+ ions and the
capping of the bioreduced AgNPs synthesized by seaweed extract.
For comparison, the seaweed filtrate was mixed with KBr powder
and pelletized after drying properly and subjected to measurement.
X-ray diffractometry
X-ray diffraction (XRD) measurement of the seaweed reduced AgNPs
was carried out using powder X-ray diffractometer instrument
(PXRD-6000 SCHIMADZU) in the angle range of 10˚C-80˚C at 2θ,
scan axis: 2:1 sym. The size of the AgNPs was calculated from the
PXRD peak positions using Bragg’s law.
Extended Dispersive analysis X-ray Spectroscopy
The presence of elemental Silver was confirmed through EDS.
Energy dispersive analysis X-ray spectrometer takes advantage of
the photon nature of the light. In the X-ray range the energy of a
single photon is just sufficient to produce a measurable pulse X ray;
the output of an ultra low noise pre-amplifier connected to the low
noise is a statistical measure of the corresponding quantum energy.
A semiconductor material is used to detect the X-rays together with
processing electronics to analyses the spectrum. The EDX
observations were carried out in STIC, CUSAT, Kerala (JOEL Model
JED-2300).
SEM observation of silver nanoparticles
The sample was prepared by placing a drop of AgNPs on carbon
coated copper grid and subsequently drying in air, before
transferring it to the microscope operated at an accelerated voltage
of 120 KV (JOEL Model JSM-63 90 LV).
In vitro assay for Cytotoxicity activity (MTT assay).
Hep-2 (Human Epidermoid larynx carcinoma) cell lines were
obtained from National centre for cell sciences Pune (NCCS). The
cells were maintained in RPMI-1640 supplemented with 10% FBS,
penicillin (100 U/ml), and streptomycin (100 μg/ml) in a humidified
atmosphere of 50 μg/ml CO2 at 37 °C.
The Cytotoxicity of synthesized AgNPs on Human Epidermoid
Larynx cancer cells was determined by the MTT assay (Mosmann et
al., 1983). Cells (1 × 105/well) were plated in 100 μl of medium/well
in 96-well plates. After 48 hours incubation the cell reaches the
confluence. Then, cells were incubated in the presence of various
concentrations of the samples in 0.1% DMSO for 48h at 37°C. After
removal of the sample solution and washing with phosphate-
buffered saline (pH 7.4), 20µl/well (5mg/ml) of 0.5% 3-(4, 5-
dimethyl-2-thiazolyl)-2, 5-diphenyl--tetrazolium bromide cells
(MTT) phosphate- buffered saline solution was added. After 4h
incubation, 0.04M HCl/ isopropanol were added. Viable cells were
determined by the absorbance at 570nm with reference at 655nm.
Measurements were performed in 3 times, and the concentration
required for a 50% inhibition of viability (IC50) was determined
graphically. The absorbance at 570 nm was measured with a UV
spectrophotometer, using wells without sample containing cells as
blanks. All experiments were performed in triplicate. The effect of
the AgNPs on the proliferation of Human Epitheloid larynx cancer
cells was expressed as the % cell viability, using the following
formula:
% cell viability = A570 of treated cells / A570 of control cells × 100%.
Int J Pharm Pharm Sci, Vol 4, Suppl 4, 710-715
Data analysis
The IC50 values (concentration at which 50% of cells were death) are
reported as mean± standard deviation of three independent
experiments. The IC50 values against the Human laryngeal Hep-2
cancer cell lines were calculated for the synthesized AgNPs
inhibiting at least 50% inhibition when tested at a concentration.
One-way analysis of variance (ANOVA) and Student t-tests were
used to compare data using Statistica version 5.0 software at a 95%
confidence limit.
RESULTS AND DISCUSSIONS
Reduction of silver ion into Ag particles during exposure to the
seaweed extract could be followed by color change. AgNPs exhibit
dark yellowish-brown color in aqueous solution due to the surface
Plasmon resonance phenomenon (Fig.1). UV-Vis spectrograph of Ag
nanoparticles has been recorded as a function of time (Fig.2).
Absorption spectra of Ag nanoparticles formed in the reaction media
at 72 hrs has absorbance peak at 435 nm, broadening of peak. The
position and the number of peaks in the absorption spectra are
dependent on the shape of the particles: for an ellipsoidal particle
there are two peaks whereas for spherical particle there is only one
peak centered at 420 nm, indicating the formation of silver
nanoparticles (13). The absorption maximum at 420 nm is
attributed to the Mie scattering by silver metal (14).
The appearance of the yellow color indicated the formation of AgNPs
in the reaction mixture, as it is well-known that AgNPs exhibit
striking colors (light yellow to brown) due to excitation of surface
plasmon vibrations in the particles (15). It was reported that some
amount of OH- groups tended to promote the reduction of silver ions
in some chemical methods (16).
FT-IR spectrum of AgNPs is shown in Fig.3. This spectrum shows the
presence of bands at 3419, 1650, 1489, 1059 cm-1. The bands at
3419cm-1 corresponds to primary amine O-H band, 1650 cm-1
corresponds to primary amine N-H band, the band at 1489 cm-1 is
assigned to methylene scissoring vibration from the protein in the
solution, and the band at 1033 cm-1 were assigned to C-N stretching
vibration of the proteins (17). The positions of these bands were
close to that reported for native proteins (18). This evidence
suggests that the protein molecules could possibly perform the
function of the formation and stabilization of AgNPs in the aqueous
medium.
XRD pattern taken using powder X-ray diffractometer instrument
(PXRD-6000 SCHIMADZU) in the angle range of 10˚C-80˚C of the
AgNPs at 2θ, scan axis: 2:1 sym is shown in Fig.4. A number of Bragg
reflections corresponding to (111), (200), (220), (311) sets of lattice
planes are observed, which can be indexed to face-centered cubic
silver. The peaks matches with the Joint Committee on Powder
Diffraction Standards (file No. 04-0783), which further proves the
formation of crystal AgNPs (19). The peaks were identified as AgNPs
according to PCPDFWIN software (PDF#030921). The XRD pattern
thus clearly shows that the AgNPs are crystalline in nature.
Furthermore, the average diameter of the silver nanoparticles is
calculated as 6.4nm by the Scherrer formula
D = 0.89λ
βcosθ
Where D is mean grain size, λ is the wavelength for Cu target, β is the
FWHM of diffraction peak and θ is the diffraction angle. Thus XRD is
commonly used for determining the chemical composition and
crystal structure of a material. The line broadening of the peaks is
primarily due to small particle size. The X-ray diffraction results
clearly show that the AgNPs formed by the reduction of silver ions
by the extract of Gelidiella sp. are crystalline in nature (20).
EDS micro-analysis is performed by measuring the energy and
intensity distribution of X-ray signals generated by a focused
electron beam on a specimen. Fig.5 shows the EDS spectrum
recorded in the spot-profile mode. The optical absorption peak is
observed at 3KeV, which is typical for the absorption of metallic
AgNPs (21). Strong signals from the silver atoms are observed, while
weaker signals from C, O, P and Cl atoms are also recorded. Those
711
 Bhimba et al.
Int J Pharm Pharm Sci, Vol 4, Suppl 4, 710-715

