Enzyme and Microbial Technology 102 (2017) 60–66

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Enzyme and Microbial Technology

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

Self assembled snowball-like hybrid nanostructures comprising Viburnum MARK

opulus L. extract and metal ions for antimicrobial and catalytic applications
⁎
Nilay Ildiza, Ayse Baldemirb, Cevahir Altinkaynakc, Nalan Özdemird, Vedat Yilmazc, ,
⁎
Ismail Ocsoyc,
a
Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Erciyes University, Kayseri, 38039 Turkey
b
Department of Pharmaceutical Botany, Faculty of Pharmacy, Erciyes University, Kayseri, 38039 Turkey
c
Department of Analytical Chemistry, Faculty of Pharmacy, Erciyes University, Kayseri, 38039 Turkey
d
Department of Chemistry, Faculty of Science, Erciyes University, Kayseri, 38039 Turkey

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

Keywords: Herein, we report the production of novel organic-inorganic nanobio-catalytic and antimicrobial agents called
Viburnum opulus “nanosnowball” (NSBs) with a rational design and elucidate the increase in the catalytic and antimicrobial
Hybrid nanosnowball activities. The NSBs resemble to guelder rose were formed of the extract of Viburnum opulus (VO) as the organic
Inhibitory property component and copper (II) ions (Cu2+) as the inorganic component. The effects of the VO extract and Cu2+
Bacterial and fungal pathogens
concentrations on the morphology of NSBs were systematically examined and characterized with several
techniques such as SEM, FT-IR, EDX and XRD. Our results demonstrated that the presence of CueO bonds in
NSBs could be indication of VO extract-Cu2+ complexes. Interestingly, the NSBs exhibited peroxidase-like
activity towards guaiacol used as a model substrate depending on Fenton-like reaction. While free VO extract did
not show antimicrobial activity at indicated concentration (2000–125 μg/mL), the NSBs showed effective
antimicrobial activity against bacterial (Escherichia coli ATCC 35218, Salmonella typhi ATCC 14028, Enterococcus
faecium ATCC 8459, Enterococcus faecalis ATCC 29212, Bacillus cereus ATCC 11778, Staphylococcus aureus ATCC
25923, except Pseudomonas aeruginosa ATCC 27853 and Haemophilus influenza ATCC 49247) and fungal
pathogens (Candida albicans ATCC 10231, C. glabrata ATCC 90030), respectively.

1. Introduction extracts have been used to form metallic nanomaterials (NMs)

Various plants have been actively used in different forms due to
intrinsic properties of their contents as antimicrobial, anticancer and
antioxidant agents. There is still high demand about plant related
studies to explore their new functions or to enhance properties of plant
based products [1–4]. Viburnum opulus L. (Caprifoliaceae family)
referred to as “snowball or guelder rose” is grown in many European
countries [5,6]. It is worthy to mention that V. opulus (VO) comprise
sources of antimicrobial compounds, such as polyacids, phenolic
glucosides, anthocyanins, tannin, terpenoide, iridoide, and some or-
ganic acids [7–10]. In addition to that, protein content of VO fruit and
seed was determined around 5% [11–13]. The polyphenolics presented
in all plants with various rates have potential antimicrobial activity
against several types of pathogens. For instance, ethanol and methanol
extracts and juices of VO fruits inhibit some Gram positive and Gram
negative bacteria growth [14,15].
The various biomolceules including DNA, cellulose and plant
[16–20]. The main purpose of plant extract usage is to provide a
simple, one-step, cheap and eco-friendly procedure in fabrication of
biocompatible NMs. Additionally, it is considered that using plant
extracts for NMs formation may enhance their aforementioned intrinsic
properties compared to extracts. Up to now, many types of single or
bimetallic NMs in various sizes and shapes have been producing with
the use of whole plant or plant tissue extracts [21–27]. The metal ion
reduction ability of plant extracts has been known since 1900s
however, reduction mechanism is not well documented yet. Use of
plant extracts as reducing and capping agents has attracted consider-
able much attention within the last three decades for the biosynthesis of
NMs [28–32].
Herein, we rationally present self-assembly of the VO extract with
coordination of metal ion for the formation of snowball-like hybrid
nanostructures and demonstrate their peroxidase like and antimicrobial
activity. We report the production of novel organic-inorganic nanobio-
catalytic and antimicrobial agents called “nanosnowball” (NSBs) with a

⁎
Corresponding authors.
E-mail addresses: vyilmaz@erciyes.edu.tr (V. Yilmaz), ismailocsoy@erciyes.edu.tr, ismailocsoy66@gmail.com (I. Ocsoy).

http://dx.doi.org/10.1016/j.enzmictec.2017.04.003
Received 23 November 2016; Received in revised form 27 March 2017; Accepted 7 April 2017
Available online 08 April 2017
0141-0229/ © 2017 Elsevier Inc. All rights reserved.
N. Ildiz et al. Enzyme and Microbial Technology 102 (2017) 60–66

Fig. 1. (a) SEM image of NSBs when 0.1 mg/mL−1 VO extract was used. Inlet: photograph of natural VO flower, (b) Apperance of pores on the high resolution image of (a), (c) NSBs was
not formed when 0.5 mg/mL−1 VO extract was used. (d) SEM image of copper phosphate nanocrystals formed without VO extract.

rational design and elucidate the increase in the catalytic and anti- 2. Materials and methods
microbial activities. The NSBs resemble to guelder rose were formed of
the extract of Viburnum opulus (VO) as the organic component and 2.1. Chemicals and reagents
copper (II) ions (Cu2+) as the inorganic component. The morphology of
those nanostructures called nanosnowballs (NSBs) was also studied as a Bovine serum albumin (BSA), copper sulfate pentahydrate, metha-
function of extract concentration. The NSBs composed of two compo- nol, phosphoric acid, NaCl, KCl, Na2HPO4, KH2PO4, HCl, NaOH and
nents, VO extract as organic component and copper ion (Cu2+) as Coomassie brilliant blue G-250 were purchased from Sigma-Aldrich.
inorganic components, were characterized with several techniques
including SEM, FT-IR, EDX and XRD. The extract-Cu2+ complexes in 2.2. Preparation of VO extract
the NSBs can be shown with Cu-O bonds. The presence of Cu2+ in NSBs
promoted them to exhibit peroxidase-mimicking activity depending on Dried fruits of VO were used to make the methanolic extract.
Fenton-like reaction mechanism against guaiacol. Powdered material was extracted with methanol at room temperature
Antibiotic resistance of microorganisms is continuously rising and for three days. Then, the extract were filtered through Whatman filter
progressing by one species to another [33], for this reason there is paper No. 1 (pore size 25 μm) and evoporated to dryness in vacuo at
basically no developed antimicrobial agent available for human use −40 °C. The dried extract was stored in refrigerator at −20 °C for
without resistance. The high resistance to different pathogen micro- further studies.
organisms attracts attention to antimicrobial agents. The health in-
dustry can prevent and control the spread of antibiotic resistance by
2.3. Synthesis of VO extract based-Cu2+ hybrid nanosnowball
investigating in research and development of new antibiotics, vaccines,
diagnostics and other tools [34]. In addition to that, the NSBs were also
The snowball-like hybrid nanostructures (NSBs) were synthesized
used as very effective antimicrobial agents towards the different
following modified reported method [35–41]. Typically, VO extract
pathogens even at low concentrations compared the extracts. We claim
with concentrations of 0.1 and 0.5 mg mL−1 were separately added into
that plant extract incorporated NSBs may expand their uses as promis-
the mixture containing 50 mL of 10 mM PBS (pH 7.4) and 0.8 mM Cu2+
ing catalytic and antimicrobial agents in a variety of technical and
ion. The each mixture was vortexed for 30 s, and then was incubated at
scientific fields. In this respect, green synthesis of hybrid nanostructure
4 °C for 3 days without disturbing. The precipitates occurred at the
or plant extract containing antimicrobial components is important, and
bottom of reaction tubes were collected and washed with water using
has potentially promising applications against the increasing amount of
centrifugation at 10,000 rpm for 15 min. The washing process was
resistant pathogen microorganisms that is a constant threat to human
repeated 3 times and the final products were dried at 50 °C for futher
health.
characterization and use.

2.4. Characterization of VO extract based-Cu2+ hybrid nanosnowball

The NSBs were characterized using Scanning Electron Microscopy

(SEM, ZEISS EVO LS10), Fourier Transform Infrared Spectroscopy

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N. Ildiz et al. Enzyme and Microbial Technology 102 (2017) 60–66

Fig. 2. (a) EDX spectra of the nanosnowball-like morphology (b) XRD patterns of the nanosnowball like morphology consistent with the peak position of the Cu3(PO4)2·3H2O (lower line,
JCPDS card (00-022-0548)_. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

(FTIR, Perkin Elmer 400 FT-IR Spectrometer Spotlight 400 Imaging tion of the each resulting mixture, the changes in absorbance values
System), Energy Dispersive X-Ray (ZEISS EVO LS10) and X-Ray based on oxidation of guaiacol were also recorded at 470 nm using a
Diffraction (XRD, Bruker AXS D8 Advance Model) analysis. UV–vis UV–vis spectrophotometer.
spectra were collected with HITACHI UV–vis spectrophotometer.

2.6. Evaluation of antimicrobial activity

2.5. Catalytic activity of VO extract based-Cu2+ hybrid nanosnowball
The catalytic activity of identical amount of VO extract and the
NSBs were tested towards guaiacol using spectroscopic method. A
typical activity measurement protocol (pH 6.8, 0.1 M KH2PO4, 25 °C)
was used. The VO extract and the NSBs were separately dissolved in
1 mL of PBS and it was followed by addition of 1 mL of 22.5 mM
hydrogen peroxide (H2O2) and 1 mL of 45 mM guaiacol. After incuba-
The Gram negative (E. coli ATCC 35218, S. typhi ATCC 14028 P.
aeruginosa ATCC 27853, H. influenza ATCC 49247) Gram positive (E.
faecium ATCC 8459 E. faecalis ATCC 29212 B. cereus ATCC 11778, S.
aureus ATCC 25923) bacterial strains and fungi (C. albicans ATCC
10231, C. glabrata ATCC 90030) were obtained from Inonu University
Medical Microbiology Laboratory collection. VO extract and the NSBs
were tested to determine their antimicrobial activities based on Clinical

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N. Ildiz et al. Enzyme and Microbial Technology 102 (2017) 60–66

Laboratory Standards Institute (CLSI) guidelines by using disc diffusion

Fig. 3. FTIR analysis of (a) free extract and (b) nanosnowball-like morphology.
Fig. 4. Catalytic activity of VO extract and NSBs.
and broth microdilution method [32–44]. Disc diffusion method was
applied for only NSBs samples at concentration that determined in the
minimal inhibitory concentration (MIC) method because VO extract
were not effective at specified concentrations. The bacterial cultures
were sub-cultured in Mueller Hinton broth (MHB, HIMEDIA, India) and
incubated at 37 °C for 14 h. Then, the bacteria cultures measured at 0.5
Mc Farland were swabbed onto petri plates containing Mueller Hinton
agar (MHA, HIMEDIA, India) using sterile cotton swab. The sterile discs
containing 30 μg/mL of sample were placed on the MHA. The plates
were incubated and inhibition zones were measured after completion of
incubation period. The MIC values were determined by the broth
microdilution method which was carried out in 96-well microtitre
plates in triplicate at a dilution of the tested samples from 2000 to
125 μg/mL for the extracts and from 700 to 0.7 μg/mL for NSBs.
Negative control formed MHB and the positive control was appropriate
antibiotics for bacteria and MHB + bacterial suspension only. Then
MHB, bacterial suspension and samples were added in each well. After
incubation at 37 °C for 18–24 h, the plates were visually examined for
bacterial growth. The well containing the lowest concentration of the
compound with no visible bacterial growth was taken as the MIC value.
The experiments were conducted in triplicates.
The in vitro antifungal susceptibility was performed by disc diffusion
and broth microdilution methods recommended by the CLSI documents
[45,46]. L-Glutamine, sodium bicarbonate-free RPMI 1640 broth added
with 0.2% glucose was used as a media to determine MICs and
inhibition zones. The same media used for disc diffusion method by
adding 1.5% agar. The MICs were obtained with the final concentration
ranged from 0.5 × 103 to 2.5 × 103 cells/mL after incubation at 35 °C
during 72 h. Broth, each compounds and fungus added to well. After
24–48 h, plates were determined by visually. The sterile disk that
contains 30 μg/mL sample impregnated then placed to the prepared
agar surface that contains fungus inoculum in disc diffusion method.
Amphotericin B was used as a positive control and dimethyl sulfoxide
(DMSO) was used as a negative control. Each experiment was
performed in duplicate and repeated three times. Results were eval-

