plants
Article
Phytochemical Profile and Biological Effects of Spruce
(Picea abies) Bark Subjected to Ultrasound Assisted and
Microwave-Assisted Extractions
Adrian Nisca 1, *, Ruxandra S, tefănescu 2, * , Diana Ionela Stegărus, 3 , Anca Delia Mare 4 , Lenard Farczadi 5 and
Corneliu Tanase 6
1
2
3
4
5
6
Citation: Nisca, A.; S, tefănescu, R.;
Stegărus, , D.I.; Mare, A.D.; Farczadi,
*
Doctoral School of Medicine and Pharmacy, “George Emil Palade” University of Medicine, Pharmacy,
Sciences and Technology of Târgu Mures, 38 Gheorghe Marinescu Street, 540139 Târgu Mures, , Romania
Department of Pharmacognosy and Phytotherapy, “George Emil Palade” University of Medicine,
Pharmacy, Sciences and Technology of Târgu Mures, 38 Gheorghe Marinescu Street,
540139 Târgu Mures, , Romania
National Research and Development Institute for Cryogenics and Isotopic Technologies—ICSI Râmnicu
Vâlcea, 4th Uzinei Street, 240050 Râmnicu Vâlcea, Romania; diana.stegarus@icsi.ro
Department of Microbiology, “George Emil Palade” University of Medicine, Pharmacy, Sciences and
Technology of Târgu Mures, 38 Gheorghe Marinescu Street, 540139 Târgu Mures, , Romania;
anca.mare@umfst.ro
Chromatography and Mass Spectrometry Laboratory, Center for Advanced Medical and Pharmaceutical
Research, “George Emil Palade” University of Medicine, Pharmacy, Sciences and Technology of Târgu Mures,
38 Gheorghe Marinescu Street, 540139 Târgu Mures, , Romania; lenard.farczadi@umfst.ro
Department of Pharmaceutical Botany, “George Emil Palade” University of Medicine, Pharmacy, Sciences and
Technology of Târgu Mures, 38 Gheorghe Marinescu Street, 540139 Târgu Mures, , Romania;
corneliu.tanase@umfst.ro
Correspondence: adrian.nisca@umfst.ro (A.N.); ruxandra.stefanescu@umfst.ro (R.S, .)
L.; Tanase, C. Phytochemical Profile
and Biological Effects of Spruce
(Picea abies) Bark Subjected to
Ultrasound Assisted and
Microwave-Assisted Extractions.
Plants 2021, 10, 870. https://doi.org/
10.3390/plants10050870
Academic Editor:
Antonella Smeriglio
Received: 31 March 2021
Accepted: 24 April 2021
Published: 26 April 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
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Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
Abstract: The bark of various tree species is a byproduct of the forestry industry that is not used at
its full potential, considering the wide range of phytochemicals that are contained in these vegetal
matrices and the health benefits that these compounds could provide for society. Our goal was
to assess and compare the phytochemical composition of some hydroalcoholic spruce (Picea abies)
bark extracts attained by ultrasound assisted extraction (UAE) and microwave-assisted extraction
(MAE) and their antioxidant and antibacterial effects. The levels of total phenolics and tannins in the
bark extracts were determined using methods based on the Folin–Ciocâlteu reagent, while specific
phenolic and volatile compounds were identified and quantified using an UPLC-PDA method and
a GC-FID method, respectively. After the chemical composition assessment, the antioxidant capacity
(AC) was evaluated by measuring the scavenging ability against two free radicals (DPPH and ABTS).
The minimum inhibitory concentration (MIC) was determined to assess the antibacterial activity
of the extracts. The results indicated that the extracts produced by UAE had higher contents of
polyphenols and tannins and also a higher content of the main phenolic compounds identified,
catechin and epicatechin, compared to the MAE extracts. In contrast the highest content of volatile
terpenoids (mainly α- and β-pinene) was found in the MAE extracts. All of the tested extracts
exhibited relatively high antioxidant activities (especially the UAE extracts) and low MICs against
Gram-positive bacteria but were mildly efficient against Gram-negative bacteria. These findings
show that the spruce bark might be an important source of bioactive compounds that can be easily
extracted from these industrial secondary products. Various uses of this vegetal material may emerge,
due to its antioxidant and antibacterial effects.
