Journal of Molecular Liquids 215 (2016) 3946

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Journal of Molecular Liquids

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

Enzymatic browning reduction in white cabbage, potent antibacterial

and antioxidant activities of biogenic silver nanoparticles
Arif Ullah Khan a,, Yun Wei a, Aftab Ahmad a, Zia Ul Haq Khan b, Kamran Tahir a, Shahab Ullah Khan c,
Nawshad Muhammad d, Faheem Ullah Khan a, Qipeng Yuan a,
a

State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing, PR China
Department of Chemistry, University of Science and Technology Bannu, 28100, Khyber pakhtoonkhwa- Pakistan
Department of Physics, University of Peshawar, 25000, Pakistan
d
Interdisciplinary Research Centre in Biomedical Materials, COMSATS Institute of Information Technology, Lahore 54000, Pakistan
b
c

a r t i c l e

i n f o

Article history:
Received 12 September 2015
Received in revised form 7 November 2015
Accepted 7 December 2015
Available online xxxx
Keywords:
Longan fruit juice
Silver nanoparticles
Enzymatic browning reduction
Antimicrobial activity
Antioxidant activity

a b s t r a c t
To produce economically benign and nontoxic enzymatic browning reducing and antimicrobial agents is the goal
of nanotechnology. In this study we synthesized environmentally friendly silver nanoparticles (AgNPs) using
Longan fruit juice as reducing and stabilizing agent. Silver nanoparticles synthesis was monitored by UVvis
spectroscopy showing localized surface plasmon resonance at 443 nm. XRD (X-ray diffraction analysis),
HRTEM (high resolution transmission electron microscopy) and EDX (energy dispersive X-ray analysis) were
used to characterize crystalline structure, size (410 nm), shape and elemental composition of silver nanoparticles. Surface capped phytochemicals were characterized by FTIR. Silver nanoparticles have signicant enzymatic
browning reduction (p b 0.001) using white cabbage as a model system. No research has been reported on the
Enzymatic browning reduction of biosynthesized silver nanoparticles. The silver nanoparticles showed prominent antibacterial activity with MIC values of 31.25 g/ml against Staphylococcus aureus and Basillus subtilus
while 62.5 g/ml against Escherichia coli. High cells constituents' release was exhibited by B. subtilus with 2
MIC value of silver nanoparticles. Silver nanoparticles exhibited no hemolytic activity against Red blood cells of
male Wistar albino rat. Silver nanoparticles also showed signicant DPPH free radical scavenging activity. This research would have an important implication for the synthesis of more efcient antimicrobial, antioxidant and
anti-enzymatic browning agents for food preservation, processing and other biomedical applications.
2015 Elsevier B.V. All rights reserved.

1. Introduction
To preserve the color of fruit juice during processing and storage is
one of the key objectives of fruit processors because changes in the
structure of fruit products change the color and nal appearance of
the product. Browning is one of the main factors that can change the
color of fruit juice and limits its' commercial shelf life [1,47]. Therefore
browning needs to be controlled during the processing stages of the
food to preserve their quality, beyond this the organoleptic and
nutritional properties will be strongly changed. For these reasons and
due to the importance of appearance as a quality parameter, the prevention of these undesirable reactions has always been a challenge for food
scientists [47,35].
Cabbage (Brassica oleracea L. capitata) is a versatile food and is increasingly becoming an important vegetable in restaurants, dinning
commons and fast food outlets because of its ease and less requirement
Corresponding authors.
E-mail addresses: khanbuct@gmail.com (A.U. Khan), yuanqp@mail.buct.edu.cn
(Q. Yuan).

http://dx.doi.org/10.1016/j.molliq.2015.12.019
0167-7322/ 2015 Elsevier B.V. All rights reserved.

for washing, cutting or shredding [21]. However, cabbage and a variety

of fruits and vegetables, such as lettuce, potato, apple and banana are
susceptible to enzymatic browning during processing and storage.
Enzymatic browning in vegetables is often associated with undesirable
brown colors, off-avors and lower nutritional value [42]. The browning
reaction needs the presence of oxygen, polyphenol oxidase (PPO) and
phenolic compounds and is generally triggered by the enzymatic
oxidation of monophenols into o-diphenols and quinones, which further undergo non-enzymatic polymerization leading to the formation
of pigments [36]. Although enzymatic browning is benecial to the
color and avor development of certain food items such as tea and coffee but it impairs the quality of fresh-cut produce [27]. About 30% loss of
quality in post-harvest food supplies including vegetables and fruits in
developing nations is due to enzymatic browning [20,34]. In recent decades, several methods have been used to prevent PPO activity in foods
[26,44,47,45,53,55]. However, U.S. Food and Drug Administration (FDA)
has restricted the use of sultes to inhibit the browning of foods,
because they have been associated with severe allergy-like reactions
in certain populations [11,44]. Moreover, heat treatments are not suitable for inhibiting this sort of reaction, and so several other methods

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A.U. Khan et al. / Journal of Molecular Liquids 215 (2016) 3946

have been tried to reduce PPO activity, including the addition of ascorbic
acid or chemical agents, the exclusion of oxygen, refrigeration and
various non-thermal treatments [29,51,52]. Similarly honeys can also
be used for enzymatic browning reduction [33].
Additionally for many decades, food borne ailments have been also
considered as serious threats to public health all over the world. In
food borne pathogens studies, four major pathogens have emerged
signicantly important in terms of human health and diseases. These include: Escherichia coli O157:H7, Listeria monocytogenes, Salmonella
typhimurium and Vibrio parahaemolyticus. These organisms have
commonly been related with food products and associated to a number
of human diseases [59]. E. coli O157: H7 is a major worldwide cause of diarrhea, hemorrhagic colitis and hemo-lytic-uremic syndrome. The disease
is often associated to the use of infected and undercooked ground beef as
well as unpasteurized fruit juices (A. K. [46,48]). L. monocytogenes has
been implicated in food borne outbreaks and subsequently isolated
from various products. Such as meat, milk, milk products, vegetables,
poultry and sh [12].
In this respect, there has been a renewal of interest in anti-browning
agents and inhibitors of microorganisms causing food spoilage, which
are cheap, economically benign, nontoxic and eco-friendly. Among
such reagents are the phytochemicals based silver nanoparticles
[2,4,19]. Plants have been known to internally bio-mineralize calcium
carbonate, silica and even magnetite. Certain plants are known to
hyper-accumulate these heavy metals within different parts of plants.
The internal accumulation of metals in plants can occur both via
complexation of the metal ion with a suitable bio-ligand in its native
oxidation state or after it's reduction to a lower oxidation state. The possibility of reduction of metal ions by plants and the presence of metal
complexing agents in them entices a materials scientist to use plants
for the purpose of synthesizing nanoparticles and controlling their size
and shape and to test the possibility of synthesizing nanoparticles of
low reduction potential metals [3]. Due to the improvement of antibiotic resistance in pathogenic bacteria, the pharmaceutical companies and
researchers are searching for novel antimicrobials and the AgNPs are
capable candidate for the same. The reduction of Ag+ ions leads to the
formation of silver atoms (Ag) which is followed by agglomeration
into oligomeric clusters. These clusters eventually lead to the formation
of colloidal AgNPs. When the colloidal particles are much smaller than
the wavelength of visible light, the solution possess characteristic
yellow color with an intense band in the 380430 nm range and other
less intense bands at longer wavelength in the UVvisible absorption
spectrum.
Silver nanoparticles can act as anti-browning agent, antioxidant, reducing agent and inhibitors of food borne pathogens. Here in we report
the phytochemicals inspired Ag nanoparticles using Longan fruit juice
as reducing, capping and stabilizing agent. These nanoparticles are
studied for their Enzymatic browning reduction in white cabbage, antioxidant (DPPH free radical scavenging), antibacterial activities against
food borne pathogens and Hemolytic activity against healthy RBCs of
Wistar albino male rat.
2. Materials and methods
2.1. Materials
Longan fruit and white cabbage were purchased from the local market. AgNO3, Vit. C, DPPH, Nutrient agar, nutrient broth and methanol
were purchased from Beijing chemical works, China. All solutions
were made in sterile double distilled (DD) water and methanol. All
apparatus were washed with aqua regia and rewashed with DD water.
2.1.1. Juice extraction
Longan esh juice was quenched using juice extractor. The juice was
centrifuged at 10,000 rpm for 10 min at 4 C to remove the solid fruit

material. The supernatant was used as a reducing, capping and stabilizing agent of Ag nanoparticles.
2.1.2. Syntheses
25 ml Longan fruit juice was mixed with 75 ml of 6 mM aqueous
solution of AgNO3 in 250 ml ask to synthesize Ag nanoparticles. The
mixture was magnetically stirred at room temperature. Ag nanoparticles
were separated from the colloidal solution by repeated centrifugation at
12,000 rpm for 10 min and 4 C. Then the Ag nanoparticles were freeze
dried using VirTis freeze mobile 6ES freeze drier.
2.1.3. Characterization
The biosyntheses of Ag nanoparticles was monitored frequently by
scanning the aliquot sample in the wavelength range of 350800 nm
in Shimadzu UV-2450 spectrophotometer. The XRD measurements
were examined by Rigaku Miniex X-ray diffracto meter. A Hitachi
EDX elemental microanalysis system and JEOL3010 high resolution
transmission electron microscope were used to determine the crystalline
nature, morphology, size and elemental analysis of the Ag nanoparticles.
Infra red (IR) spectrum was obtained using the KBr pellet technique on
an ABB MB3000 spectrophotometer.
2.2. Ability of Ag nanoparticles to reduce enzymatic browning
The ability of Ag nanoparticles to reduce enzymatic browning was
determined with a spectrophotometric assay [8] with a little modications. 2 g of fresh homogenates of white cabbage were taken in separate
test tubes and added 5 ml of double distilled (DD) water. Test samples
were incubated for 1 h with and without Ag nanoparticles. Ag nanoparticles concentration varied from 1 mg to 0.125 mg/g (1, 0.5, 0.25 and
0.125 mg) of total homogenate. After incubation, an aliquot of each
homogenate was extracted with aqueous methanol. The extract was
centrifuged at 11,200 rpm for 5 min and 4 C then the supernatant
was passed through a 0.22 m Teon lter before conducting the
spectroscopy. The absorbance was monitored at 420 nm against an
aqueous methanol blank. Enzymatic browning reduction was presented
in browning units, where a difference of 0.01 absorbance unit from
control per gram homogenate was considered to be equivalent to 1
browning unit. Therefore, samples with the highest numbers indicate
the greatest enzymatic browning reduction.
2.2.1. Micro organisms
Three food-borne pathogens including E. coli ATCC 8739 (Gram negative), Staphylococcus aureus ATCC 6538 and Basilus subtilus ATCC 6633
(Gram positive) were used in antimicrobial assay. These strains were
maintained on agar slants at 4 C in the College of life science and technology Beijing University of chemical technology, Beijing for antimicrobial tests. Micro organisms were incubated overnight at 37 C in
Mueller-Hinton Broth (Oxoid) at pH 7.4. Cephalexin (50 l of 4 mg/ml)
in sterile DD water was used as reference drug.
2.2.2. Antimicrobial screening
2.2.2.1. Screening for antibacterial activity. The antibacterial activity was
determined through agar well diffusion method [16]. All bacterial
strains were grown in nutrient broth at 37 C for 24 h incubation till
turbidity became equivalent to McFarland 0.5 turbidity standard. The
inocula of the B. subtilus, S. auroeus and E. coli were streaked on to the
condensed Muller Hinton agar (Oxoid) in petri plates by a sterilized cotton swab in order to make sure an uniform thick lawn or layer of growth
following incubation. Wells of 8 mm in diameter were formed with the
help of sterilized cork borer on to nutrient agar plates. The silver nanoparticles (50 l of 4 mg/ml) in sterile DD water were put into the wells
and the plates were allowed to stay for 2 h at room temperature. Finally,
the plates were incubated at 37 C for 2024 h and the resulting

A.U. Khan et al. / Journal of Molecular Liquids 215 (2016) 3946

diameters of zones of inhibition were measured. The experiments were

repeated three times and the data were calculated as means SD.
2.2.3. Determination of minimum inhibitory concentration (MIC)
Minimum inhibitory concentration (MIC) of Ag nanoparticles was
determined by agar dilution method [22,24]. The sterilized Muller
Hinton Agar (oxoid) was cooled to 50 C and 19 ml of this was added
to sterilized test tubes which contained 1 ml of different concentrations
of Ag nanoparticles. This mixture was smoothly shaken, mixed and
poured into pre-labeled sterile Petri dishes. Petri dishes having only
growth media were prepared in the same way so as to serve for comparison with Petri plates containing Ag nanoparticles. The concentrations of
the Ag nanoparticles used in this test ranged from 15.62 - 2000 g/ml
(15.62, 31.25, 62.5, 125, 25, 500, 1000, 2000 g/ml). The suspensions
of the respective microorganisms having density adjusted to 0.5
McFarland turbidity standards were inoculated onto the series of agar
plates using standard loop. The plates were then incubated at 37 C for
24 h. The lowest concentration which inhibited the growth of the respective microorganisms was taken as MIC. All tests were carried out
in triplicate.
2.3. Assessment of the cell constituents' release
The release of the cell constituents into the media was assessed as
previously described method [40] with a little modication. In brief,
working cultures of the three bacteria tested were collected by centrifugation (10 min, at 5000 g/min) after being grown for 24 h in the nutrient
broth for bacteria cells from 50 mL, washed 3 times with 0.1 M PBS
(pH 7.0), and the pallet was re-suspended in 50 mL 0.1 M PBS. The
cell suspensions were incubated at 37 C under agitation for 2 h in the
presence of silver nanoparticles at three different concentrations
(2 MIC, MIC, and control). 3 mL of sample suspensions were collected
and centrifuged for 2 min at 12,000 g/min and afterward, 2 mL of the supernatant was processed to measure UV absorption at 260 nm to determine the concentration of the cells constituents released in the media.
2.4. Hemolytic activity assay
The in-vitro hemolytic property of silver nanoparticles was assessed
by measuring the hemoglobin release from the red blood cells (RBCs) by
treatment with silver nanoparticles. The blood was collected from male
Wistar albino mouse in a tube containing EDTA. The tube was centrifuged at 1500 r/min for 10 min. The supernatant and buffy coat was
carefully removed and the pallet was washed three times with phosphate buffered saline (PBS) at pH 7.4. Silver nanoparticles in different
concentrations (12.5, 25, 50, 75 and 100 g) in PBS were put into
separate tubes and the cells (5% v/v) in PBS were added to each tube
and the nal volume was made up to 1 ml. RBCs in PBS and 1% TritonX100 solution were taken as negative and positive controls respectively.
These reaction mixtures were incubated in shaking incubator at 37 C
for 1 h with gentle shaking. Then the tubes were centrifuged at
1500 r/min for 10 min and the supernatant was monitored at 540 nm
against their blank. The percentage hemolytic activity was determined
as previously reported [54].

by UV 1100 spectro-photometer (MAPADA instruments) at 517 nm

against methanol as a blank. Vit. C was used as standard and DPPH
methanol reagent without sample was used as control. The percentage
of inhibition was calculated by the following formula.
%of inhibition Absorbancecontrol Absorbancetest =Absorbancecontrol 
 100
2.6. Statistical analysis
All experiments were performed in triplicate. The data was recorded
as mean standard deviation (SD) for the measurements. The data was
statistically analyzed by the statistical package (GraphPad prism V5).
3. Results and discussion
The synthesis of silver nanoparticles was observed based on visual
observation of the color change from dark brown to black within
30 min, followed by monitoring the rate of Ag nanoparticles biosynthesis using UVvis spectrophotometer (Shimadzu 2450). The appearance
of black color with aqueous extract is the clear indication of formation of
Ag nanoparticles. This color appears due to localized surface plasmon
resonance in Ag nanoparticles. The characteristic localized surface
plasmon resonance (LSPR) peak was not observed at the initial stage
but after 30 min the free electrons of silver nanoparticles give rise to
LSPR peak [25,32,39].
3.1. UVvis spectroscopy
The rate of Ag nanoparticles biosynthesis was monitored using UVvisible spectroscopy which is a very useful technique for the analysis
of nanoparticles [15]. The UV- vis spectra of reaction medium was
recorded as a function of time using aq.AgNO3 and Longan fruit juice
(Dimocarpus longan Lour) (Fig. 1). It is clear from Fig. 1 that with the
increase in contact time the intensity of localized surface plasmon
resonance (LSPR) peak (443 nm) increases because the concentration
of silver nanoparticles increases. The LSPR depends upon the size,
shape dispersion and the surrounding media of silver nanoparticles.
LSPR gives rise to peak which is well-documented for various metallic
silver nanoparticles ranging from 2 nm to 100 nm [15].
3.2. XRD graph showing crystalline Ag nanoparticles
The crystalline character of Ag nanoparticles was further conrmed
by X-ray diffraction analysis at 2080 (Fig. 2). Different numbers of

%Hemolysis Abs540 nm in the AgNPs solutionAbs540 nm in PBS

=Abs540 nm in 1%Triton X100 Abs540 nm in PBS  100

2.5. DPPH free radical scavenging assay

DPPH radical scavenging assay for Ag nanoparticles was performed
as previously described [10] with a little modication. 0.5 ml of 1 mM
DPPH were separately mixed with Different concentrations
(0.0311 mg/ml) of Ag nanoparticles and incubated in dark for
30 min. After incubation the absorbance of the samples was determined

41

Fig. 1. Time dependent UVVis spectra of Ag nanoparticles.

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A.U. Khan et al. / Journal of Molecular Liquids 215 (2016) 3946

Fig. 2. XRD pattern of Ag nanoparticles showing crystal structure of Ag nanoparticles.

Braggs' reection with 2theta values of 38.31, 44.44, 64.7 and 77 represent to the (111), (200), (220) and (311) set of lattice planes (Fig. 2).
Which may be indexed to the face centered cubic structure of Ag nanoparticles, which are in agreement with JCPDS le number 00-004-0783.
The peak corresponding to (111) is more intense than the other planes
suggesting that (111) is the predominant orientation as conrmed by
the HRTEM measurements.
3.3. EDX prole showing crystalline silver nanoparticles
The EDX prole of Ag nanoparticles showed strong signals for Silver
atoms as shown in Fig. 3. It clearly shows that the Ag nanoparticles are
in atomic state, which is caused by the reduction of silver ions. The
EDX analysis obtained in this study conrmed the presence of Silver
nanoparticles and mostly showed strong signal energy peaks for silver
atoms in the range of 3 keV which is typical for the absorption of metallic and spherical silver nano crystals due to surface Plasmon resonance
[18,28,30]. The unassigned peaks are of cupper grid. It is clear from
the EDX analysis that silver nanoparticles have C and O atoms which
means that organic compounds present in the Longan fruit juice capped
the silver nanoparticles and stabilized them.
3.4. HRTEM image showing the size and dispersion of Ag nanoparticles
High resolution transmission electron microscopy (HRTEM) was
performed to characterize the size, shape and morphology of synthesized silver nanoparticles. It is clear from the HRTEM image that the
morphology of the silver nanoparticles is mostly spherical which is in
agreement with the shape of LSPR band in UVvis spectra. The average

Fig. 3. EDX showing elemental composition of Ag nanoparticles.

Fig. 4. HRTEM of silver nanoparticles showing spherical shape and high dispersion of silver
nanoparticles.

particle size measured from the HRTEM image is 410 nm, which is in
good agreement with the particle size calculated from XRD analysis. It
is also cleared from the HRTEM image that the silver nanoparticles are
spherical, number is more, highly dispersed and there is no agglomeration among them (Fig. 4). Which are in close agreement with EDX
ndings. The smaller, spherical particle size and high dispersity of silver
nanoparticles leads to more active sites and large surface area which are
responsible for its efcient anti-enzymatic browning, antibacterial, and
antioxidant properties [5,37].
3.5. FTIR spectrum showing various groups capping Ag nanoparticles
FTIR spectrum (Fig. 5) was shown possible Bio-molecules taken part
in capping and stabilization of the Ag nanoparticles synthesized by the
Longan fruit juice. FTIR spectrum shows absorption peaks at 3365,
2911, 1633, 1521, and 1035 cm1 (Fig. 5). The absorption bands at
3370 cm1 and 2911 cm1 represent Hydrogen bonded OH stretch
and sp2 CH (alkane) vibration respectively. The peak at 1633 cm 1
was attributed to CC_C symmetric stretching vibration of aromatic
rings. The absorption peak at 1521 cm1 was attributed to NH bend

Fig. 5. FTIR spectra indicating different bio-molecules capping and stabilizing Ag

nanoparticles.

A.U. Khan et al. / Journal of Molecular Liquids 215 (2016) 3946

43

regression model in the current study explained 95% of the variability

in enzymatic browning reduction across 4 concentrations of Ag nanoparticles. We detected a high correlation between enzymatic browning reduction and Ag nanoparticles concentrations (r2 = 0.9777, F = 113.3,
p b 0.001). Our results suggest that the concentration of Ag nanoparticles
could probably indicate the strength of Ag nanoparticles to reduce
enzymatic browning. Ag nanoparticles are reducing agents and are
preferentially oxidized in foods, thus preventing or minimizing oxidative
avor and color deterioration. Ag nanoparticles can control enzymatic
browning, function as antimicrobial agent in food and as antioxidant
and reducing agent. Silver nanoparticles have three functions in controlling enzymatic browning in foods. They act as free radical scavengers,
change the red-ox potential of the system and act as reducing agents.
3.7. Antibacterial activity

Fig. 6. Enzymatic browning reduction (EBR) index of Ag nanoparticles.

Table 1
Antibacterial activity (zone of inhibition) of silver nanoparticles and standard.
Microorganisms

S. areus
B. subtilus
E. coli

Zone of inhibition in mm
Silver nanoparticles

Standard

18 (0.5)
20 (0.4)
18 (0.5)

9 (0.4)
9 (0.5)
11 (0.5)

mm = millimeter, standard = cephalexin, 35 C, 2024 h, 50 l of 8 mg/ml, standard

deviations.

vibration. The FTIR peak at 1035 cm1 might be contributed by the CN

bonds. It is clear from the FTIR spectrum that OH group present in the
Longan fruit juice is responsible for the reduction of silver ions to silver
nanoparticles through the oxidation of alcohol to aldehydic group. The
analysis of IR spectrum also provided an idea about biomolecules bearing different functionalities which are present in the underlying system.
3.6. Enzymatic browning reduction
The concentration of Ag nanoparticles ranged from 1 mg - 0.125 mg/g
showed varying abilities to reduce browning in white cabbage homogenates (Fig. 6). In this study the Enzymatic browning reduction for Ag
nanoparticles with browning units ranging from 3.3 0.15 to 8.41
0.38 units (higher values indicate stronger potential in enzymatic
browning reduction). Regression analysis revealed linear relationships
between enzymatic browning reduction and Ag nanoparticles concentration. Enzymatic browning reductions were found to increase with
increasing concentration of Ag nanoparticles (Fig. 6). The overall

The silver nanoparticles showed signicant antibacterial activities

against S. aureus, B. subtilus and E. coli (table 1). The zone of inhibition
against S. Aureus, B. subtilus and E. coli was 18( 0.5) mm, 20( 0.4)
mm and 18(0.5) mm respectively. Our results showed high antibacterial activities against the studied bacterial strains than the recently published report (M. Harishny et al.). While that of the standard Cephalexin
were 9 (0.4) mm, 9 (0.5) mm and 11(0.5) mm against S. areus, B.
subtilus and E. coli respectively. The presence of ionic silver for bacterial
contact affects the antibacterial activity of silver containing composites
[6]. It is suggested that the negatively charged bio-macromolecular
components (phosphate and disulde or sulfhydride of enzymes) and
nucleic acids can interact with positively charged Silver ions, causing
deformation and structural changes in bacterial cell walls and membranes that lead to disruption of metabolic process followed by cell
death. It is suggested that the negatively charged bio-macromolecular
components (phosphate and disulde or sulfhydride of enzymes) and
nucleic acids can interact with positively charged Silver ions, causing
deformation and structural changes in bacterial cell walls and
membranes that lead to disruption of metabolic process followed by
cell death [7,31]. It is believed that surface area of Silver nanoparticles
also have a great role in the antibacterial activity because surface area
most likely act as a reservoir for releasing Silver ions. The antibacterial
mechanism of silver nanoparticles is related to the membrane damage
caused by the free radicals from the surface of nanoparticles [23]. Moreover Silver nanoparticles can accumulate in the bacterial cytoplasmic
membrane causing a notable increase in membrane permeability with
subsequent cell death [49]. The images of zone of inhibition against
different bacteria are shown in Fig. 7.
3.8. MIC of Ag nanoparticles
Because of signicant antibacterial activity of Ag nanoparticles, it
was further processed to determine the MIC (minimum inhibitory
concentration).The MIC values were 62.5 g for E. coli and 31.25 g for
both S. areous and B. subtilus (Table 2). The lethality of silver for bacteria

Fig. 7. Images indicating clear zone of inhibition of antibacterial activity of Ag nanoparticles against Basilus subtilus, Staphylococcus areous and Escherichia coli.

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A.U. Khan et al. / Journal of Molecular Liquids 215 (2016) 3946

Table 2
MIC of Ag nanoparticles against the tested bacterial strains.

Table 4
Hemolytic property of silver nanoparticles on RBCs at different concentrations.

Bacterial strains

MIC

Sample (n = 3)

Hemolytic activity (%) (OD540 nm)

Staphylococcus aureus
Basilus subtilus
Escherichia coli

31.25 g
31.25 g
62.5 g

Control
1% Triton X-100
AgNPs (12.5 g)
AgNPs (25 g)
AgNPs (50 g)
AgNPs (75 g)
AgNPs (100 g)
AgNPs (125 g)

1.22 0.18
102 0.0
1.2 0.11
1.25 0.15
1.25 0.0
1.27 0.12
1.3 0.11
1.32 0.13

g = microgram, 35 C, 2024 h,

can also be in part explained by thiol group reactions that inactivate enzymes [9,13]. Also, Steuber and colleagues suggested a mechanism for
Ag + action in Vibrio alginolyticus involving the direct displacement of
FAD from the holo-enzyme NaNQR, which results in loss of enzyme
activity [50]. In summary, silver treatment inhibits DNA replication,
expression of ribosomal and other cellular proteins and interferes with
the bacterial electron transport chain [57].

hemolytic activity against RBCs even at higher concentrations (up to

1024 g/ml) [41].
Experiments are in triplicates and the results are presented as
mean standard deviation. OD540 = optical density at 540 nm.

3.9. Cells constituents' release

3.11. Antioxidant activity of Ag nanoparticles

In addition, we assessed and measured the cell contents release to

prove that the cells membranes were broken enough that their constituents are released into their media. Following the application of silver
nanoparticles, the cell constituents' release was assessed by measuring
the absorbance of the supernatants of three bacteria (S. aureus,
B. subtilus and E. coli) at 260 nm. Table 3 shows the results of cells
constituents' release of the bacteria understudy after being treated
with silver nanoparticles at the concentrations of 0, MIC, and 2 MIC
for 2 h. The results showed that after the addition of silver nanoparticles
to the bacterial cultures, the cell constituents' release have increased
with an increase of the concentration of silver nanoparticles as compared to the control. The most efcient cell constituents' release was
shown by B. subtilus following the treatment with a concentration of
2 MIC of the silver nanoparticles, showing an absorbance of 0.775.
The authors reported that a high loss or release of cell constituents indicate an irreversible damage to the cytoplasmic membranes. Thus, the
experimental results may give evidence that silver nanoparticles could
take an irreversible injure or damage to the cytoplasmic membranes,
which might be resulted with a more deteriorated shape of the treated
cells.

Since the reducing power of compounds is directly proportional to

antioxidant activity, antioxidant activity of Ag nanoparticles was
assessed by DPPH scavenging assay by using Vit. C as positive control.
DPPH is a stable compound and accepts hydrogen or electrons from silver nanoparticles. The results obtained in the DPPH assay showed effective free radical inhibition by Ag nanoparticles (Fig. 8). In this dose
dependent assay six different concentrations were used and the antioxidant activity was found to be increased with increasing concentrations
of AgNPs. Same results were shown with enhanced DPPH scavenging
activity by selenium, platinum, silver nanoparticles ([14,43,58,17])
and by torolex and chitosan coated gold nanoparticles [38,58]. According to some researchers the antioxidant property has been reported to
be related with the development of reducing power. Reductones,
which have strong reducing power, are generally believed not only to
react directly with peroxides but also to prevent peroxide formation
by reacting with certain precursors [56]. Antioxidants control the activity of free radicals hence they will support the body's immune system,
allowing the system to signicantly deal with viruses and other
elements that invade the body.
4. Conclusion

3.10. Hemolytic activity

Hemolytic activity of silver nanoparticles was carried out in order to
assess the biocompatibility on normal cells, especially RBCs. The hemolytic assessment was performed using different concentrations of silver
nanoparticles (12.5, 25, 50, 75, 100 and 125 g/ml) on RBCs. It was
found that silver nanoparticles have no hemolytic activity against
RBCs on different tested concentrations (Table 4). The obtained results
were found to be correlated with the previously reported research
[54]. Ruden et al. also reported that silver nanoparticles exhibit no

Silver nanoparticles are synthesized using Longan fruit juice as

reducing and stabilizing agent by a nontoxic, economically benign and
reproducible method. The prepared AgNPs are spherical in shape and
well dispersed with an average size of 410 nm, as clear from HRTEM
results. AgNPs showed signicant Enzymatic browning reduction with

Table 3
Effect of silver nanoparticles concentration on the cells constituents' release of bacterial
strains.
Bacteria

Silver nanoparticles
concentration

Cells constituents' release

(OD260 nm)a

Staphylococcus aureus

0
MIC
2 MIC
0
MIC
2 MIC
0
MIC
2 MIC

0.008 0.001b
0.424 0.002
0.686 0.017
0.078 0.006
0.457 0.015
0.775 0.022
0.021 0.004
0.370 0.003
0.665 0.032

Basilus subtilus

Escherichia coli

a
The experiments are in triplicates and the results are presented as mean standard
deviation. OD260 nm optical density at 260 nm, b Means within the same strain.

Fig. 8. Dose dependant antioxidant activity of silver nanoparticles.

A.U. Khan et al. / Journal of Molecular Liquids 215 (2016) 3946

an Enzymatic browning reduction index of 8.4 (p b 0.001). In addition

the silver nanoparticles exhibited an efcient antibacterial activity
against food borne pathogens and cause effective cells constituents'
release in bacteria. Furthermore, silver nanoparticles did not exhibit
hemolytic activity on RBCs and found nontoxic to normal healthy
cells. Silver nanoparticles also exhibited prominent DPPH free radical
scavenging activity. This study provides a cost effective and ecofriendly protocol to synthesize more effective and non-toxic Enzymatic
browning reducing, antioxidants and antibacterial agents for use in food
preservation and processing industry as well as other biomedical
applications. The use of silver nanoparticles may provide a future alternative to current toxic and expansive Enzymatic browning reducing,
antioxidants and antibacterial agents.
Acknowledgments
The authors would like to acknowledge nancial support from
National Natural Science Foundation of China (no. 21376017 and no.
(21176018).
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