foods

Article
Radical Scavenging and Antimicrobial Properties
of Polyphenol Rich Waste Wood Extracts
Anita Smailagić 1 , Petar Ristivojević 2 , Ivica Dimkić 3 , Tamara Pavlović 3 ,
Dragana Dabić Zagorac 1 , Sonja Veljović 4 , Milica Fotirić Akšić 5 , Mekjell Meland 6
and Maja Natić 2, *
1 Innovation Center of the Faculty of Chemistry, University of Belgrade, P.O. Box 51, 11158 Belgrade, Serbia;
[email protected] (A.S.); [email protected] (D.D.Z.)
2 Faculty of Chemistry, University of Belgrade, P.O. Box 51, 11158 Belgrade, Serbia; [email protected]
3 Faculty of Biology, University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia;
[email protected] (I.D.); [email protected] (T.P.)
4 Institute of General and Physical Chemistry, University of Belgrade P.O. Box 551, 11001 Belgrade, Serbia;
[email protected]
5 Faculty of Agriculture, University of Belgrade, 11080 Belgrade, Serbia; [email protected]
6 Norwegian Institute of Bioeconomy Research-NIBIO Ullensvang, NO-5781 Lofthus, Norway;
[email protected]
* Correspondence: [email protected]




Received: 12 February 2020; Accepted: 2 March 2020; Published: 10 March 2020


Abstract: The main focus of this study is to assess radical scavenging and antimicrobial activities
of the 11 wood extracts: oak (Quercus petraea (Matt.) Liebl., Q. robur L., and Q. cerris L.), mulberry
(Morus alba L.), myrobalan plum (Prunus cerasifera Ehrh.), black locust (Robinia pseudoacacia L.), and
wild cherry (Prunus avium L.). High-performance thin-layer chromatography (HPTLC) provided
initial phenolic screening and revealed different chemical patterns among investigated wood extracts.
To identify individual compounds with radical scavenging activity DPPH-HPTLC, assay was applied.
Gallic acid, ferulic and/or caffeic acids were identified as the compounds with the highest contribution
of total radical scavenging activity. Principal component analysis was applied on the data set
obtained from HPTLC chromatogram to classify samples based on chemical fingerprints: Quercus spp.
formed separate clusters from the other wood samples. The wood extracts were evaluated for their
antimicrobial activity against eight representative human and opportunistic pathogens. The lowest
minimum inhibitory concentration (MIC) was recorded against Staphylococcus aureus for black locust,
cherry and mulberry wood extracts. This work provided simple, low-cost and high-throughput
screening of phenolic compounds and assessments of the radical scavenging properties of selected
individual metabolites from natural matrix that contributed to scavenge free radicals.

Keywords: wood waste; phenolic profile; planar chromatography; DPPH-HPTLC assay;

antimicrobial activity

1. Introduction
Ageing processes of some alcoholic beverages are one of the most important practices during
their production. This contributes to improved sensory characteristics such as aroma, color, taste and
astringency. The most commonly used material in cooperage is oak heartwood barrels. Alternative
wood species such as chestnut, cherry and mulberry are also used in Balkan cooperages, in different
forms such as wood chips and staves [1]. Nowadays, notable studies have showed that agri-food
wastes and by-products, including waste from barrel production, represent an inexhaustible source of
valuable biologically active compounds. Additionally, this waste represents a low-cost material, which

