Bioprocess Biosyst Eng

DOI 10.1007/s00449-016-1666-x

ORIGINAL PAPER

Antibacterial, anti-biofilm and anticancer potentials of green

synthesized silver nanoparticles using benzoin gum
(Styrax benzoin) extract
Juan Du1 • Hina Singh1 • Tae-Hoo Yi1

Received: 7 June 2016 / Accepted: 2 August 2016

Ó Springer-Verlag Berlin Heidelberg 2016

Abstract This study described a simple and green Keywords Benzoin gum
Biosynthesis
Silver
approach for the synthesis of silver nanoparticles (AgNPs) nanoparticles
Antibacterial
Anti-biofilm
Cytotoxicity
employing benzoin gum water extract as a reducing and
capping agent and their applications. The AgNPs were
characterized by ultraviolet–visible spectrophotometer, Introduction
X-ray diffraction pattern, field emission transmission
electron microscopy, dynamic light scattering, zeta poten- Nanoparticles (NPs) cause attraction of researchers because
tial and fourier transform infrared spectroscopy. The of their unique properties, owing to their small size
AgNPs showed promising antimicrobial activity against (1–100 nm), large surface-to-volume ratio and increased
various pathogens (Gram-negative, Gram-positive and reactivity [1]. Specifically, AgNPs play a significant role in
fungus) and possessed high free radical scavenging activity the field of biology and medicine, and have been com-
(104.5 ± 7.21 % at 1 mg/ml). In addition, the AgNPs monly used as antimicrobial and antifungal agents for a
exhibited strong cytotoxicity towards human cervical can- wide variety of products, such as medical dressing, pack-
cer and human lung cancer cells as compared to the normal aging, cosmetics, paints, spray and textile fabrics and
mouse macrophage cells. Moreover, the AgNPs possessed toothbrushes [2]. Generally, AgNPs are prepared by
anti-biofilm activity against Escherichia coli, and com- chemical methods which are usually carried out at high
patibility to human keratinocyte HaCaT cells, which sug- temperatures with chemical reducing agents [3, 4]. During
gests the use of dressing with the AgNPs in chronic wound the chemical synthesis process, reducing agent donates
treatment. Therefore, AgNPs synthesized by benzoin gum electrons to the Ag?, resulting in reverting Ag? to Ag0.
extract are comparatively green and may have broad The biological approaches of synthesis AgNPs employing
spectrum potential application in biomedicine. plant extracts and microorganisms is termed as economic
and environmental friendly biomaterial and received
tremendous attention [5]. The active ingredients contained
in plant extracts act as reducing agents. It is found that the
catalytic and antibacterial properties of AgNPs depend on
& Tae-Hoo Yi their size, shape and capping agents [6, 7]. Reducing
[email protected] agents, reaction media, temperature and Ag precursors
Juan Du influence the size and shape of AgNPs, since they define
[email protected] the pressure of the reaction. If the reaction pressure is high,
Hina Singh the process of nucleation and growth of the nanoparticles is
[email protected] greatly accelerated, and the opposite effect will occur at
1
low pressure, causing various shapes and sizes of synthe-
Department of Oriental Medicinal Biotechnology, College of
sized AgNPs.
Life Science, Kyung Hee University, Global Campus, 1732
Deokyoungdaero, Giheung-gu, Yongin-si, Human beings and animals are often infected by
Gyeonggi-do 446-701, Republic of Korea pathogenic microorganisms in the living environment,