Fig. 5. Diameter of inhibitory zones for NBSs by disc diffusion method.

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N. Ildiz et al. Enzyme and Microbial Technology 102 (2017) 60–66

Fig. 6. MIC values of NBSs by broth microdilution method.

uated visually. The NSBs have quite spherical shapes and very narrow size
distrubition with diameters of ∼8.0 μm size. Interestingly, the SEM
image of NSBs resembles to natural VO flower. Also surprisingly, no
3. Results and discussion
formation of NSBs was observed when 0.5 mg/mL−1 VO extract was
used. We claim that high concentration of extract triggers formation of
3.1. Characterization of VO-Cu2+ hybrid nanosnowball
metal-extract complex and prevents growth step for NSBs formation.
The chemical structures of VO extract and NSBs were characterized
In this study, NSBs were formed of VO extract and Cu2+ ions. It is
using FTIR. The very broad and weaks bending vibration of OePeO
well known that VO extract contains different types of compounds, such
groups appeared in the range of 500 ∼538 cm−1 (Fig. 3a) while very
as valeric acid, tannins, iridoid [47], proanthocyanidins [48], arbutin,
sharp and strong bending vibration was at ∼558 cm−1 (Fig. 3b).
valerianic acid, flavonoids [49]; chlorogenic acid [7], L-malic acid,
It could be assumed that molecules in the extract were involved in
ascorbic acid, phenolic compounds [50], catechin, epicatechin, cyani-
the NSBs with different conformations. It is worthy to mention that
din-3-glucoside, cyanidin-3-rutinozit, quercetin [51], polysaccharides
extract molecules were packed in the NSBs with different conforma-
[52], amino acids and proteins. Among those compounds, molecules
tions, which increase localized extract molecules concentration and
containing amine and diols groups may interact with copper phosphate
may lead to strong and shifted vibration peaks. The vibration bands of
to form NSBs. The formation mechanism of protein-inorganic hybrid
NH2 groups were at ∼1608 cm−1 and ∼1630 cm−1, for free extract
snowballs was described before [35,39,40]. The mechanism consisted
and NSBs, respectively. The stretching bands of CH2 and CH3 groups
of three main steps (1) nucleation for formation of primary nanocrys-
were at ∼2930 cm−1 and ∼2950 cm−1 for free extract and NSBs,
tals, (2) growth for formation of large petals with Cu2+ through
respectively. The two peaks at ∼1717 cm−1 and ∼3256 cm−1 were
coordination reactions (3) completion for end point of growth process
assigned to C]O groups and OH groups from phenolic compounds in
[53].
the free extract, respectively.
The morphology of NSBs was investigated as function of VO extract
concentrations (0.1 mg/mL−1 and 0.5 mg/mL−1). Although both VO
extract concentrations caused occurrence of blue precipitate, which can
3.2. Catalytic activity of VO extract and the NSBs
be considered as formation of NSBs, only 0.1 mg/mL−1 VO extract
formed NSBs (Fig. 1a). The pores on the surface of NSBs were observed
The catalytic activity of VO extract and the NSB were measured
with the high-resolution image (Fig. 1b). Also surprisingly, no forma-
against guaiacol in the presence of the H2O2. The absorbance changes of
tion of NSBs was observed when 0.5 mg/mL−1 VO extract was used
the product were spectrophotometrically recoded at 470 nm (Fig. 4).
(Fig. 1c). Likewise, the NSB like morphology was not observed in the
While VO extracts showed the slight catalytic activity due to the less
absence of VO extract as SEM image of copper-phosphate nanocrystals
number of negatively charged groups in thier content, the much
shown in Fig. 1d.
catalytic activity was exhibited via the NSBs which have plenty of
The structure and morphology of the NSB were systematically
negatively charged groups and Cu2+ ions. The negatively charged
characterized via several techniques, SEM, EDX, XRD and FTIR. The
groups may oxidize the substrate by producing Cu1+ ions in the NSBs
presence of Cu metals in the NSBs by the technique of EDX as shown
depending on the Fenton like reaction [54,55].
Fig. 2a. The diffraction peaks in the NSB corresponding to Cu3(PO4)2.
3H2O nanocrystals were consistent with the JCPDS card (00-022-0548)
as presented in Fig. 2b.

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N. Ildiz et al.
3.3. Determination of antimicrobial activity
In terms of antimicrobial properties, Cesoniene et al. [14] and
Sagdic et al. [15] demonstrated that VO fruit extracts show mild-
antimicrobial activity based on concentrations against Gram positive
and Gram negative bacteria. Currently, no studies have evidently
reported the formation of plant extracts-incorporated hybrid nanos-
tructures. In this study, free VO extracts did not show an effective
antimicrobial activity up to 2000 μg/mL concentration. However the
NSBs displayed much antimicrobial activity against microrganisms
except P. aeruginosa ATCC 27853 and H. influenza ATCC 49247.
Additionally, the NSBs caused the effective cell inactivation of Candida
albicans and C. glabrata. Results are shown in Figs. 5 and 6.
We proposed that the plenty of negatively charged groups in the
may promote the interaction between the NSBs and bacterial cell
membrane, which leads to inhibit the cell replication and eventual cell
death due to deformation of the cell membrane. It is also considered
that the NSBs acted as Fenton like agent in based on the Fenton
chemistry in order to produced Cu1+ ions and various radicals, which
may induce the cell death through oxidative stress and membrane
damage.
4. Conclusion
We were inspired from previously reported studies on formation of
enzyme-inorganic hybrid nanoflowers showing much enhanced cataly-
tic activity as a new enzyme immobilization approach. We designed and
reported, for the first time, the synthesis of VO extract based Cu2+
hybrid nanostructures called “snowballs” and their effective catalytic
and antimicrobial properties compared to free VO extracts. We
proposed that the presence of Cu-O bonds in the NSBs could be
indication of the molecule of extract-Cu2+ complexes. The NSBs acted
as Fenton’s like reagent and their catalytic performance was evaluated
by oxidation of guaiacol to colored 3,3-dimethoxy-4,4-diphenoquinone
in the presence of hydrogen peroxide (H2O2). The NSBs also were
utilized as antimicrobial agent against several bacterial and fungal
pathognes. We claim that our findings are quite promising to effectively
and rationally design and use plant extracts in a variety of technical and
scientific fields including textile, biosensor and biomedicine.
Conflict of interest
The authors all declare that they have no conflict of interest.
Acknowledgements
The authors would like to thank Zeytinburnu Medicinal Plant
Garden due to providing plant materials. This work was supported by
the Scientific Research Projects Coordination Unit of Erciyes University
[Project number: THD-2016-6590]. This article was partially presented
as an oral presentation at the 4th International BAU Drug Design
Congress in 13–15 October 2016, Istanbul, Turkey.
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