Foods 2020, 9, 319; doi:10.3390/foods9030319 www.mdpi.com/journal/foods

Foods 2020, 9, 319 2 of 15

can be used as material for the production of the extracts. Recently, various extraction techniques
were reviewed and compared with classical extraction procedures used for recovery of the antioxidant
compounds from wastes [2]. Using simple, fast and inexpensive eco-friendly extraction methods for
phenolic compounds represents an efficient method and advantage for further implementation in the
food, pharmaceutical and cosmetic industries [3–5].
From the production of wood barrels, it is estimated that more than 200 tons of wood waste is
available annually in Serbia [6]. Forests and other wooded land occupy ~2.5 million hectares, which is
about one third of the territory of the Republic of Serbia. These natural populations of Serbia contain
a large number of economically important forest tree species (oak, beech, black locust, spruce, pine
and fir) together with autochthonous and introduced wild fruit trees species (wild cherry, cherry
plum, mulberry, wild pear, wild apple, cornelian cherry, hazel and walnut) which are used for timber
production, afforestation and erosion prevention, for grafting, in human diet, in medicine, in industrial
processing and in landscape architecture [7,8].
In Serbia, barrels are mostly made of oak (pedunculate oak, Quercus robur, or sessile oak, Quercus
petraea (Matt.) Liebl. L.) and Turkey oak (Quercus cerris L.) but sometimes black locust (Robinia
pseudoacacia L.), myrobalan plum (Prunus cerasifera Ehrh.), mulberry (Morus alba L.) and even wild cherry
(Prunus avium L.) are used as a cheaper substitute. Oak is the most widespread deciduous tree in Serbia,
a national tree with strong historical and religious importance. Myrobalan plum is a native tree in
Southeast Europe, has great genetic importance for horticultural breeding, and has spread throughout
the whole country in all kinds of micro-climatic and pedologic conditions. Mulberries are very common,
since ex-Yugoslavia used to be the fifth largest silk producer in the world with more than 2.5 million
white mulberry trees [9]. Wild cherry, a noble tree, is widely distributed by birds; its seeds are used
for generative rootstock production and its fruits are suitable for table consumption and as a local
medicine [10,11]. Black locust is mostly used in construction works, as technical or ornamental wood,
and is most commonly used as firewood. It has special value as a honey species for beekeeping [12].
Wood waste has a potential to be reused in the food and pharmaceutical industry due to its
richness in potentially bioactive phenolic compounds with high antioxidant and antimicrobial activity.
In our previous research [6], ellagic acid was abundant in sessile and pedunculate oak wood. It was
also found in Turkey oak, black locust and myrobalan plum, but in much lower quantities. Mulberry
contained the largest concentration of p-hydroxybenzoic acid and stilbenoids in comparison with
other wood species, while myrobalan plum showed the highest content of protocatechuic acid and
5-O-caffeoylquinic acid. Wild cherry was characterized by richness in flavonols, flavanones, flavones,
isoflavones and flavanonols [6,13,14], with taxifolin as the most abundant phenolic compound [6].
Extracts from sessile and pedunculate oak, black locust, myrobalan plum, wild cherry and mulberry
showed notable antioxidant capacity, with the highest radical scavenging activity in the latter extract.
Turkey oak showed the lowest radical scavenging activity [6]. According to the literature, phenolic
acids were identified as the major contributors to the antioxidant capacity in wood samples, including
gallic, protocatechuic, p-coumaric and ellagic acid and all the ellagitannins, due to their characteristic
structure [15]. The following phenolic acids: ferulic acid, caffeic acid, protocatechuic acid, gallic acid,
p-coumaric acid and chlorogenic acid, also present in some wood species, exhibit strong free radical
scavenging properties on silica plates [16].
It is proposed that phenolic compounds can damage the bacteria cell membrane by interacting
with the proteins of the cell membrane, or can be involved in interaction with cellular enzymes [17],
which may directly or indirectly cause metabolic dysfunction and finally bacterial death [18]. Phenolic
compounds are able to inhibit bacterial quorum sensing signal receptors, enzymes and secretion of
toxins [19]. The type, structure and concentration of phenolic compounds, as well as the microorganism
used, will influence the bacterial growth. Large doses of phenolic compounds may be toxic for bacteria,
but lower doses can be used as substrates [17].
Some phenolic compounds present in several wood species showed antimicrobial activity.
Taxifolin exhibited antibacterial activity against six known clinical pathogens: Escherichia coli, Listeria
Foods 2020, 9, 319 3 of 15

sp., Pseudomonas aeruginosa, Bacillus sp., and S. aureus [20]. Oxyresveratrol, the most abundant stilbene
in mulberry, was active against the methicillin-resistant S. aureus [21].
Among flavonoids present in wild cherry wood, flavonols were distinguished by effective
antimicrobial activity against resistant bacteria [22]. Methanolic extract (80%, v/v) from oak bark
(Q. robur L.) showed moderate bactericidal, fungicidal, bacteriostatic and fungistatic activity on S. aureus,
Enterobacter aerogenes (today known as Klebsiella aerogenes) and C. albicans [23]. Q. robur bark showed
strong antibacterial activities against Pseudomonas aeruginosa, M. flavus and E. coli, and moderate
effects against other bacterial species [24]. Heartwood and resin of cherry wood exhibited cytochrome
inhibition and antifungal activity [25], while cherry wood extracts possessed noticeable antimicrobial
activity against 9 out of the 11 wine organisms tested [17]. Oak wood has abundant ellagitannins,
which are toxic to microorganisms, and provides good resistance to fungal degradations [26].
Antimicrobial resistance presents a global problem since resistant pathogens can cause life-threatening
conditions that become incurable with one or more known drugs. The mechanisms of the antibacterial
activities of many plant-derived flavonoids are different than those of conventional drugs, which open
new possibilities in enhancement of antibacterial therapy [27]. In addition, many synthetized drugs
have side-effects, which are small in the case of plant-derived compounds [27]. Due to all these reasons,
the development of alternative drugs derived from natural resources is an attractive option.
Radical scavenging activity using DPPH-HPTLC (high performance still layer chromatography)
assay and antimicrobial activity on wood waste extracts are not investigated so far. Thus, the main aim
of this research was to assess radical scavenging and antimicrobial activities of the wood waste extracts
from mulberry (M. alba L.), myrobalan plum (P. cerasifera Ehrh.), black locust (R. pseudoacacia L.), wild
cherry (P. avium L.), and different species of oaks (Q. petraea (Matt.) Liebl., Q. robur L. and Q. cerris
L.) and consider their usage in the pharmaceutical and food industries. Phenolic compounds were
separated by using HPTLC, while radical scavenging activity was determined using DPPH-HPTLC.

2. Materials and Methods

2.1. Chemicals
Ethyl acetate was purchased from Merck (KGaA, Darmstadt, Germany); formic acid, hexan
2,2-diphenyl-1-picrylhydrazyl (DPPH) and phenolic standards from Sigma-Aldrich (Steinheim,
Germany); and 2-aminoethyl diphenylborinate (NTS) from Fluka (Steinheim, Germany). Gallic
acid, ferulic acid and caffeic acid were supplied by Sigma Aldrich (Steinheim, Germany).

2.2. Samples and Preparation of Wood Extracts

Eleven different wood staves of different geographical origins were analyzed (Table 1). In total
three samples of Pedunculate oaks (Quercus robur L.), three of sessile oaks (Quercus petraea (Matt.) Liebl),
and one sample of Turkey oak (Quercus cerris L.), black locust (Robinia pseudoacacia L.), myrobalan plum
(Prunus cerasifera Ehrh.), wild cherry (Prunus avium L.), and mulberry (Morus alba L.) were included.
Nine staves were stored for the whole year in the open air at cooperage industry VBX-SRL. D.O.O. in
Kraljevo, Central Serbia., while two samples (sessile oak from Kuršumlija and Turkey oak) were not
seasoned [6]. The wood age of the oak wood staves was over 60 years, while the wood age of non-oak
wood staves was more than 40 years.
Firstly, the staves were grinded in a mill for wood and sieved until granulation of 0.5–1.5 mm was
obtained. The sawdust (2.5 g) was extracted with 25 mL of ethanol (60%, v/v), in Erlenmeyer flasks,
with constant stirring in a magnetic stirrer for seven days in darkness and room temperature (20 ±
2 ◦ C) [6]. The extracts were centrifuged twice (5 min at 8000 rpm). For investigation of antimicrobial
activity, the extracts were evaporated with a rotary evaporator and diluted in methanol until the
concentration of 50 mg mL−1 was reached. The extraction yield of each extract was calculated from the
weight of the extract residue obtained after solvent removal and the weight of waste wood employed
in the extraction procedure.
Foods 2020, 9, 319 4 of 15

Table 1. Selected wood waste extracts of different forest trees for DPPH-HPTLC (high-performance
thin-layer chromatography) and antimicrobial testing assay.

Sample No. Tree Geographical Origin Extraction Yield (%)

1 Slavonija (Croatia) 4.44
Pedunculate oak—Quercus
2 Gornji Radan (Serbia) 4.40
robur L.
3 Olovo (Bosnia and Herzegovina) 4.12
4 Kučaj (Serbia) 5.06
Sessile oak—Quercus petraea
5 Kuršumlija (Serbia) 3.05
(Matt.) Liebl.
6 Ravna Gora (Serbia) 4.58
7 Turkey oak—Quercus cerris L. Kuršumlija (Serbia) 1.63
Black locust—Robinia
8 Kraljevo (Serbia) 6.37
pseudoacacia L.
Myrobalan plum—Prunus
9 Vrnjačka Banja (Serbia) 5.80
cerasifera Ehrh
10 Wild cherry—Prunus avium L. Ravna Gora (Serbia) 3.15
11 Mulberry—Morus alba L. Vrnjačka Banja (Serbia) 7.29

2.3. High-Performance Thin-Layer Chromatography and Image Analysis

HPTLC Silica gel 60F254 plates were used for both HPTLC fingerprint and DPPH-HPTLC assay
(Merck, Germany). The oak and wild cherry samples (5 µL), black locust, myrobalan plum and
mulberry (2 µL), and four standard compounds: gallic acid, ferulic acid, caffeic acid and p-coumaric
acid (2 µL, c = 1000 ppm), were applied as bands (8 mm) using Linomat 5 system (Camag, Muttenz,
Switzerland).
The mobile phase consisted of a mixture of ethyl acetate:hexan:formic acid:water (11:2:1:0.5 v/v/v/v).
The plates were developed at room temperature (20 ◦ C) in a twin-trough-chamber (CAMAG) saturated
with the vapors of mobile phase for 15 min, at a developing distance of 70 mm. The obtained HPTLC
chromatograms were derivatized with 2-aminoethyldiphenylborate solution (NTS - 0.2% in ethanol) in
order to intensify the fluorescence of compounds.
For DPPH-HPTLC assay, a developed HPTLC chromatogram was immersed manually for
3 seconds (s) in DPPH·methanol solution (0.2%) and then photographed every 30 s for 15 min. Images
of the plates were captured with mobile phone (Huawei P Smart) equipped with a 13-pixels camera.
All developed plates were photographed both before and after derivatization and saved as TIF files.
Images of the HPTLC chromatograms were analyzed using free available Image J software.
The obtained results for each sample were cropped and denoised by using median filter with three
pixels width filter. Further, images were transformed and the tracks were outlined with a rectangular
selection tool. The line profile plots were generated with Plot Profile option for each sample. Profile
plot displays a 2-D graph of the intensities of pixels along a line.

2.4. Principal Component Analysis

The line profiles were obtained using ImageJ software [28]. Principal Component Analysis (PCA)
was applied using PLS ToolBox, v.6.2.1 (Eigenvector Research, Inc. 196 Hyacinth Road Manson, WA
98831, USA), for MATLAB (7.12.0(R2011a) (http://www.eigenvector.com/software/pls_toolbox.htm,
Eigenvector Research, Inc., Wenatchee, WA). The data were pre-processed using correlated optimized
warping (COW), standard normal variate (SNV) and mean centering to improve multivariate models.

2.5. Bacterial Strains and Growth Conditions

Antibacterial activity was tested using eight indicator strains in line with their growth requirements
(Table 2). Suspensions were adjusted to McFarland standard turbidity (0.5) (BioMérieux, Marcy-l’Étoile,
France), which corresponds approximately to 1 × 108 CFU mL−1 .
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poured into Petri dishes over the solid medium. Molds (5 mm in diameter) were removed after soft
9
agars solidification and 20 µL of each extract (1 mg/well) was added. Vancomycin and nystatin were
9
used as a positive control (antibiotic/mycotic, viz., 0.2 mg/well), for bacterial strains and C. albicans,
respectively. As a negative control, 20 µL of methanol was used. The Petri dishes were incubated at
37 ◦ C, for 24 h. After the incubation, bacterial susceptibility and zones of inhibition were measured
and expressed in mm.

2.7. MIC Assay

A broth microdilution method was used to determine the minimum inhibitory concentration
(MIC) and minimum bactericidal concentrations (MBC) of the selected 11 wood waste extracts. Extracts
were tested in the concentration range from 0.02 to 2 mg mL−1 by performing two-fold serial dilutions
with the appropriate medium in 96-well microtiter plates. Negative control (control of bacterial and
yeast growth) and sterility control (blank, only appropriate medium) were also tested. The final
concentration of the solvent control (methanol) in the first wells was 10%. Vancomycin, streptomycin
and nystatin were tested as positive controls in concentration range from 0.001 to 0.4 mg mL−1 . Beside
negative and sterility controls, each well was inoculated with 20 µL of bacterial/yeast culture (approx.
1 × 106 CFU mL−1 ), reaching a final volume of 200 µL. In addition, 22 µL of resazurin indicator was
added to each well. Microtiter plates were incubated for 24 h at 37 ◦ C. In the presence of living bacterial
cells, blue colored resazurin was being irreversibly reduced to pink colored and highly red fluorescent
resorufin [31]. The lowest concentration of each extract which showed no change in color of resazurin
was defined as MIC value. MBC/MFC values were determined by sub-culturing the dilutions from
wells without color changes on agar plates. Plates were incubated 24 h at 37 ◦ C and bacterial/yeast
growth was monitored. The lowest concentration without growth was defined as MBC/MFC value.
The results were expressed in mg mL−1 .

3. Results and Discussion

3.1. Line Profiles of Investigated Extracts

Investigated wood samples contained several characteristic phenolic compounds at RF values of:
0.28, 0.35, 0.43, 0.74, 0.86 (gallic acid), 0.91 (ferulic acid), 0.88 (caffeic acid) and 0.91 (p-coumaric acid)
 without growth was defined as MBC/MFC value. The results were expressed in mg mL−1.

3. Results and Discussion

3.1.
Foods Line9, Profiles
2020, 319 of Investigated Extracts 6 of 15

Investigated wood samples contained several characteristic phenolic compounds at RF values

of: 0.28, 0.35, 0.43, 0.74, 0.86 (gallic acid), 0.91 (ferulic acid), 0.88 (caffeic acid) and 0.91 (p-coumaric
(Figure 1). Based on HPTLC profiles, wood extracts contained bands with RF values from 0.28 to 0.91.
acid) (Figure 1). Based on HPTLC profiles, wood extracts contained bands with RF values from 0.28
There are five different patterns in the investigated samples: Quercus samples showed one band of
to 0.91. There are five different patterns in the investigated samples: Quercus samples showed one
weak intensity with RF value at 0.85, while sample 1 (Pedunculate oak—Q. robur L.) had the highest
band of weak intensity with RF value at 0.85, while sample 1 (Pedunculate oak—Q. robur L.) had the
intensity
highestpeak of this
intensity compound.
peak Further, black
of this compound. locust
Further, (sample
black locust 8) showed
(sample 8) greenish bands with
showed greenish RF at
bands
0.75, 0.82 and 0.87, clearly different from standard phenolic acids (Figure 2a).
with RF at 0.75, 0.82 and 0.87, clearly different from standard phenolic acids (Figure 2a).

Foods 2020, 9, x FOR PEER REVIEW 8 of 17

Figure
Figure 1. Line
1. Line profiles
profiles of of investigatedwood
investigated woodextracts
extractsbased
based on HPTLC
HPTLC analysis:
analysis:(A)(A)oak
oaksamples
samples(no.
(no.
1–7);
1–7); (B) (B) non-oak
non-oak samples
samples (no.
(no. 8–11):black
8–11): blacklocust
locust(Robinia
(Robinia pseudoacacia
pseudoacacia L.)
L.) (no.
(no.8),
8),myrobalan
myrobalanplum
plum
(Prunus
(Prunus cerasifera
cerasifera Ehrh.)
Ehrh.) (no.9),9),wild
(no. wildcherry
cherry(Prunus
(Prunus avium
avium L.)
L.) (no.
(no. 10)
10)and
andmulberry
mulberry(Morus
(Morusalba L.)L.)
alba
(no
(no 11).11).

Figure 2. 2.HPTLC
Figure HPTLCchromatograms
chromatograms of samples:Q.Q.
of samples: robur
robur (no.(no.
1–3),1–3), Q. petraea
Q. petraea cerris Q.
(no.Q. 4–6),
(no. 4–6), (no.cerris
7),
7), Robinia
(no.Robinia pseudoacacia (no. 8), Prunus cerasifera (no. 9), Prunus avium (no. 10),
pseudoacacia (no. 8), Prunus cerasifera (no. 9), Prunus avium (no. 10), mulberry (no. 11) and four mulberry
(no.standard
11) andcompounds
four standard compounds
(gallic (gallic
acid (no. 12), acid
ferulic acid(no.
(no.12),
13),ferulic
caffeicacid
acid (no. 13),and
(no. 14) caffeic acid (no.
p-coumaric
and p-coumaric
14) acid(no. acid(no.
15)); (a) under UV 15));
light (a)
at under
366 nm;UV (b)light at 366
under UV nm;
light (b) under
at 254 nm;UV (c) light at 254 nm;
DPPH-HPTLC
chromatogram. chromatogram.
(c) DPPH-HPTLC

Wild cherry contained one characteristic peak at 0.36, whereas myrobalan plum had a different
pattern from other wood samples with three characteristic peaks at RF values of 0.28, 0.35 and caffeic
acid (Figure 1). A different profile of myrobalan plum in comparison with other wood samples could
be seen also by HPLC [6], where it contained significantly larger amounts of protocatechuic acid and
5-O-caffeoylquinic acid than other wood samples. The peak profiles for mulberry showed it
Foods 2020, 9, 319 7 of 15

Wild cherry contained one characteristic peak at 0.36, whereas myrobalan plum had a different
pattern from other wood samples with three characteristic peaks at RF values of 0.28, 0.35 and caffeic
acid (Figure 1). A different profile of myrobalan plum in comparison with other wood samples could
be seen also by HPLC [6], where it contained significantly larger amounts of protocatechuic acid and
5-O-caffeoylquinic acid than other wood samples. The peak profiles for mulberry showed it contained
peaks at 0.43, 0.84, gallic, ferulic and/or p-coumaric acids.
Mulberry sample showed hardly visible blue band with RF at 0.86, recognized as gallic acid, while
black locust and oak wood samples contained gallic acid in higher amounts. In addition, wild cherry
contained p-coumaric and ferulic acid in greater quantities than mulberry, and caffeic acid in greater
quantities than myrobalan plum, which was observed neither on HPTLC plates nor line profiles.

3.2. DPPH-HPTLC Assay

It was previously shown that the total antioxidant activity of each extract through the DPPH assay,
mulberry and myrobalan plum wood extracts had significantly higher DPPH values in comparison to
the other samples [6]. The single compounds with radical scavenging activity and their contribution to
.
the total radical scavenging activity were investigated by DPPH -HPTLC assay. Substances exhibiting
radical scavenging properties (yellow bands against a purple background) were located between RF
.
values at 75 and 92 (Figure 2c). The most dominant zones in the HPTLC-DPPH fingerprints were
compounds with RF values at 0.87, 0.91 and 0.92, which could be recognized as gallic, ferulic and/or
caffeic acids (zone 4). Extracts no. 8–11 showed strong radical scavenging activities, mainly due to the
previously detected phenolic compounds, while Quercus samples revealed one weak band with RF at
75 against the purple background. P. cerasifera contained two bands at 0.84 and caffeic acid, and were
recognized as radical scavengers. The, M. alba sample showed radical scavengers with RF values at
0.84, gallic, ferulic and/or p-coumaric acids. These compounds have been recognized before as strong
radical scavengers on silica plates [16].

3.3. Principal Component Analysis

Visual inspection of HPTLC chromatograms is a subjective method and mainly depends on the
analyst’s perception. On the other hand, multivariate chemometrics analysis applied on the HPTLC
chromatogram provides an objective classification of the investigated samples for identification of
phenols most responsible for classification, as well as identification of outliers. The HPTLC system
was optimized to separate and identify all phenols from different wood extracts.
Principal component analysis (PCA) is a commonly used multivariate technique. It accounts
for most of the variation of total variability, visualizes the structure of data by grouping objects into
two or three dimensions, and identifies important variables responsible for discrimination between
wood samples. PCA as an initial multivariate technique was applied on the data matrix (11 samples
× 389 variables) obtained from HPTLC chromatograms, where variables represent the intensities of
pixels along the 389 length lines. The first two Principal Components (PCs) accounted for 33.35% and
20.09% of the total variability, respectively. The first five principal components describe 87.85% of total
variability. From the PC score plot (Figure 3a), six Quercus samples were positioned on left side of
PC score plot, while other four wood samples were misclassified and positioned on right side of PCs
score plot. The loading plots (Figure 3b,c) demonstrated the significant contribution of polyphenolic
compounds to the total variability. The most influential phenolic compounds discriminating between
Quercus and the other wood samples were compounds with RF values at 0.35, 0.43, 0.86 and 0.91.
In contrast to other types of wood samples Quercus samples contained low amounts of phenolic
compounds with RF values at 0.35, 0.43, 0.86 and 0.91. Polar compounds with low RF values could be
some phenolic acids and/or glycosides. These phenolic compounds may be identified as characteristic
taxonomical markers between wood species.
Foods 2020, 9, 319 8 of 15
Foods 2020, 9, x FOR PEER REVIEW 10 of 17

3. Principal
Figure 3. Principalcomponent
componentanalysis
analysis (PCA)
(PCA) of HPTLC
of HPTLC chromatogram:
chromatogram: (A) (A)
TheThe PC score
PC score plot;plot; (B)
(B) and
andThe
(C) (C)loading
The loading
plots.plots. 1–3 Pedunculate
1–3 Pedunculate oaks (Quercus
oaks (Quercus robur
robur L.), L.), 4–6—sessile
4–6—sessile oaks (Quercus
oaks (Quercus petraea
petraea (Matt.)
(Matt.)7—Turkey
Liebl), Liebl), 7—Turkey oak (Quercus
oak (Quercus cerris L.).cerris L.).

3.4. Well-Diffusion Method

3.4. Well-Diffusion Method
Antimicrobial
Antimicrobial potential
potential ofof the
the extracts
extracts waswas tested
tested against
against eight
eight representative
representative humanhuman and and
opportunistic pathogens. Besides clear zones of inhibition, bacteriostatic/fungistatic
opportunistic pathogens. Besides clear zones of inhibition, bacteriostatic/fungistatic effect of tested effect of tested
extracts
extracts was
wasalso observed.
also observed.WoodWood waste extracts
waste in general
extracts showedshowed
in general the highest
the antimicrobial potential
highest antimicrobial
against S. mutans, S. pyogenes L. monocytogenes
potential against S. mutans, S. pyogenes and L. monocytogenes strains in tested concentration of4).
and strains in tested concentration of 1 mg/well (Figure 1
The wild (Figure
mg/well cherry extract
4). The(10) inhibited
wild cherrythe growth
extract S. mutans the
(10)ofinhibited S. aureusofyielding
andgrowth S. mutans theand
largest zones
S. aureus
of inhibition
yielding (21.7 and
the largest 19.8,ofrespectively),
zones inhibition (21.7 compared
and 19.8, to respectively),
other extracts,compared
towards to to the mentioned
other extracts,
pathogens. Additionally, only wild cherry and mulberry extracts (10,11) showed
towards to the mentioned pathogens. Additionally, only wild cherry and mulberry extracts (10,11) moderate bactericidal
effect
showed against E. faecalis.
moderate This indicator
bactericidal effectstrain dueE.
against to its higherThis
faecalis. resistance was strain
indicator excluded duefortofurther MIC
its higher
testing. Mentioned extracts also showed high bacteriostatic effect against MRSA.
resistance was excluded for further MIC testing. Mentioned extracts also showed high bacteriostatic Additionally, the wild
cherry extract showed
effect against MRSA. clear bactericidal
Additionally, theeffect
wildonly against
cherry C. albicans
extract showed L. monocytogenes,
andclear bactericidal while
effect other
only
extracts acted more bacteriostatically. On the other hand, other wood extracts showed
against C. albicans and L. monocytogenes, while other extracts acted more bacteriostatically. On the overwhelmingly
bacteriostatic/fungistatic
other hand, other wood effect extractsagainst
showedalmost including E. coli. All pathogens
all pathogens,bacteriostatic/fungistatic
overwhelmingly were
effect against
susceptible to tested vancomycin and nystatin mycotic.
almost all pathogens, including E. coli. All pathogens were susceptible to tested vancomycin and
nystatin mycotic.
Foods 2020, 9, 319 9 of 15
Foods 2020, 9, x FOR PEER REVIEW 11 of 17

Figure 4. 4. Antimicrobial
Antimicrobial potential
potential of wood of wood
waste waste
extracts extracts in
in well-diffusion well-diffusion
method. method.
*V/N—Vancomycin/
*V/N—Vancomycin/Nystatin.
Nystatin. Values within columns Values within
represent columns
a mean represent
of inhibition a mean
zones of inhibition
and expressed in mm.zones and
Q. robur
expressed
(no. in petraea
1–3), Q. mm. Q.(no.robur (no.Q.
4–6), 1–3), Q. (no.
cerris petraea
7), (no. 4–6),pseudoacacia
Robinia Q. cerris (no. 7), 8),
(no. Robinia pseudoacacia
Prunus (no. 8),
cerasifera (no. 9),
Prunus avium
Prunus cerasifera
(no.(no.
10),9), Prunus avium
mulberry (no. 10), mulberry (no. 11).
(no. 11).

3.5. MIC Assay

3.5. MIC Assay
For
For evaluation
evaluationofofnew new antimicrobials,
antimicrobials,the the
assessment of minimum
assessment of minimum inhibitory concentration
inhibitory (MIC)
concentration
is usually
(MIC) the firstthe
is usually stepfirst
[27].step
The[27].
MICTheis the
MICminimum concentration
is the minimum that causesthat
concentration visible inhibition
causes visible
of bacterial growth. Plant extracts with MIC < 100 µg mL −1 and purified compounds with MIC <
inhibition of bacterial growth. Plant extracts with MIC < 100 μg mL and purified compounds with
−1

10 µg<mL −1
MIC 10 μgare
mLconsidered promising
−1 are considered [27]. However,
promising bactericidal
[27]. However, activity,activity,
bactericidal determined by MBC by
determined value
MBCin
time-kill assays, isassays,
value in time-kill also anisimportant parameter
also an important in assessing
parameter the antimicrobial
in assessing activity. MBC
the antimicrobial and MBC
activity. MIC
parameters complement each other, and MBC below four times MIC value
and MIC parameters complement each other, and MBC below four times MIC value suggests the suggests the bactericidal
action of a tested
bactericidal actioncompound
of a tested[27].
compound [27].
The −1 −1
The obtained MIC values
obtained MIC valueswere
wereininrange
rangefrom
from0.02
0.02mg
mgmLmL−1 of of extract
extract 11,
11,totoMRSA
MRSAto to22mg
mgmLmL−1
in
in the
the case
case of
of activity
activity of
of extract
extract 99 (myrobalan
(myrobalan plum) against C.
plum) against C. albicans
albicans (Table
(Table3).
3).
Foods 2020, 9, 319 10 of 15

Table 3. Minimum inhibitory, minimum bactericidal and minimum fungicidal concentrations (MIC/MBC/MFC) of 11 wood extracts towards selected human pathogens
(mg mL−1 ).

Indicator Strains/MIC
1 2 3 4 5 6 7 8 9 10 11 Str Van Nys
(mg mL−1 )
S. mutans 0.25 0.13 0.25 0.25 0.25 0.25 0.13 0.25 1.00 0.05 0.13 0.020 0.006 NT
S. pyogenes 0.03 0.13 0.03 0.05 0.03 0.13 0.08 0.05 0.03 0.03 0.03 0.002 0.001 NT
S. aureus 0.08 0.05 0.05 0.03 0.03 0.03 0.03 0.09 0.06 0.05 0.09 0.009 0.002 NT
MRSA 0.06 0.06 0.13 0.06 0.03 0.05 0.03 0.03 0.13 0.13 0.02 - - NT
L. monocytogenes 0.50 0.50 0.75 0.75 0.19 0.63 0.50 0.13 0.06 0.06 0.03 0.019 0.002 NT
E. coli 0.75 0.75 1.50 1.50 0.75 1.50 - 0.75 - 0.75 0.75 0.009 0.200 NT
C. albicans - - - - - - - - 2.00 0.25 - NT NT 0.006
Indicator strains/MBC
1 2 3 4 5 6 7 8 9 10 11 Str Van Nys
(mg mL−1 )
S. mutans 2.00 2.00 2.00 2.00 2.00 2.00 0.50 2.00 2.00 0.06 0.50 0.050 0.150 NT
S. pyogenes 0.50 0.50 1.00 1.00 0.50 0.50 1.00 0.25 2.00 0.50 1.00 0.050 0.050 NT
S. aureus 0.25 0.13 0.25 0.25 0.13 0.25 0.50 0.13 0.25 0.13 0.13 0.025 0.003 NT
MRSA 0.50 0.50 0.50 0.50 0.25 0.50 0.63 0.25 1.00 0.25 0.03 - - NT
L. monocytogenes 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.50 1.00 0.13 0.25 0.025 0.006 NT
E. coli 1.00 1.00 2.00 2.00 1.00 2.00 - 1.00 - 1.00 1.00 0.013 0.400 NT
C. albicans - - - - - - - - - 0.50 - NT NT 0.025
Str—Streptomycin; Van—Vancomycin; Nys—Nystatin; NT—not tested; (-)—not determined. Values are marked with lowest value (blue), middle value (yellow), and the highest value
(red). Q. robur (no. 1–3), Q. petraea (no. 4–6), Q. cerris (no. 7), Robinia pseudoacacia (no. 8), Prunus cerasifera (no. 9), Prunus avium (no. 10), mulberry (no. 11).
Foods 2020, 9, 319 11 of 15

The lowest MIC values (viz., 0.03 mg mL−1 ) were recorded against MRSA (extracts 5, 7 and 8),
S. aureus (extracts 4–7) and S. pyogenes (extracts 1, 3, 5, 9–11). S. mutans also showed high sensitivity
to some of the tested extracts with MICs below 0.2 mg mL−1 . MIC values for L. monocytogenes were
in range from 0.03–0.75 mg mL−1 , while extracts 9–11 significantly inhibited the growth rate of this
pathogen. Compared to Gram-positive isolates, E. coli was less sensitive to the tested extracts. Candida
albicans showed poor sensitivity to the action of all extracts, with the exception of extract 10 with
obtained MIC value of 0.25 mg mL−1 . Alañón et al. [17] also concluded that yeasts had a stronger
resistance to wood extracts than bacteria, since only toasted American oak wood and wild cherry
wood extracts inhibited their growth. MICs for vancomycin, streptomycin and nystatin were lower
compared to the tested extracts (0.001–0.4 mg mL−1 ). Additionally, MRSA showed resistance to all
antibiotics on the highest concentration tested (0.4 mg mL−1 ). Interestingly, non-seasoned sessile oak
(sample 5) showed lower MIC against MRSA and L. monocytogenes than seasoned oaks (samples 1, 2, 3,
4, 6).Comparing the results for Q. robur with the results for oak bark (Q. robur) [24], higher values for
MIC were found against L. monocytogenes and E. coli, but lower values against S. aureus. In addition,
the values of MIC for streptomycin were significantly lower than Elansary et al. [24] obtained. MBC
and MFC values of tested extracts varied from 0.03–2 mg mL−1 . The lowest MBC was recorded against
S. aureus for extracts 2, 5, 8, 10 and 11.
There was a strong simultaneous activity against all pathogens tested of extract 10 from the wild
cherry wood. This could be explained by its richness of phenolic compounds which was observed in
previous research [6]. For example, kaempferol is a potential candidate against different pathogenic
microbes, and effective against fluconazole-resistant Candida albicans and Methicillin-resistant S. aureus
(MRSA) [22]. In addition, galangin exhibited selective anti-cytochrome and antifungal activity [25], and
showed antimicrobial activity against S. aureus [25,32], and methicillin-sensitive and methicillin-resistant
S. aureus, Enterococcus spp., and P. aeruginosa [33]. Flavone apigenin showed strong activity against
Gram-negative bacteria [34], while quercetin and apigenin derivatives showed strong antibacterial
properties against Gram-negative and Gram-positive bacteria [35]. Some phenolic acids (gallic, caffeic and
ferulic acids) showed antibacterial activity against Gram-positive (S. aureus and Listeria monocytogenes) and
Gram-negative bacteria (E. coli and P. aeruginosa) with a greater efficiency than conventional antibiotics
such as gentamicin and streptomycin [36]. Contrarily, chlorogenic acid, which was not abundant in wood
species, showed no activity against Gram-positive bacteria [36]. Interestingly, noticeable antimicrobial
activity of cherry wood against wine organisms was observed before [17], but, to our knowledge,
its antimicrobial activity against human and opportunistic pathogens has not been investigated so far.
However, MIC values for extracts 1–7 against S. aureus, L. monocytogenes and E. coli were similar
to MICs obtained from some other Quercus spp. bark extracts (Table 4), but MICs recorded towards
C. albicans were lower compared to the results of this study.

Table 4. Summarized MICs values for other waste extracts obtained from literature data.

Bark Extracts Origin MIC (mg mL−1 ) Sa Mr Lm Sm Sp Ec Ca References

Picea abies 0.13 - 0.16 - - 0.08 0.97
[37]
Larix decidua 0.21 - 0.15 - - 0.33 0.60
Quercus acutissima 0.23 - 0.27 - - 0.17 0.40
Quercus macrocarpa 0.22 - 0.29 - - 0.13 0.34 [24]
Quercus robur 0.23 - 0.25 - - 0.10 0.31
Quercus robur 0.08 - - - - 0.08 - [38]
Quercus ilex 0.13 - - - 0.51 0.26 - [39]
Quercus infectoria - 1.25 - - - - - [40]
Maclura tinctoria - - - 0.08 - - - [41]
Prunus africana 0.07 0.16 - - - - - [42]
Prunus avium 6.25 - - - - 12.50 - [43]
Prunus cerasoides 5.00 1.00 - - - - 1.00 [44]
Morus mesozygia 0.16 - - - - 0.04 0.16 [45]
Sa—S. aureus; Mr—MRSA; Lm—L. monocytogenes; Sm—S. mutans; Sp—S. pyogenes; Ec—E. coli; Ca—C. albicans;
(-)—not tested.
Foods 2020, 9, 319 12 of 15

On the other hand, P. avium stem bark extracts from Nigeria showed lower antimicrobial activity
against S. aureus and E coli, with MICs 6.25 mg mL−1 and 12.5 mg mL−1 , respectively [43]. Compared
to extracts 9 and 10, Prunus cerasoides showed similar antibacterial activity towards MRSA [44].
Unlike extract 11, originating from M. alba, bark extracts originating from Morus mesozygia showed
significant antimicrobial activity against C. albicans, with obtained MIC of 0.16 mg mL−1 (Table 4).
Interestingly, higher susceptibility of C. albicans was also observed for Picea abies and Larix decidua
bark extracts [37]. In the literature, no significant correlation was found between antimicrobial activity
and total phenolic content [17,46], as well as between antimicrobial activities and antioxidant capacity.
However, structure-function of the phenolic extracts have more influence on the antimicrobial activity
than the total phenol content [17]. Finally, according to Cowan [47], a wide variety of specialized
metabolites show antimicrobial activity in vitro, such as tannins, terpenoids and alkaloids, also found
in wood.

4. Conclusions
Wood waste from forest trees is a source of different bioactive metabolites which could find
application in the food and pharmaceutical industries. Radical scavenging and antimicrobial activities
of the wood waste extracts appeared to be a valuable bio-functional source. In general, HPTLC
fingerprint identify the main phenolic acids present in investigated samples and revealed chemical
patterns among investigated wood extracts. DPPH-HPTLC assay identified gallic, ferulic and/or caffeic
acids as compounds with the highest contribution to total radical scavenging activity. Based on PCA plot,
six Quercus samples were separated from other extracts showing strong radical scavenging activities.
Wood samples were the most active against MRSA, S. aureus and S. pyogenes. The lowest MIC
and MBC values were detected in mulberry extract against MRSA. Activities were also distinguished
against MRSA (extracts of non-seasoned sessile oak (5), Turkey oak, black locust and mulberry) and
S. aureus (Turkey oak and all sessile oak extracts). The largest zones of inhibition of the growth of
S. mutans and S. aureus were observed for wild cherry extract. Among sessile and pedunculate oak
extracts, non-seasoned sessile oak extract (5) was distinguished by lower MIC against MRSA and
L. monocytogenes. Extracts of myrobalan plum, wild cherry and mulberry significantly inhibited the
growth rate of L. monocytogenes. E. coli was less sensitive to the tested extracts. C. albicans showed poor
sensitivity to the action of all extracts, with the exception of the wild cherry extract.
Wild cherry wood extract can be commercially important due to good simultaneous activity
against all pathogens, and is a valuable source for various formulations: Wild cherry and mulberry
wood extracts with given antimicrobial activities can be especially useful in preserving perishable
foods with short shelf life.

Author Contributions: Conceptualization, M.N. and P.R.; methodology, P.R.; software, D.D.Z.; investigation, P.R.,
A.S., I.D. and T.P.; resources, S.V.; writing—original draft preparation, A.S., P.R. and T.P.; writing—review and
editing, M.N., M.F.A. and M.M.; supervision, M.F.A. and M.M. All authors have read and agreed to the published
version of the manuscript.
Funding: This research was funded by the Ministry of Education, Science and Technological Development,
Republic of Serbia, grant number 172017, and Research Council of Norway (project No 11060-“NORWEGIAN
FRUIT GENETIC RESOURCES—HEALTHY, TASTE & NO WASTE”).
Acknowledgments: Authors would like to thank Ivanka Ćirić for technical assistance during the course of analysis.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.

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