Keywords: Picea abies; phenolic compounds; antioxidant; terpenoids; catechin; antibacterial activity
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Plants 2021, 10, 870. https://doi.org/10.3390/plants10050870
https://www.mdpi.com/journal/plants
Plants 2021, 10, 870
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1. Introduction
For the entirety of human history, different tree barks were used for various purposes, including medicinal purposes such as the treatment or the prevention of many pathologies [1,2].
Despite the health benefits provided by these raw materials, most of the bark’s parts that result
after wood processing are still inefficiently used for thermal energy [3,4]. Thus, a lot of recent
research in this field has been focused on the evaluation of the phytochemical content and
biological activities of the extracts attained from various tree species barks [2,5,6], with several
studies indicating numerous classes of bioactive compounds, mainly polyphenols [4,7].
Polyphenols are secondary metabolites that are often found in the vegetal foodstuff [8],
but lately, their presence was indicated in different tree barks [9,10]. They have an essential
role in the protection of the plant against external threats, such as bacterial and fungal
pathogens, herbivores, and insects [11,12].
Moreover, these secondary metabolites are not only essential for plant protection and
environmental integration of the containing species, but also possess various biological
activities that may benefit human health, such as antioxidant [13–15], antibacterial [14,16],
antiviral [17–19], antifungal [20–22], and antitumor [9,10,23] effects.
One of the most important properties of phenolic compounds is their antioxidant
capacity, provided by their phenolic hydroxyl groups that can donate electrons to neutralize
free radicals. These reactive molecular species may cause DNA damage or lipids’ oxidation
in humans [2,9], processes that are often linked to different pathologies such as diabetes,
neurodegenerative disorders, cardiovascular and respiratory diseases, and cancers [24]. By
neutralizing these free radicals, polyphenols are considered useful for the prophylaxis of
these health problems [25]. Moreover, the antibacterial activity and synergistic effects of
polyphenols along with synthetic antibiotics [24–28] may become useful in the management
of various bacterial infections with multidrug-resistant bacterial strains that are emerging
at a fast pace nowadays [29,30].
However, in order to use these phytochemicals, for their beneficial effects, they must
be extracted from the vegetal matrices first. For a long time, traditional methods like
maceration and Soxhlet extraction were used for extraction. Still, these methods proved
to be time and energy consuming [31]. Nowadays, these conventional methods are enhanced using microwave radiations and ultrasounds to reduce time, energy, and solvent
consumption [10]. The ultrasound assisted extraction (UAE) is commonly used as it does
not require complex equipment and is simple to use, while efficiently solving the problems
of the classic methods, but compared to other modern methods this one is the most time
and solvent consuming [32]. This method is based on the cavitation process caused by the
ultrasounds passing through the solvent. The process leads to the formation of cavitation
bubbles near the surface of the vegetal matrix. When these bubbles collapse they damage
the cellular walls’ structure, increasing the contact surface of the matrix and solvent by
increasing the cellular wall permeability, thus enhancing the diffusion process and extraction yield [33,34]. The microwave assisted extraction (MAE) on the other hand uses the
energy of the microwave radiation to heat the entire sample volume (solvent and matrix)
at once. The fast raise of internal temperature in the vegetal matrix leads to an increased
intracellular pressure which damages the cellular walls, releasing the internal cellular
content, while the raise in solvent temperature accelerates the diffusion process [33]. This
method has a high extraction and time efficiency and needs low solvent amounts, but can
be used only for the extraction of low molecular weight, polar compounds that are stable
at high temperatures [35].
Picea abies L. (Norway spruce) is a species of the Pinaceae family; previous studies identified in its bark various phenolic compounds such as gallic, vanillic, syringic, p-coumaric, ferulic, synapic acids, taxifolin, quercetin, and catechin [36,37], but also terpenoids (β-sitosterol,
γ-sitosterol, thunbergol, α-pinene, β-pinene, and limonene) and stilbenes (piceid and astrignin) [38,39]. The presence of such compounds in spruce bark might explain the extracts’
antioxidant, antibacterial, antifungal and antitumoral (against human melanoma cells) activities [9,40,41].
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Although, data about the cytotoxicity of P. abies bark extracts could not be found,
a study that analyzed the P. mariana bark hot water extracts did not show any cytotoxic
effects on RAW 264.7 macrophages and, even though high levels of phenolic compounds
may exert pro-oxidant effects in vitro, due to the overproduction of H2 O2 , quinones, and
semiquinones, which might lead to cellular death, these effects are unlikely to happen
in vivo, because of the biotransformation of these compounds [42,43].
Considering the data presented in the literature, the bark of P. abies might be an important forestry waste material, containing several bioactive compounds that could have
beneficial health implications, due to their biological effects.
Therefore, this study focused on the evaluation of the phytochemical profile and
biological effects of the extracts attained by different extraction methods and from P. abies
barks collected in different locations. A comparison between the two extraction methods
and between the different collecting sites was performed. For the chemical composition assessment, the Folin–Ciocâlteu method was performed to measure the levels of polyphenols
and tannins, while UPLC-PDA and GC-FID methods were performed to identify the main
phenolic compounds and volatile terpenoids, respectively. As for the biological activity,
the antioxidant capacity (AC) and antibacterial activity were assessed by the DPPH and
ABTS assays and by the microdilution assay, respectively.
2. Results and Discussions
2.1. Assessment of the Content of Total Polyphenols (TPC)
The results of the spectrophotometric determination of the spruce bark extracts TPC
are presented in Figure 1a, as mg of GAE per g of vegetal material.
Figure 1. (a) Total polyphenol content (TPC) of spruce bark extracts; (b) total tannin content (TTC) of spruce bark extracts;
significant differences are represented by different letters (significance level α = 0.05); M1 US—P. abies (Maramures, ului
Mountains National Park site 1) extract produced by UAE, M2 US—P. abies (Maramures, ului Mountains National Park site 2)
extract produced by UAE, MT US—P. abies (Tas, ca) extract produced by UAE, MD US—P. abies (Dolhasca) extract produced
by UAE, M1 M—P. abies (Maramures, ului Mountains National Park site 1) extract produced by MAE, M2 M—P. abies
(Maramures, ului Mountains National Park site 2) extract produced by MAE, MT M—P. abies (Tas, ca) extract produced by
MAE and MD M—P. abies (Dolhasca) extract produced by MAE.
As it can be observed, the MD US variant had the highest TPC (43.64 ± 10.52 mg/g),
but the content was significantly higher only when compared to MT M and MD M. The
lowest TPC was recorded for the MT M variant (19.54 ± 1.79 mg/g), having significantly
lower levels of phenolic compounds in comparison to the other variants excepting MD
M. It can be also noted that the extracts attained via UAE are associated with higher TPC.
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Every variant obtained using this extraction method had higher TPC than the variants
obtained by MAE, however the only significant differences occurred between the MT (US
and M) and MD (US and M) experimental variants. Although slight differences occurred
between the different collecting sites, there were no significant differences registered
regarding the TPC.
Other studies regarding the TPC of spruce bark extracts attained by UAE achieved
similar results. For example a study that used almost identical parameters for the UAE
of the P. abies bark, identified a TPC of around 40 mg GAE/g, indicating the similarity of
the results [44]. Another baseline study, that also focused on the UAE of the P. abies bark,
achieved a TPC of the extracts of 28.73 ± 0.08 mg GAE/g, thus recording a lower level of
phenolic compounds compared to most of our experimental variants. However, in this
baseline study an aqueous extraction was performed and not a hydro-alcoholic one, using
a higher extraction temperature (70 ◦ C) and a lower extraction time (15 min) compared to
our extraction parameters [45].