weaker signals are likely to be due to X-ray emission from the
Gelidiella sp. From the EDS spectrums, it is clear that AgNPs reduced
by Gelidiella sp have the weight percentage of silver as 30.15%.
SEM technique was employed to visualize the size and shape of AgNPs.
The formation of AgNPs as well as their morphological dimensions in
the SEM study demonstrated that the average size was from 40-50 nm.
The shapes of the AgNPs were proved to be spherical (Fig.6.). Similar
phenomenon was reported by Chandran et al (22).
Cytotoxicity of silver nanoparticles
The invitro cytotoxicity of the AgNPs was evaluated Hep-2 cell lines
at different concentrations. Our cytotoxicity analysis of the sample
shows a direct dose-response relationship; cytotoxicity increased at
higher concentrations. The IC50 value was plotted by taking the
concentration of AgNPs on X-axis versus percentage of cell viability
on Y- axis (Fig.7). The samples demonstrated a considerable
cytotoxicity against the Hep-2 cell lines. The result showed that
Hep2 cells proliferation were significantly inhibited by AgNPs with
an IC50 value of 31.25 μg/ml of the concentration. As shown in
Table.No.1, in the lowest tested concentration (3μg/ml), AgNPs were
able to inhibit the cell line’s growth by less than 10%. In contrast the
presence of 15.62μg/ml of AgNPs significantly inhibited the cell
line’s growth (> 60%). Previous study shows that phytochemicals
depletes intracellular antioxidants thereby induced cancer cell death
(23).

Fig. 1: Photography of a) Gelidiella sp. extract. b) Formation of AgNPs

Fig. 2: UV-VIS absorption spectra of Ag nanoparticle synthesized from Gelidiella sp. at 1mM silver nitrate.

100

90
.58
9

7
.1

80
7
7

1
2

9
9

.8
2

9
3

9
.0

70
4

4
2

.7
5

0
2

7
.0
6
1

60
1
7

1
.1

50
4
8

2
1
T

.7

5
%

.4
9

5
1

5
4

8
3

40
3.3
3 7

0
.2
0
1

30
7
8

20
67.0

2
4

6.3

10
2
5

4
4
189.0

-10
4000 3500 3000 2500 2000 1500 1000 500
Wavenumbers (cm-1)

Fig. 3: FTIR spectra of capped silver nanoparticles synthesized using aqueous extract of Gelidiella sp.

712
 Bhimba et al.
Int J Pharm Pharm Sci, Vol 4, Suppl 4, 710-715

Fig. 4: XRD pattern of synthesized silver nanoparticles at 2θ

a) b)
Fig. 5: a) EDS spectra of AgNPs. Silver X-ray emission peaks are labeled. Strong signals from the atoms in the nanoparticles are observed
in spectrum and confirms the reduction of silver ions to AgNPs and b) the elemental composition of the AgNPs

Fig. 6: Scanning Electron Microscopy image of AgNPs

Table 1: Tumor cell proliferation inhibitory activity of synthesized AgNPs using aqueous extract of Gelidiella sp. on Hep-2 cell lines
S. No. Concentration (µg/ml) Dilutions Absorbance at 570nm %Cell viability %Cell inhibition
1 1000 Neat 0.05 9.80±0.41 90.20
2 500 1:1 0.11 21.56±0.13 78.44
3 250 1:2 0.14 27.45±0.26 75.55
4 125 1:4 0.19 37.25±0.82 62.75
5 62.5 1:8 0.23 45.09±0.35 54.91
6 31.25 1:16 0.28 54.90±0.91 45.10
7 15.625 1:32 0.33 64.70±0.73 35.30
8 7.8125 1:64 0.37 72.54±0.56 27.46
9 3.906 1:128 0.46 90.19±0.18 09.81
10 Cell control - 0.51 100 0.00

713
 Bhimba et al.
Fig. 7: Effect of AgNPs on Hep2 cell viability
Fig. 8: Photography of a) Cytotoxic changes observed Vs b) Control
CONCLUSION
In conclusion, the bio-reduction of aqueous silver ions by the
aqueous extract of Gelidiella sp. has been demonstrated. This
approach towards the synthesis of AgNPs can be easily scaled up
economically and eco-friendly. Applications of the synthesized
AgNPs in bactericidal, fungicidal and cytotoxic applications, makes
this method potentially for invivo method.
ACKNOWLEDGEMENT
We are grateful to Dr. Rengaswami, Director, CAS Botany, Madras
University for identifying the seaweed. The authors thank, Dr.
Sugumaran, Department of Animal Biotechnology, Veterinary
College, Vepery, Chennai for TEM analysis, Chemistry Lab for XRD
analysis. We also thank Mr. Magesh Peter, NIOT, Chennai for helping
us in the initial progress of work.
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