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and the marked increased resistance of pathogen to modified eagle medium (DMEM), and penicillin–strep-
antibiotics in the past two decades is a growing concern tomycin were purchased from Gibco BRL (Grand
for doctors and medical officials world-wide [8]. One of Island, NY, USA). Raw 264.7 (mouse macrophage),
the modes by which bacteria exert this persistent Hela (human cervical cancer), A549 (human lung can-
infections resistant to systemic antibiotic therapy is cer) and HaCaT (human keratinocyte) cells were
their ability to develop biofilms [9]. Biofilms are bac- employed from Korean Cell Line Bank (KCLB, Seoul,
terial communities encased in a self-produced extra- Korea). Media used for bacterial cultures were procured
cellular polymeric substance (EPS) to create more from Oxoid Ltd. (Basingstoke, England). Benzoin gum
complex structures [10]. Chronic wounds are a severe powder (Styrax benzoin), which purchased form
worldwide problem, and they are ideal environment for Mountain Rose Herbs (Eugene, Oregon, USA), was
bacterial colonization. Compared to planktonic cells originally harvested from Indonesia. The pathogenic
biofilm cells may play an important role, causing bacterial cultures Candida tropicalis ATCC 18807,
infection of both acute and chronic wounds. Around Pseudomonas aeruginosa ATCC 10145, E. coli ATCC
60 % of chronic wounds contain biofilms, which rep- 43894 and Staphylococcus aureus ATCC 6538 were
resent a challenge to the treatment [11]. The AgNPs purchased from American Type Culture Collection
have a broad antibacterial activity, and have been (ATCC, Manassas, VA, USA).
commonly used as an essential additive in number of
products such as medical dressing, packaging, cosmet- Preparation of the benzoin gum extract
ics, paints, spray and textile fabrics for their inhibitory and synthesis of AgNPs
effects on microbes [2]. Apart from the antimicrobial
properties, AgNPs exhibit a cytotoxic behavior to nor- 50 g of benzoin gum powder was boiled in a conical flask
mal and cancer cells. Considering this, in this study we containing 500 ml of deionized water for 20 min. The
aim to find a fast, simple and nontoxic strategy to water extract was centrifuged, supernatant obtained was
synthesis the AgNPs which exhibit anti-biofilm activity filtered through a 0.45 lm PVDF syringe filter (SmartPor,
and have low cytotoxic effects and generate a better Seoul, Korea). Then, aqueous solution of AgNO3 at a final
biocompatibility to normal cells. concentration of 1 mM was added to the extract. The
Benzoin gum is a balsam obtained from Styracaceae mixture was stirred on a magnetic stirrer at 60 °C. The
trees of the family Styracaceae and produced mainly in synthesis of AgNPs was indicated by color change of the
Asia [12]. Benzoin gum has been widely used in flavor and reaction mixture. The AgNPs were collected by high speed
fragrance industry owing to its pleasant, sweet balsamic centrifugation at 22,000 rpm for 30 min. The pellet
odor, and also is used in pharmaceutical and traditional obtained was washed thoroughly and air dried for charac-
Chinese medicine preparations as an ingredient and carrier terization and further studies.
[13]. The main constituents of benzoin gum are coniferyl
benzoate, benzoic acid, p-coumaryl benzoate, cinnamyl Characterization of the AgNPs
cinnamate, vanillin and siaresinolic acid [13, 14]. In this
study, we synthesized the AgNPs by benzoin gum water The morphology of the AgNPs was analyzed using FE-
extract, evaluate the ability of antibacterial and antibiofilm TEM (JEM-2100F, JEOL, Tokyo, Japan), operated at a
of the AgNPs and their compatibility with human ker- voltage of 200 kV. UV–vis spectrums of the AgNPs were
atinocyte HaCat cells, for its possible application in recorded between the wavelength range from 300 to
chronic wounds; and evaluate their cytotoxic effects in 800 nm (Optizen POP; Mecasys, Daejeon, Korea). The
human cervical cancer cells, human lung cancer cells and functional groups capped on surface of the AgNPs were
mouse macrophage cells, for its possible application in identified using a FTIR spectroscopy (Spectrum One
cancer treatment. System, Perkin-Elmer, Waltham, MA, USA). Benzoin
gum water extract powder was prepared and performed
FTIR. The hydrodynamic size and zeta potential of the
Materials and methods AgNPs were investigated by DLS analysis using a Mal-
vern Zetasizer Nano ZS90 (Malvern Instruments,
Materials Worcestershire, UK). The selected-area electron diffrac-
tion (SEAD) and XRD (D8 Advance, Karlsruhe, Bruker)
Silver nitrate (AgNO3), crystal violet solution, MTT, were used to detect the crystal structure of the synthesized
dimethyl sulfoxide (DMSO), methanol, DPPH and AgNPs. Moreover, energy dispersive X-ray spectroscopy
sodium bicarbonate were purchased from Sigma-Aldrich (EDX) and elemental mapping of the AgNPs were
(St. Louis, USA). Fetal bovine serum (FBS), Dulbecco’s performed.

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Antimicrobial testing Cell culture and MTT assay

Disk diffusion method on Mueller–Hinton agar (MHA) The cytotoxicity of the AgNPs on HaCaT, Raw 264.7, Hela
plates was used to determine the antimicrobial activity of and MCF-7 cells was studied. Cells were maintained in
the AgNPs. Briefly, 100 ll fresh overnight inoculums of C. DMEM medium supplemented with 1 % penicillin–strep-
tropicalis, E. coli, S. aureus and P. aeruginosa cultures tomycin and 10 % FBS at 37 °C in a humidified atmo-
were spread on MHA plates. The sterile paper discs of sphere containing 5 % CO2. Stock solution of AgNPs was
8 mm in diameter loaded with 30 ll of the AgNPs (500 prepared in sterile deionized water and diluted using the
and 1000 ppm) were placed on the surface of the inocu- medium (without FBS). HaCaT Cells (2 9 103/well) and
lated plates. The plates were incubated for 24 h at 33 °C Raw 264.7, Hela and MCF-7 cells (104/well) in 0.2 ml
and the inhibition zone (diameter in mm) of each well was medium/well were plated in triplicate in 96 well cell cul-
measured. The assay was carried out in triplicate for all the ture plates (SPL Lifescience, Pocheon, Korea) and incu-
test organisms. bated for 72 h. Cells were treated with different
concentrations of the AgNPs and incubated for a further
Anti-biofilm testing 24 h, non-treated cells were used as control. Following the
incubation, MTT at a final concentration of 0.1 mg/ml was
The anti-biofilm activity of the AgNPs was determined by added to each well and further incubated for 3 h. Viable
colorimetric method against strain E. coli. Fresh overnight cells were determined by measuring UV absorption at
inoculum of E. coli (400 ll/well) was added in triplicate to 570 nm. The effect of the AgNPs was expressed as the cell
wells of 48-well tissue culture plates (SPL Lifescience, viability %, using the following formula: cell viability
Pocheon, Korea). Biofilms were formed after culturing for % = Abstreated cells/Abscontrol cells 9 100.
48 h, the medium were removed from the wells and dif-
ferent concentrations of the AgNPs ranging from 1 to
10 lg/ml in sterile water were added to the wells, sterile Results and discussion
water were used as control. After 24 h incubation at 33 °C,
the solutions were removed and wells were washed with Characterization of the AgNPs
sterile water. The plates were air dried for 1 h and 400 ll
of 1 % (v/v) crystal violet solution were added in each well The color of benzoin gum extract was changed from clear
and incubated for 30 min. Thereafter, the crystal violet to deep brown within 5 h (Fig. 1a, b), indicated the for-
solution was removed and wells were washed with sterile mation of AgNPs, as a result of the surface plasmon res-
water. For the quantification of adherent cells, the crystal onance phenomenon [15]. UV–Vis spectra of the mixture
violet was solubilized with 200 ll of 95 % (v/v) ethanol. were shown in Fig. 1c and d, a peak at around 450 nm was
A FilterMax F5 Microplate Reader (Molecular Devices, observed, confirmed the formation of the AgNPs. Using
Sunnyvale, CA, USA) was used to measure the absorbance DLS, the AgNPs in aqueous solution showed a narrow size
at 575 nm. The effect of the AgNPs was expressed as the distribution ranging from 12 to 38 nm, average hydrody-
biofilm formation %, using the following formula: biofilm namic size and zeta potential were observed to be
formation % = Abstreated/Abscontrol 9 100. 18.7 ± 1.2 nm and -27.1 ± 0.5 mV, respectively
(Fig. 1d, e). The zeta potential value gave an important
Antioxidant assay idea regarding surface charge and indicated that the AgNPs
were stable and discrete in aqueous solution. FE-TEM
Free radical scavenging activity was determined by DPPH images revealed the majorities of the AgNPs formed were
assay. 20 ll of different concentrations of the AgNPs were spherical in shape (Fig. 2a, b). The SAED pattern (Fig. 2c)
placed in a 96-well plate, 180 ml of DPPH (0.2 mM) in showed four rings which can be indexed to (111), (200),
methanol was also added. DPPH solution without silver (220) and (311) lattice planes of face-centered cubic silver,
nanoparticles served as the blank. Control was prepared as respectively. In the XRD pattern (Fig. 3) of the AgNPs,
above solutions without AgNPs. The plate was standing at diffraction peaks at 2h values of 38.05°, 44.17°, 64.42° and
37 °C for 30 min, and the absorbance was measured by the 77.31° were identified and matched with JCPDS databases
Microplate Reader at wavelength of 520 nm. Arbutin was of the standard silver (file No. 04-0783). Those 2h values
used as positive control. The effect of the AgNPs was can be indexed to respective (111), (200), (220) and (311)
expressed as DPPH radical scavenging activity %, using the diffraction planes, in line with the result of SAED. The
following formula: scavenging capacity % = 100 - elemental mapping (Fig. 4a, b) and EDX (Fig. 4c) showed
[(Abssample - Absblank)/Absorbance control] 9 100. The study the distribution of silver element and characteristic signals
was done in triplicate. of silver atoms, respectively. The presence of signals of

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Fig. 1 Digital images of

benzoin gum extract (a) and the
synthesized silver nanoparticles
(b); UV–vis spectra of the
AgNPs (c) and the control
which contains benzoin gum
extract and AgNO3, DLS
(d) and surface zeta potential
(e) result

Fig. 2 FE-TEM images (a, b), and SAED pattern (c) of the synthesized AgNPs

copper and carbon in EDX was because of the use of FE- functional groups [17]. The peaks around 2957 and 2872,
TEM grid [16]. 2923 and 2852/cm is attributed to C–H asymmetric and
The FTIR spectrum obtained from benzoin gum water symmetric stretching vibration of methylene [18]. The
extract powder and the AgNPs were shown in Fig. 5a and minor as well as sharp peak at 1642/cm is due to C=C
b, respectively. The broad peak around 3419 and stretching vibration of alkenyl groups in benzoin gum
3421 cm-1 can be assigned to O–H stretching vibration of extract [19]. The peaks at 1598 and 1583/cm is attributed to

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Additionally, the broad peak around 1070 and 1077/cm can

be assigned to C–O–C stretching vibration [23]. Among
them, the peaks of 1642 and 1558/cm in benzoin gum
extract lost intensity in the AgNPs, band 1450/cm is shifted
to 1376/cm, and band 1441/cm is shifted to 1207/cm,
which indicate the involvement of the corresponding
groups mentioned above during the reduction of Ag? to
Ag0, suggesting the role of coniferyl benzoate and p-cou-
maryl benzoate in the synthesis of the AgNPs.

Antibacterial activity of the AgNPs

AgNPs were known for the capable of antimicrobial

Fig. 3 XRD spectra of synthesized AgNPs
activity, to overcoming the limited resource of antibiotics,
AgNPs had been studied and shown the potential as a new
C=C–C stretching vibration of aromatic rings [20]. The generation of antimicrobials [24]. In this study, antimi-
peak around 1558/cm can be assigned to carboxy- crobial activity of the AgNPs were tested against a range of
late group stretching vibration. The band observed at pathogenic microorganisms including Gram-positive S.
1451/cm is due to C–H stretching vibration of methylene aureus, Gram-negative E. coli, P. aeruginosa, and fungal
bridge [21]. The strong band observed at 1411/cm is strain C. tropicalis (Fig. 6). The inhibition zones of the
attributed to C–H stretching vibration of vinyl [22]. AgNPs against pathogenic strains are shown in Table 1.

Fig. 4 The elemental mapping

of synthesized AgNPs: TEM
image used for mapping (a) and
silver distributions (b); EDX
spectrum of synthesized AgNPs
(c)

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Fig. 5 FTIR spectrum of benzoin gum water extract powder (a) and the AgNPs (b)

Fig. 6 Antimicrobial activities

of synthesized AgNPs (30 ll) at
500 and 1000 ppm
concentrations in water against
P. aeruginosa (a), S. aureus (b),
E. coli (c) and C. tropicalis (d)

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Table 1 Antimicrobial activity of the AgNPs against selected Therefore, we checked the cytotoxic effect of the AgNPs
pathogenic strains on human keratinocyte HaCaT cells. The AgNPs exhibited
Pathogenic microorganism Zone of inhibition (mm) no significant cytotoxic effect up to 10 lg/mL concentra-
tion (Fig. 7b). This good biocompatibility indicated that
AgNPs (500 ppm) AgNPs (1000 ppm)
the AgNPs display anti-biofilm activity without being
P. aeruginosa 11.0 ± 0.7 12.6 ± 0.6 harmful to human keratinocyte HaCat cells at this con-
S. aureus 9.9 ± 0.5 11.7 ± 0.6 centration, and were further able to be used for prevention
E. coli 9.5 ± 0.5 13.7 ± 0.3 and treatment of bacterial infections in chronic wounds.
C. tropicalis 9.3 ± 0.6 10.6 ± 0.4 The anticancer activity of the AgNPs was evaluated by
cytotoxicity on Raw 264.7 (non-cancerous), Hela (human
The experiments were done in triplets and the results were interpreted
in terms of standard deviation of mean diameter of zone of inhibition cervical cancer) and A549 (human lung cancer), at differ-
ent concentrations (1, 2, 5 lg/mL). The AgNPs exhibited
The results indicated that the antibacterial activity ability biocompatible nature with Raw 264.7 cells at low con-
of AgNPs against all the tested bacterial strains, and the centrations (1 and 2 lg/mL); however, they were found to
activity was increased with the increasing of AgNPs be cytotoxic to A549 and Hela in a dose dependent manner
concentrations. under identical culture conditions (Fig. 8). The cell via-
bility % of Hela and A549 cells was decreased to
Anti-biofilm assay 54 ± 3.85 and 40.0 ± 2.45 %, respectively, after treat-
ment with AgNPs at quite low concentration of 1 lg/ml.
The majority of the bacteria in natural habitats live as The cell viability of Raw 264.7, Hela and A549 cells was
biofilms, which have up to 1000 times more resistant to decreased to 56.4 ± 5.7, 40.5 ± 5.3 and 44.8 ± 2.4 %,
antimicrobial agents than those in a planktonic state [25]. respectively, after the concentration increased to 5 lg/mL.
However, most of the antibiotics and the methods for These results in this study indicated that the AgNPs may
antimicrobial work have been developed for planktonic have potential as inhibitors of Hela and A549 cells growth
bacteria. There is a great need to meet the demands of in low concentrations. AgNPs are known to be cytotoxic to
effective anti-biofilm therapy. Herein, we studied the effect cancer cells, through the various mechanisms [26, 27].
of AgNPs on biofilm formation of strain E. coli with Recently reports suggested that the generation of ROS play
respect to wide distribution of the strain. E. coli grown in a pivotal role in the AgNPs induced cytotoxicity in cancer
wells were treated with concentrations of AgNPs ranging cells. The possible mechanisms is AgNPs trigger intracel-
from 1 to 10 lg/mL. Treatment for 24 h resulted in lular ROS by inhibiting the synthesis of intracellular
approximately 10.9, 44.1, 54.0 and 65.5 % decrease in antioxidant systems, and the generated ROS favors the
biofilm formation at 1, 2, 5 and 10 lg/mL concentration, DNA damage leading to cell death [28, 29]. According to
respectively (Fig. 7a), indicate that the AgNPs were able to this, the free radical scavenging activity of the AgNPs may
inhibit the biofilm formation of E. coli. contribute to the cytotoxicity against A549 and Hela cells.
The low cytotoxic effect of the synthesized AgNPs against
Cytotoxic effect of the AgNPs Raw 264.7 and HaCaT cells may because of the functional
groups capped on surface of the AgNPs, those groups may
Despite of its excellent antibacterial and anti-biofilm biocompatibility and low toxicity. Thus, the AgNPs could
activities, the potential biological applications of AgNPs be a good candidate for the treatment of the cancer cells via
were limited due to their cytotoxicity against human cells. cytotoxic mechanisms.

Fig. 7 Effect of AgNPs on

biofilm formation of strain
E. coli (a), MTT cytotoxicity
assay of AgNPs against human
keratinocyte HaCat cells (b).
Experiments were performed in
triplicates; mean ± SD are
shown

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radical scavenger. These results indicate that the AgNPs

could work as potent antioxidants.

Conclusion

Synthesis of AgNPs using benzoin gum extract is an

ecofriendly and relatively new manner. The synthesized
AgNPs were characterized by several instrumental
techniques, and the efficacy of the AgNPs was further
Fig. 8 MTT cytotoxicity assay of AgNPs against Raw 264.7, A549 studied. The AgNPs showed excellent antimicrobial
and Hela cell lines (values are mean ± SD of three determinations)
ability against all the tested pathogenic strains. The
AgNPs exhibited the anti-biofilm activity, and the cell
viability studies in human keratinocyte HaCat cells
showed good biocompatibility at concentrations up to
10 lg/ml, suggesting its potential application as an agent
in chronic wound healing. In addition, the AgNPs pos-
sessed antioxidant property and remarkable toxicity to
Hela and A549 cells even at concentrations of 1 lg/ml,
suggesting its possible use anticancer agent. However,
further researches are needed to study the mechanism
and develop the technology into therapeutic and pre-
ventive strategies.

Acknowledgments This work was conducted under the industrial

infrastructure program (No. N0000888) for fundamental technologies
Fig. 9 DPPH free radical scavenging assay of AgNPs and arbutin which is funded by the Ministry of Trade, Industry & Energy
(values are mean ± SD of three determinations) (MOTIE, Korea).

Antioxidant efficacy of the AgNPs Compliance with ethical standards

Conflict of interest The authors declare that they have no competing

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molecules that are forms as natural byproduct during the
body’s metabolic reactions, the dramatically increase of
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