Sládková A. et al. conducted a study on P. abies bark extracts attained by MAE,
indicating a maximum TPC of 321.1 mg GAE per 100 g dry bark. Although these results
showed a lower yield of phenolic compounds compared to our results for the extracts
obtained by MAE, it must be mentioned that a different MAE protocol was used [46].
These data show that the UAE might be a more efficient extraction technique to
achieve higher yields of phenolic compounds from the bark of P. abies.
2.2. Assessment of the Content of Total Tannins (TTC)
The results regarding the TTC are represented in Figure 1b, as mg pyrogallol per g of
vegetal material. It has been noticed that the highest TTC was recorded for the M1 M variant
(19.34 ± 4.88 mg/g); however, no significant difference occurred between the experimental
variants except MT M, for which a significantly lower level of tannins (7.56 ± 0.65 mg/g)
was identified. Although almost no significant differences were registered after comparing
the extracts, the results showed that slightly higher TTCs were associated with the extracts
obtained by UAE, except the M1 variants, for which, the MAE seemed to lead to a slightly
higher tannin yield, but the difference was not significant. As in the case of TPC, minor
differences occurred between the collecting sites, but these differences were not significant.
Cos, arcă S.L. et al. quantified the tannin content of their aqueous spruce bark extracts
attained by UAE and achieved a concentration of 11.4 mg pyrogallol/g, thus indicating a
lower level of TTC compared to our experimental variants [9]. Although the methods used
in this baseline study were similar to the methods used in the present study, the difference
in the extraction time, temperature and solvent might be the reason for the disparity of the
results. Data about the tannin content of spruce bark extracts attained by MAE could not
be found in the literature.
Other studies regarding the tannin content of spruce bark were found, but these
studies focused on the aqueous extraction of the spruce bark at pilot scales [47,48].
2.3. Terpenoids Content
After the GC-FID analysis, the volatile compounds presented in Table 1 were identified
and quantified, the results being expressed as µg compound per g vegetal material. It was
observed that the main terpenoids identified in the spruce bark were α-pinene and β-pinene,
the M1 M variant having the significantly highest α-pinene content (2198.33 ± 0.25 µg/g),
while the MD M variant had the highest β-pinene content (4312.24 ± 4.07 µg/g). As for the
less abundant identified compounds, the M2 M variant had the highest content of camphene,
α-phellandrene, myrcene, and tricyclene, the MT M variant contained the highest yield
of 3-carene and limonene, while the MD M variant also had the highest sabinene content
out of all tested extracts. The most part of the results showed that higher yields of volatile
compounds were associated with the variants obtained with the MAE method, however
exceptions were identified too. For example, higher levels of camphene and myrcene were
measured in the MT US variant compared to MT M and 3-carene content was higher in the
UAE variants compared to the MAE counterparts, excepting the MT variants. Moreover,
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α-phellandrene and limonene contents were higher in the M1 US and M2 US, respectively,
compared to their MAE counterparts.
Table 1. Terpenoids present in the bark of P. abies (mean ± st. dev. µg compound/g vegetal material).
Compound
M1 US
M1 M
M2 US
M2 M
MT US
MT M
MD US
MD M
α-pinene
β-pinene
camphene
3-carene
α-phellandrene
limonene
sabinene
myrcene
tricyclene
1938.77 ± 3.05 a
2140.04 ± 3.59 a
51.11 ± 0.13 a
60.39 ± 0.14 a
9.18 ± 0.17 a
98.96 ± 0.14 a
22.12 ± 0.03 a
100.1 ± 0.1 a
19.31 ± 0.17 a
2198.33 ± 0.25 b
2997.66 ± 5.07 b
71.60 ± 0.09 b
55.26 ± 0.09 b
8.14 ± 0.08 b
101.07 ± 0.06 b
23.09 ± 0.04 b
105.46 ± 0.05 b
19.37 ± 0.11 a
1960.84 ± 2.97 c
3747.53 ± 22.01 c
80.77 ± 0.36 c
109.41 ± 0.23 c
14.22 ± 0.16 c
109.21 ± 0.05 c
26.15 ± 0.04 c
255.1 ± 0.04 c
2005.08 ± 2.53 d
3808.67 ± 6.88 d
99.74 ± 1.13 d
81.09 ± 0.03 d
15.09 ± 0.03 d
107.06 ± 0.08 d
26.61 ± 0.06 d
255.66 ± 0.37 c
29.18 ± 0.06 c
1368.23 ± 3.21 e
3915.73 ± 10.04 e
65.96 ± 0.67 e
100.59 ± 1.06 e
12.75 ± 0.34 e
113.55 ± 0.11 e
67.3 ± 0.03 e
1566.11 ± 7.79 f
3936.65 ± 32.46 e
57.17 ± 0.07 f
112.71 ± 4.08 c
13.16 ± 0.08 e
113.82 ± 0.30 e
1806.31 ± 8.09 g
4215.63 ± 6.22 f
71.12 ± 0.26 b
110.18 ± 0.15 c
2014.84 ± 7.18 d
4312.24 ± 4.07 g
90.56 ± 0.46 g
101.31 ± 1.80 e
13.96 ± 0.12 c
26.05 ± 0.06 b
232.4 ± 4.24 d
11.12 ± 0.06 d
67.79 ± 0.07 f
229.99 ± 0.09 d
11.95 ± 0.08 e
12.16 ± 0.09 f
101.11 ± 0.03 b
75.02 ± 0.14 g
215.26 ± 0.19 e
12.11 ± 0.07 e
105.46 ± 0.47 f
81.01 ± 0.18 h
220.21 ± 0.06 f
13.20 ± 0.07 f
M1 US—P. abies (Maramures, ului Mountains National Park site 1) extract produced by UAE, M2 US—P. abies (Maramures, ului Mountains
National Park site 2) extract produced by UAE, MT US—P. abies (Tas, ca) extract produced by UAE, MD US—P. abies (Dolhasca) extract produced by UAE, M1 M—P. abies (Maramures, ului Mountains National Park site 1) extract produced by MAE, M2 M—P. abies (Maramures, ului
Mountains National Park site 2) extract produced by MAE, MT M—P. abies (Tas, ca) extract produced by MAE and MD M—P. abies (Dolhasca)
extract produced by MAE; significant differences are represented by different letters (significance level α = 0.05).
In the case of volatile terpenoids content, the collecting site had a significant influence.
For example, it can be seen in Table 1 that the spruce bark collected from Dolhasca (MD)
had the significantly highest content of β-pinene, while the spruce bark collected from
site 1 of Maramures, ului Mountains National Park had the highest α- pinene content even
when compared to the second site of the same area.
In a study conducted on spruce bark extracts attained by Soxhlet and super-critical
CO2 extractions, the terpenoids were identified and quantified, but none of the compounds
identified in this study corresponded to their results [49]. However, two other studies
that focused on the emissions of volatile organic compounds from the spruce bark have
indicated that high levels of α- and β-pinene were emitted from the spruce barks, but
also lower quantities of limonene, 3-carene, camphene, sabinene, α-phellandrene, and
tricyclene were identified [50,51], these results are also similar with our findings.
2.4. Individual Polyphenol Content
The major peaks occurring at retention times of 4.53 min and 5.25 min were catechin
and epicatechin. The calculated contents of catechin and epicatechin are found in Table 2.
For the UAE samples higher contents of catechin and epicatechin were identified, while
for the MAE variants, a lower yield of these compounds was registered. Consistent with
the results obtained in TPC and tannin content, M1 and MD samples contain the highest
concentrations of both catechin and epicatechin, closely followed by MT samples. High
catechin and epicatechin concentrations were expected, since they are the main monomers
of spruce bark proanthocyanidins [47,48].
Table 2. Polyphenolic compounds identified and semiquantified in the extracts (mg compound/g
vegetal material).
Compound M1 M
Catechin
Epicatechin
1.91
6.72
M1 US
M2 M
M2 US
MT M
MT US
MD M
MD US
7.47
27.09
0.86
7.41
6.43
16.86
0.94
2.02
5.47
25.59
1.63
20.93
6.94
25.71
M1 US—P. abies (Maramures, ului Mountains National Park Site 1) extract produced by UAE, M2 US—P. abies
(Maramures, ului Mountains National Park site 2) extract produced by UAE, MT US—P. abies (Tas, ca) extract
produced by UAE, MD US—P. abies (Dolhasca) extract produced by UAE, M1 M—P. abies (Maramures, ului
Mountains National Park site 1) extract produced by MAE, M2 M—P. abies (Maramures, ului Mountains National
Park site 2) extract produced by MAE, MT M—P. abies (Tas, ca) extract produced by MAE and MD M—P. abies
(Dolhasca) extract produced by MAE.
None of the samples had detectable levels of gallic, sinapic, and vanillic acids, neither taxifolin, although previous reports suggested that these compounds are among the
polyphenols found in spruce bark extracts [44]. These polyphenolic compounds are, however, susceptible to degradation during sample preparation and extraction. Moreover,
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our results clearly suggest that the UAE is more efficient for the extraction of catechin
and epicatechin from the spruce bark. In contrast Albuquerque et al. showed that the
microwave assisted extraction is more efficient for the extraction of catechin compared
to the ultrasound assisted extraction [52]. This discrepancy may appear, because a different vegetal matrix was used in the baseline study and also a more controlled environment
was used for the MAE. However, the degradation of catechin and epicatechin in our MAE
variants is unlikely due to the fact that these compounds suffer modifications only at higher
temperatures than those achieved by us in the MAE [53].
2.5. Antioxidant Capacity (AC)
2.5.1. DPPH Assay
To assess the AC the DPPH method was used, and the results of the assay, expressed
as inhibition percentages of the DPPH free radical, are represented in Figure 2a. These
inhibition percentages correspond to 0.15 mg vegetal material and 0.1 mg DPPH reagent
per tested sample.
Figure 2. (a) Antioxidant capacity (AC) assessed by the DPPH method; (b) antioxidant capacity (AC) assessed by the
ABTS method; significant differences are represented by different letters (significance level α = 0.05); M1 US—P. abies
(Maramures, ului mountains national park site 1) extract produced by UAE, M2 US—P. abies (Maramures, ului Mountains
National Park site 2) extract produced by UAE, MT US—P. abies (Tas, ca) extract produced by UAE, MD US—P. abies
(Dolhasca) extract produced by UAE, M1 M—P. abies (Maramures, ului Mountains National Park site 1) extract produced by
MAE, M2 M—P. abies (Maramures, ului Mountains National Park site 2) extract produced by MAE, MT M—P. abies (Tas, ca)
extract produced by MAE and MD M—P. abies (Dolhasca) extract produced by MAE.
It was shown that the extracts attained by UAE exhibit higher ACs compared to their
MAE counterparts. The M2 US variant had the highest AC (52.64% ± 2.35%) between
all extracts obtained by UAE, however the differences were not statistically significant.
However, when compared to MAE variants, M2 US had a significantly higher AC. M1 M
was the only MAE variant that had a comparable AC with the UAE variants, these results
indicating a correlation between the AC and TPC. This correlation is further reinforced by
the fact that MT US and MD US had significantly higher Acs than their MAE counterparts,
but also significantly higher TPC values, suggesting that higher polyphenolic content
may lead to a stronger antioxidant activity. The results of the Pearson test presented in
Figure 3a have also confirmed that a correlation exists between the TPC and the inhibition
percentages registered in the DPPH assay (R2 = 0.8006).
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Figure 3. (a) Correlation between the TPC and inhibition percentages (DPPH assay) R2 = 0.8006; (b) Correlation between
the TPC and inhibition percentages (ABTS assay) R2 = 0.8484.
Previous studies have also indicated the DPPH radical scavenging ability of the spruce
bark extracts attained by the UAE [9,37], but unfortunately no data were found describing
this activity in spruce bark extracts obtained by MAE.
2.5.2. ABTS Assay
The inhibition percentages registered for the ABTS assay indicate the AC corresponding to 1.25 mg spruce bark and 0.17 mg ABTS reagent per tested sample. These results
are presented in Figure 2b, showing that the extracts obtained by UAE have a superior
AC compared to their MAE counterparts. Although between M1 M and M1 US, and M2
M and M2 US no significant difference occurred by comparing their AC resulted after
the ABTS assay, the differences between the MT M and MT US, and MD M and MD US
are significantly different, indicating a similar trend as in the results of the previously
performed antioxidant activity assay. Moreover, like in the previous assay, the variants
that had higher polyphenol contents have led to a superior AC, the Pearson test indicating
even a stronger correlation between the TPC and the inhibition percentages resulted in the
ABTS assay (R2 = 0.8484), as shown in Figure 3b.
The correlations registered between the TPC and the Acs recorded for both assays indicate
that polyphenols are mainly responsible for the antioxidant properties of the bark extracts,
even though the bark is a complex matrix containing a mix of phytochemical compounds.
Regarding the differences between the AC of samples collected from different sites,
slight variations were registered, however none of them were statistically significant.
A previous study also highlighted the AC of ethanolic spruce bark extracts obtained
by UAE, tested with the ABTS reagent [54]; however, no studies measuring the AC of
spruce bark extracts obtained by MAE with the ABTS assay, were found. For both assays
(DPPH and ABTS), the results of this study and those of the baseline studies, could not be
directly compared because of the different methodologies used and the different ways to
express the final results.
As previously stated our results suggested a positive correlation between the TPC
and AC of the tested extracts, however other studies that analyzed the relation between
the two variables found no significant correlation [55–57]. It is necessary to mention
that the two studies that analyzed this correlation on spruce bark extracts used deep
eutectic solvents, while the third study focused on Rosmarinus officinalis ethanolic extracts.
Moreover, studies that compared the TPC and AC of extracts attained by UAE and MAE
showed that the MAE is more efficient for extracting polyphenols and results in extracts
with superior AC [58–60]. In contrast, our findings showed that the UAE is more efficient
regarding the TPC and AC. The discrepancy between our results and the data found in
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literature might be due to the different methodologies used for extraction, or even because
the vegetal matrices used in the baseline studies were not barks. For example, in one of
our previous works conducted on pine bark, it was also shown that the UAE is more
efficient for extracting polyphenols and for achieving extracts with higher antioxidant
capacities [61].
2.6. Antibacterial Activity
The antibacterial activity of the spruce bark extracts was assessed against Grampositive and Gram-negative bacterial strains, by determining the MIC of the extracts, in
order to evaluate the efficiency of each experimental variant against each tested strain. The
results of the antibacterial activity are presented in Table 3.
Table 3. MICs of the tested extracts expressed as mg vegetal material/mL extract.
MIC (mg bark/mL Extract)
Bacterial Strain
M1 M
M1 US
M2 M
M2 US
MT M
MT US
MD M
MD US
Staphylococcus aureus
ATCC 25923
3.125
6.25
6.25
6.25
6.25
6.25
3.125
3.125
Methicillin-resistant
Staphylococcus aureus
(MRSA) ATCC 43300
3.125
6.25
6.25
3.125
6.25
6.25
3.125
6.25
Escherichia coli
ATCC 25922
100
50
>100
>50
100
50
100
50
Klebsiella pneumoniae
ATCC 700603
>100
>50
>100
>50
100
>50
100
>50
Pseudomonas aeruginosa
ATCC 27853
100
50
100
50
100
50
50
50
M1 US—P. abies (Maramures, ului Mountains National Park site 1) extract produced by UAE, M2 US—P. abies (Maramures, ului Mountains
National Park site 2) extract produced by UAE, MT US—P. abies (Tas, ca) extract produced by UAE, MD US—P. abies (Dolhasca) extract produced by UAE, M1 M—P. abies (Maramures, ului Mountains National Park site 1) extract produced by MAE, M2 M—P. abies (Maramures, ului
Mountains National Park site 2) extract produced by MAE, MT M—P. abies (Tas, ca) extract produced by MAE and MD M—P. abies (Dolhasca)
extract produced by MAE.
2.6.1. Gram-Positive Bacteria
As it can be observed M1 M and MD M were the most efficient variants against Grampositive bacterial strains (Staphylococcus aureus and methicillin resistant Staphylococcus
aureus-MRSA), having the lowest MIC (3.125 mg/mL) against both strains. The M2 US
and MD US variants also had the lowest MIC value, but only against one of the two tested
strains. The Gram-positive bacterial strains were inhibited by all tested variants, roughly
with the same efficiency.
2.6.2. Gram-Negative Bacteria
A modest antibacterial activity was identified against the tested Gram-negative bacterial strains (Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coli). For the
extracts attained by UAE all MICs were equal to, or above the concentration of the raw,
undiluted extract (50 mg/mL). M1 US, MT US, and MD US exhibited antibacterial activity against E. coli, none of the UAE extracts were efficient against K. pneumonia, and
all of them showed antibacterial properties against P. aeruginosa at the highest concentration (50 mg/mL). Similarly, the extracts obtained by MAE had MICs equal or above the
maximum concentration tested (100 mg/mL) as it can be seen in Table 3.
In a previous study, it has been shown that aqueous spruce bark extracts obtained
by classical extraction and UAE had antibacterial effects against Gram-positive bacteria, but these extracts were also efficient against K. pneumoniae and P. aeruginosa at an
MIC of 15 mg/mL or lower [45]. Another study showed that spruce bark extracts obtained by Soxhlet and supercritical carbon dioxide extractions may inhibit the growth
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of Enterococcus faecalis and Streptococcus thermophilus, because of the content in catechin,
taxifolin, astringin, and isorhapontin [62]. These results suggest that our spruce bark extracts may exhibit this antibacterial activity because of the catechins present in the natural
extracts, as other studies have also shown the antibacterial properties of these compounds
against bacterial strains such as S. aureus and E. coli [63,64].
3. Materials and Methods
3.1. Materials
The spruce barks used to attain the tested extracts, were gathered in the beginning
of spring from several sites in Romania. The collecting sites were Tas, ca (46◦ 53′ 34′′ N,
26◦ 02′ 05′′ E), Neamt, county, Dolhasca (47◦ 25′ 49′′ N, 26◦ 36′ 34′′ E), Suceava county and
two sites in the area of Maramures, ului Mountains National Park (47◦ 47′ 05′′ N, 24◦ 34′ 20′′ E),
Maramures, County. The itinerary method was used to gather bark samples from randomly selected individuals from each site. The vegetal material was collected from a trunk
height of approximately 1.5 m. A voucher specimen was deposited at the Herbarium
of the Pharmaceutical Botany Department, George Emil Palade, University of Medicine,
Pharmacy, Sciences and Technology of Tîrgu-Mureş, with reference number: 116/3–116/6.
The final step of the plant material preparation consisted in the grinding of the spruce bark
samples collected with a GRINDOMIX GM 200 mill (Retsch GmbH, Haan, Germany).
Gram-positive and Gram-negative bacterial strains were used to assess the antibacterial activity of the natural extracts. Staphylococcus aureus (ATCC 25923), methicillin-resistant
Staphylococcus aureus (ATCC 43300), Escherichia coli (ATCC 25922), Klebsiella pneumoniae
(ATCC 700603), and Pseudomonas aeruginosa (ATCC 27853) strains were used and provided
by the Microbiology Department from the “G.E. Palade” University of Medicine, Pharmacy,
Sciences and Technology from Târgu-Mures, .
Reagents like ethanol 96% (v/v) used to obtain 50% and 70% solutions utilized for
extractions and Na2 CO3, used to prepare the solutions utilized for assessing total phenol
and tannin content, were purchased from Chemical Company SA, