Biosynthesis of silver nanoparticles using

aqueous bark extract of Picea abies L. and

their antibacterial activity

Corneliu Tanase, Lavinia Berta,

Anca Mare, Adrian Man, Adina Iulia
Talmaciu, Ioana Roșca, Eleonora Mircia,
Irina Volf & Valentin I. Popa
European Journal of Wood and Wood
Products
Holz als Roh- und Werkstoff

ISSN 0018-3768
Volume 78
Number 2

Eur. J. Wood Prod. (2020) 78:281-291

DOI 10.1007/s00107-020-01502-3

1 23
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 Author's personal copy
European Journal of Wood and Wood Products (2020) 78:281–291
https://doi.org/10.1007/s00107-020-01502-3

ORIGINAL

Biosynthesis of silver nanoparticles using aqueous bark extract

of Picea abies L. and their antibacterial activity
Corneliu Tanase1 · Lavinia Berta1 · Anca Mare1 · Adrian Man1 · Adina Iulia Talmaciu2 · Ioana Roșca1 ·
Eleonora Mircia1 · Irina Volf2 · Valentin I. Popa2

Received: 13 March 2019 / Published online: 1 February 2020

© Springer-Verlag GmbH Germany, part of Springer Nature 2020

Abstract
In recent years, synthesis of nanoparticles using vegetal extract has gained much interest in nanobiotechnology. The aim
of this study is the biosynthesis of silver nanoparticles (AgNPs) from silver nitrate using spruce (Picea abies L.) bark as
a bioresource of cost-effective nonhazardous reducing and stabilizing compounds. The effects of various parameters such
as concentration of reactants, ratio extract/silver nitrate and time of incubation on the controlled formation of AgNPs were
explored. The AgNPs were characterized by X-ray diffraction, Fourier transform infrared spectroscopy, ultraviolet-visible-
near infrared spectroscopy and scanning electron microscopy. A stock solution of silver nitrate (0.1 M) was prepared. Dif-
ferent concentrations of silver nitrate (1, 2.5, 4, and 5 mM) were prepared from the above solution, then added to 5 mL of
spruce bark extract. The synthesis of AgNPs was confirmed by the change in the color of the mixtures from brown to grey.
It was noticed that the synthesized nanoparticles have sizes between 0.1 and 0.5 µm (100–500 nm) with an average diameter
of 0.226 µm (226 nm). The antibacterial activity of synthesized AgNPs was investigated in vitro. It was found that the most
potent antibacterial activity appeared in AgNPs compared to the aqueous extract or silver nitrate.

1 Introduction 2013; Velusamy et al. 2016; Sorbiun et al. 2018a; Taghavi

In general, metal nanoparticles can be prepared and sta-
bilized by physical and chemical methods. However, with
the rapid development of the industry, the use of biologi-
cal methods to protect the environment has been proposed.
Thus, the biosynthesis of metal nanoparticles, and in par-
ticular AgNPs, has attracted more and more attention of
researchers. There have been numerous studies to investigate
the biosynthesis of silver, gold, or other metals nanopar-
ticles, using plant extracts as reducing agent (Mittal et al.
Tanase Corneliu and Berţa Lavinia contributed equally to this
work.
* Anca Mare
mareanca@gmail.com
1
“George Emil Palade” University of Medicine, Pharmacy,
Science and Technology of Târgu Mureș, Gh. Marinescu
Street no. 38, 540139 Târgu Mureş, Romania
2
“Gheorghe Asachi” Technical University of Iasi, Faculty
of Chemical Engineering and Environmental Protection,
73 Prof. Dr. Doc. Dimitrie Mangeron Street, 700050 Iasi,
Romania
Fardood et al. 2017; Dogru et al. 2017; Ocsoy et al. 2018)
The use of plant extracts in the nanoparticles biosynthe-
sis is advantageous because most of them have a rich con-
tent of compounds such as flavonoids, terpenoids, tannins
and alkaloids responsible for ion metals reduction (Sorbiun
et al. 2018b, c; Parlinska-Wojtan et al. 2016; Kuppusamy
et al. 2016; Ahmed et al. 2016). In addition, the use of plant
extracts for metal nanoparticles biosynthesis is cheap, easy
to scale-up at industrial level and with a low environmental
impact. It is a suitable method, particularly when it is desired
to obtain pure nanoparticles with a controlled size and mor-
phology, which must be free from toxic contaminants such
as those used in therapeutic applications (Mittal et al. 2013).
Literature data have shown that the size, morphology, sta-
bility and properties of metal nanoparticles are strongly
influenced by the experimental conditions, the kinetics of
the interaction of metal ions with reducing agents, and the
nanoparticle stabilizing adsorption processes (Sharma et al.
2009).
Studying different properties of the AgNPs is an impor-
tant research field in nanotechnology. AgNPs can be used
in various applications such as medicine, cosmetic and
food industries (food packaging), bioengineering catalysts,

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electrochemistry or disinfection in environmental appli-
cations (Keat et al. 2015). For example, in the medicine,
AgNPs have been used, based on antimicrobial properties
for a large number of microorganisms (Seifi Mansour et al.
2019; Elumalai et al. 2019; Sorbiun et al. 2018a) or in the
manufacture of medical devices (cardiovascular and bone
implants), which have been recognized as being very effec-
tive in reducing the risk of pathogenic invasion (Amin et al.
2012; Mitiku and Yilma 2017). Another direction was the
use of AgNPs as an impregnating agent in textile fibers used
for making medical equipment (Tran et al. 2013). In recent
years, AgNPs have also been used in cosmetics due to their
antiseptic properties. They have been used as preservation
additives and as bioactive agents in facial therapies (Kokura
et al. 2010). Another application of AgNPs is in the manu-
facture of smart food packaging that ensures the system-
atic release of silver ions in the environment, preventing
the development of pathogens and microbial agents, thus
ensuring better conservation (Gottesman et al. 2011). As
applications in the field of environmental protection, AgNPs
have been used in various water/air treatment and purifica-
tion methods (Tran et al. 2013).
Spruce (Picea abies L.) is a widely used material in the
wood industry. The spruce bark resulted as a waste prod-
uct during wood processing is usually used for combustion.
Spruce bark extracts could be used for biological activity
like antitumor, antibacterial, antifungal or anti-inflammatory
(Yang et al. 2008; García-Pérez et al. 2012; Le Normand
et al. 2014; Coșarcă et al. 2019) due to polyphenolic com-
pounds content.
The main objectives of the current study were (1) biosyn-
thesis of AgNPs using spruce bark polyphenolic extract as a
novel bioresource; (2) determination of the influence of the
main factors on the biosynthesis process (concentration of
the reducing agent, time and ratio extract/silver nitrate); (3)
characterization of the AgNPs biosynthesized by specific
analyzes (FTIR, XRD and SEM) and (4) evaluation of the
antibacterial activity of the biosynthesized AgNPs.
2 Materials and methods
2.1 Chemicals
Silver nitrate (­ AgNO3), Folin–Ciocalteu reagent, and sodium
carbonate ­(NaCO3) were purchased from Sigma-Aldrich.
All other chemicals used in this research were of analytical
grade.
2.2 Materials
Spruce bark (Picea abies) used for experimental research
comes as waste from wood processing in a Romanian
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European Journal of Wood and Wood Products (2020) 78:281–291
company. The plant bark was identified by Corneliu Tanase
from Botany Department of the Faculty of Pharmacy at the
University of Medicine, Pharmacy, Sciences and Technol-
ogy. Prior to extraction, the bark was dried at room tem-
perature (18–20 °C) under normal aeration conditions, then
ground with a mill VEB NOSSENER MASCHINENBAU
1980 (Germany). The wood powder was passed successively
through a series of five sieves with diameters between 0.25
and 1.25 mm (< 0.25, 0.315, 0.40, 0.63, 1.00 and 1.25 mm)
in order to determine the particle size distribution.
For antibacterial activity, the following strains were used:
Gram positive bacteria—methicillin-sensitive Staphylo-
coccus aureus (MSSA) ATCC 25923, methicillin-resistant
Staphylococcus aureus (MRSA) ATCC 43300; Gram nega-
tive bacteria—Escherichia coli ATCC 25922, Klebsiella
pneumoniae ATCC 13883 and Pseudomonas aeruginosa
ATCC 27853. These bacterial strains were selected from
the Department of Microbiology (Faculty of Medicine-Uni-
versity of Medicine, Pharmacy, Sciences and Technology
from Târgu Mureș), considering that these are all patho-
genic strains involved in a broad spectrum of mild to severe
infections (skin, respiratory, urinary, sepsis, meningitis
etc.). In addition, all these strains present different resist-
ance mechanisms against antibiotics, and they can develop
new resistance mechanisms, being an important challenge
for treatment.
2.3 Ultrasound assisted extraction (UAE)
For the extraction of polyphenolic compounds from the
spruce bark, a Sonorex thermostatic ultrasonic bath RK
100H (Bandeline Electronic GmbH& Co.KG, Berlin, Ger-
many, frequency of 35 kHz, power of 80–320 W, a 1A inten-
sity and the possibility of adjusting the working temperature
from 30 to 80 °C) was used. The spruce bark (5 g) was
placed in an Erlenmeyer reaction vessel to which 50 mL of
water was added in a solid/liquid ratio of 1:10 (g sample/mL
water) so that the plant material is rapidly and completely
wet. The extraction process was performed at 60 °C for 30
min. After extraction, the obtained extracts were centrifuged
using a Hettich Rotofix 32 A centrifuge at 4000 rpm for 4
min, the supernatant being collected and used in the subse-
quent steps of the experiment.
2.4 Determination of total polyphenol content
(TPC)
The TPC of the spruce bark extracts was determined spec-
trophotometrically using the Folin–Ciocalteu method. For
the quantitative determination of the total polyphenol con-
tent, a GBS Avanta UV–VIS spectrophotometer with vari-
able wavelength ranging from 190 to 800 nm was used. The
method involves mixing 1 mL of extract with 0.5 mL of
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European Journal of Wood and Wood Products (2020) 78:281–291 283
Folin–Ciocalteu reagent, 2 mL of 10% sodium carbonate
solution and 5 mL of distilled water. The solution obtained
was left to stand for 90 minutes in the absence of light. The
TPC, expressed in mg GAE/g biomass, was calculated based
on the spectrophotometer reading for the wavelength of 765
nm, taking into account the calibration curve of the standard
gallic acid solution. Triplicate measurements were taken for
all samples.
2.5 Biosynthesis of silver nanoparticles using
polyphenolic extracts
The extracts were used without further pretreatment. For
the synthesis of silver nanoparticles, the extract was mixed
with a 1 mM silver nitrate solution. To enhance the reduc-
tion in ­AgNO3 ions, the prepared solutions were kept under
constant stirring and heated to a maximum temperature of
70 °C for variable reaction times.
The performance of the reduction reaction and the quan-
tities of the synthesized AgNPs were studied by the varia-
tion of parameters such as the concentration of the reducing
agent, the reaction time and the pH. Thus, the influence of
the concentration of silver ions (from the ­AgNO3 silver salt
used) was studied for 1 mM, 5 mM and 50 mM. In addition,
the accumulation of AgNPs in time was monitored for 24 h,
the absorbance reading of 2 in 2 h.
Another factor studied was the ratio of silver nitrate and
extract, ranging from 1:10, 1:50 and 1:100 mL of ­AgNO3
extract/mL. The influence of these factors was tested at
pH = 9 by adding 0.5 mL NaOH (1 mM).
2.6 Characterization of AgNPs
2.6.1 UV–VIS analysis
The presence of AgNPs in the obtained solutions was moni-
tored by spectrophotometric analysis (GBS Avanta UV–VIS
spectrophotometer) at wavelengths ranging from 380 to 580
nm. Triplicate measurements were taken for all samples.
2.6.2 Morphological and structural characterization
of silver nanoparticles by XRD, FTIR and SEM
The mixture resulting from the reaction between ­AgNO3
and the extract was purified by repeated centrifugation (2–3
centrifugations) at 4000 rpm for 30 min. The obtained sus-
pensions were first analyzed by laser diffraction (Shimadzu
SALD 7001 laser diffractometer) to determine the AgNPs
size distribution.
The dimensional analysis was based on a series of
approximations of the characteristics of AgNPs analyzed,
namely: the particles to be measured are spherical, the
particle suspension is diluted, the optical properties of the
particles and the environment are known, the particles are
homogeneous. After the dimensional analysis, the suspen-
sions were dried in the oven for 24 h at 40 °C; the resulting
powder being used for subsequent SEM analyzes (scanning
electron microscopy).
For SEM analysis/device SU 1510 HITACHI), a small
amount of AgNPs was dried, placed on the SEM slide and
covered with metallic gold spray (spray coating apparatus
108 Cressington auto).
FTIR spectra were recorded using a Bio Rad spectropho-
tometer in the spectral range of 400–4000/cm with a reso-
lution of 4/cm and 32 scans using samples in KBr pellets.
Triplicate measurements were taken for all samples.
2.6.3 Tests on antimicrobial activity and synergistic effect
of silver nanoparticles
The antibacterial activity of AgNPs was evaluated against
MSSA, MRSA, Escherichia coli, Klebsiella pneumoniae
and Pseudomonas aeruginosa using the microplate method
(Tanase et al. 2018). Shortly, binary dilutions of the tested
compounds were mixed in 96-well plates with equal amount
of 2× Muller–Hinton broth, plus bacterial inoculum, and
incubated at 35 °C for 24 h. The minimum inhibitory con-
centration MIC was interpreted in the first well where the
bacterial growth was completely inhibited.
The synergistic effect of silver nanoparticles was tested
for gentamicin using the disk diffusion method. A 0.5
McFarland inoculum (prepared from fresh overnight bacte-
rial cultures) was disseminated on Muller Hinton agar. 10
µg gentamicin disks were placed on the surface of the plate
and 10 µL of silver nanoparticles suspension was added on
the surface of the gentamicin disk. Gentamicin disks without
silver nanoparticles were used as control. After the plates
had been incubated for 18 h, the inhibition diameters were
measured.
2.7 Statistical analysis
All the experiments were carried out in triplicates and the
results were expressed as mean ± standard deviation.
3 Results and discussion
3.1 Characterization of the aqueous extract
The spruce bark was characterized in terms of humidity and
particle size distribution. Thus, the spruce bark used in the
experimental studies was characterized by a moisture con-
tent of 10.44% and a particle size distribution after grind-
ing, consisting of 55% particles with a size of 0.25–1 mm,
30% particle size less than 0.25 mm and 15% particle size
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between 1 and 2 mm. The average particle diameter of the
crushed particles was 0.6 mm. The aqueous extract used had
a TPC of 17.21 mg GAE/mL extract. The extract obtained
was preliminarily characterized in order to determine the
properties and potential uses of these bioactive compounds.
Thus, spruce bark extracts contain considerable quantities
of bioactive aromatic compounds, especially catechin, taxi-
folin, gallic acid and vanillic acid (Tanase et al. 2013, 2018).
3.2 Characterization of AgNPs
The AgNPs were studied by UV-VIS spectroscopy and char-
acterized by morphological, dimensional and structural fea-
tures using electron microscopy (SEM—for morphological
characterization), laser diffraction (XRD—for dimensional
analysis) and Fourier transform IR spectroscopy (FTIR—for
structural analysis).
3.2.1 UV–VIS analyze
The preliminary formation of AgNPs can be confirmed
by the color change of the solution obtained after mixing
the metal salt with the plant extract. The color change of
the aqueous suspension (1:10 extract: A ­ gNO3 1 mM) from
brown to gray (Fig. 1) was specific for the appearance of
AgNPs (visible after 30 min). Results of experimental stud-
ies show that by maintaining solutions in contact for a longer
time, it may take up to 18 h to completely reduce silver ions
(Banerjee et al. 2014).
Influence factors of the biosynthesis process were moni-
tored spectrophotometrically. Figure  2 shows that after
the reduction reaction of silver ions in the presence of the
extract, polydisperse nanoparticles were formed, and their
presence was confirmed by a broad absorption peak at a
wavelength of about 460 nm.
Fig. 1  Color modification indicating the formation of silver nanoparticles; a extract + AgNO3 solution at time t = 0; b extract + AgNO3 solution
at time t = 3 h
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3.2.2 Determination of the optimal reaction time
After confirmation that spruce bark extract has a reduction
effect of A­ g+ ions from the silver nitrate to A ­ g0 (Fig. 2),
the study was conducted to determine the optimal reaction
time with which the highest accumulation of AgNPs can be
obtained. Thus, Fig. 3 shows that the reaction is more rapid
in the first 2–3 h and the content of the AgNPs in aque-
ous suspension (absorbance value recorded) is continuing
to slowly increase to 18 h, and tends to stabilize to 24 h.
Further, it should be noted that the shape of the formed curve
changes and the maximum intensity being reached around
the wavelength of 440 nm in the first 2 h, moving to 460 nm
over the next few hours. The UV-is spectra indicate that the
increase in retention time results in gradual growth of the
nanoparticles to larger sizes (Mulfinger et al. 2007). This
could be a possible explanation for the shift of lambda max
during 1–18 h (Fig. 3).
3.2.3 Determination of the optimal concentration
of the reducing agent and nitrate/extract ratios
Another factor studied was the concentration of the silver
nitrate used. The tests performed showed that a higher con-
centration of ­AgNO3 (5 mM or 50 mM) leads to inhibition of
the synthesis process (Fig. 4). The same was observed when
higher nitrate/extract ratios (1:50 and 1:100) were used, as
can be seen in Fig. 5. Therefore, the ratio of 1:10 mL of
extract/mL of ­AgNO3 to the concentration of 1 mM and a
required reaction time of 3 h were considered as optimal
conditions for AgNPs biosynthesis. The same conditions
were also considered to be favorable when bark aqueous
extract from Syzygium alternifolium (Yugandhar et al. 2015)
or Pterocarpus santalinus (Zayed et al. 2015) were used as
silver ions reducing agents.
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Fig. 2  UV spectrum of aque-
ous solution of spruce bark
extract + 1 mM ­AgNO3 (3 h
reaction time, ratio 1:10)
Fig. 3  UV spectra recorded for
aqueous solution of spruce bark
extract + 1 mM ­AgNO3 (1:10
mL/mL) for variuos reaction
times
3.2.4 Morphological, structural and compositional
characterization of AgNPs
The dimensional analysis of AgNPs was performed by
diffraction using a Shimadzu SALD 7001 laser diffrac-
tometer. This method allows to determine the particle size
of colloidal dispersions containing particles or molecules
in Brownian motion. In Fig. 6, it can be observed that the
synthesized AgNPs range from 0.1 to 0.5 µm (100–500
nm) with an average diameter of 0.226 µm (226 nm).
Similar results have been reported for AgNPs synthesis
using hemp extracts in the study by Manjamadha and
Muthukumar (2016).
3.2.5 Analysis by FTIR spectroscopy
FTIR analyzes were conducted to identify the bio-molecules
­ g+ ions in the
that might be responsible for reducing the A
extract, but also those that may be involved in stabilizing
of formed AgNPs. FTIR spectrum corresponding to spruce
bark extract (Fig. 7a) indicates the presence of aldehyde
groups (peak at 1386/cm), carboxyl and carbonyl groups
(peak at 1608/cm) and hydroxyl specific groups (peak at
3402/cm) characteristic for phenolic compounds that might
be involved in reducing ­Ag+ to ­Ag0 ions. According to
Yugandhar et al. (2015) and Awwad et al. (2013), intense
peaks around the 3300–3400/cm and 1500–1650/cm wave-
lengths (visible in the spectrum characteristic of AgNPs,
Fig. 7b) can be attributed to OH groups in phenolic and
carboxyl structures. This suggests that the groups of the
compounds present in the reaction medium form a complex
layer or combinations around the nanoparticles that act as
stabilizing agents, preventing their agglomeration and pre-
cipitation. The possible mechanism of spruce bark extract
mediated synthesis and stabilization of AgNPs is shown
in Fig. 8. Thus, A­ gNO3 dissociates into silver (­ Ag+) and
−
nitrate ­(NO3 ) ions. After the protons are released from the
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Fig. 4  Influence of the concen-
tration of silver nitrate solution
on AgNPs accumulation
Fig. 5  Influence of the ratio
extract/silver nitrate on the
AgNPs accumulation
flavonoid, silver ions are reduced. These ions are grouped to
form AgNPs (Some et al. 2019).
SEM analyzes were performed to determine the mor-
phology of the obtained AgNPs, also providing informa-
tion on their size. Figure 9 shows that AgNPs look like
compact blocks, hindering the determination of their struc-
ture. This can be explained by the fact that during the dry-
ing process AgNPs agglomeration takes place in the form
of metallic films, which leads to the deterioration of their
individual structure. Therefore, in the future, it is proposed
to extend the experimental studies to find techniques for
drying and recovery of synthesized AgNP, to prevent their
agglomeration.
Antibacterial activity of AgNPs was determined by
microplate method in 12 dilutions of the studied solutions.
The results showed that the solutions containing AgNPs
express inhibitory activity against all tested bacteria at
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European Journal of Wood and Wood Products (2020) 78:281–291
lower concentrations compared to the aqueous extract,
respectively the silver nitrate solutions (used as control),
especially for MSSA, E. coli and K. pneumoniae (Table 1).
The synergistic tests were performed in order to evalu-
ate whether the silver nanoparticles enhance the antibac-
terial effect of gentamicin. All the bacterial strains were
tested, but only Klebsiella spp. exhibited a larger diameter
for the inhibition zone in the presence of AgNPs (Fig. 10).
For the rest of the bacterial strains, there were no differ-
ences between the inhibition diameters for gentamicin/
gentamicin with silver nanoparticles suspension (Fig. 10).
Literature data confirms these results. The antimicro-
bial susceptibility of gram-positive and gram-negative
bacteria depends on the structural characteristics of the
studied species, shape and size of the nanoparticles,
inoculation medium and exposure time (Srikar et  al.
2016). AgNPs synthesized using plant extracts present
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Fig. 6  Dimensional characterization of synthesized AgNPs

Fig. 7  FTIR spectrum of: a spruce bark extract and b AgNPs

strong antimicrobial activity and the inhibitory capac-
ity is dependent on the AgNPs concentration. It was also
found that AgNPs size plays an important role in reduc-
ing the microbial growth. Even if AgNPs have a high
degree of toxicity, the small nanoparticles allow faster
release of silver ions, which penetrate more easily into the
microbial cell, acting on its DNA. The DNA replication
may be affected because its molecules turn into condensed
form, leading to bacterial disfunction (Manjamadha and
Muthukumar 2016; Yan et al. 2018). The antibacterial
mechanism is explained by the interaction of AgNPs with
the bacterial DNA, which present sulfur and phosphorus

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groups as major components. This effect has been dem-
onstrated on strains of S. aureus and E. coli. (Mitiku and
Yilma 2017). Oxidative stress caused by the AgNPs (gen-
erating ROS—reactive oxygen species), increasing the
bacterial membrane permeability, interacting with bacte-
rial enzymes are also antibacterial mechanism, damag-
ing the bacterial cell (Shaikh et al. 2019). These are also
the mechanisms that can explain how AgNPs enhance
antibiotic activity. By increasing the membrane perme-
ability, AgNPs allow other molecules, such as antibiot-
ics, to realize a higher concentration inside the bacterial
cell, turning an inefficient drug into an efficient alternative
treatment option. By generating ROS, AgNPs can increase
the efficiency of those antibiotics that use ROS as antibac-
terial mechanism (Morones-Ramirez et al. 2013; Garza-
Cervantes et al. 2017; Katva et al. 2018; Liu et al. 2019;
Bankier et al. 2019).
The antibacterial mechanism is different, depending
on the bacterial genus or species. For example, it was
found that AgNPs have inhibitory effect on H. pylori,
and two theories of action have been speculated: small
amounts of AgNPs penetrate into the bacteria and inhibit
Fig. 8  Illustration of the proposed mechanism for the spruce bark extract mediated synthesis and stabilization of AgNPs
Fig. 9  SEM images of AgNPs synthesized using spruce bark extract
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European Journal of Wood and Wood Products (2020) 78:281–291
the respiratory chain, while another theory considers that
AgNPs inhibit urease from H. pylori (Amin et al. 2012).
The existing literature suggests that AgNPs may exhibit
antibacterial effect through a combination of mechanisms,
more likely than a single one; this can also explain the fact
that bacterial resistance against silver compounds is very
rare (Karimi et al. 2016; Yuan et al. 2017; Leid et al. 2012;
Siddiqi et al. 2018).
Although there are numerous data showing the anti-
bacterial effect of AgNPs, their toxicity is also recog-
nized (Matharu et al. 2018; Davenport et al. 2015). For
this reason, further investigations are needed until AgNPs
can be used in the treatment of patients. However, until
then, AgNPs can be used for water treatment, food indus-
try, disinfection, etc. By demonstrating an antimicrobial
capacity of AgNPs, and their potential to enhance antibi-
otic activity, a new research direction can be developed
in order to reduce the bacterial antibiotic resistance. The
present results suggest that the AgNPs synthesized using
plant extracts could eventually be considered as antibiotic
alternatives/adjuvants.
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European Journal of Wood and Wood Products (2020) 78:281–291 289

Table 1  Minimum inhibitory Bacteria MICs–AgNPs MICs–aqueous MICs–silver

concentrations (mg/mL) of solution (mg/ extract (mg/mL) nitrate (mg/
AgNPs against the tested mL) mL)
bacteria
(A) Staphylococcus aureus (MSSA) ATCC 25923 3.25 3.25 15
(B) Methicillin-resistant Staphylococcus aureus 7.5 7.5 30
(MRSA) ATCC 43300
(C) Escherichia coli ATCC 25922 3.25 15 15
(D) Klebsiella pneumoniae ATCC 13883 3.25 15 15
(E) Pseudomonas aeruginosa ATCC 27853 7.5 7.5 15

Fig. 10  Synergistic effect of AgNPs and gentamicin against: a Escherichia coli, b Klebsiella pneumoniae, c Pseudomonas aeruginosa, d MRSA

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4 Conclusion
The results state the potential use of spruce bark extract to
reduce ­Ag+ ions from the silver nitrate salt to ­Ag0 ions with
the formation of AgNPs.
Spectrophotometric studies have shown that a ratio of
1:10 mL extract/mL silver salt at a concentration of 1 mM
are the best conditions for performing the AgNPs synthesis,
using the spruce bark extract as reducing agent.
The results obtained showed that solutions containing
AgNPs have an inhibitory activity against all tested bacteria
(MSSA, MRSA, Escherichia coli, Klebsiella pneumoniae,
and Pseudomonas aeruginosa) compared to the aqueous
extract and silver nitrate solutions used as control.
The synergistic tests were performed in order to evalu-
ate whether the silver nanoparticles enhance the antibacte-
rial effect of gentamicin. Klebsiella spp. exhibited a larger
diameter for the inhibition zone in the presence of AgNPs.
This inhibitory capacity supports the potential use of
AgNPs in medical and pharmaceutical fields.
Acknowledgements  This work was supported by the University
of Medicine and Pharmacy of Tîrgu Mure Research Grant number
15609/12/29.12.2017.
Compliance with ethical standards  org/10.4103/pm.pm_120_17
Conflict of interest  The authors declare that they have no conflict of
interest.
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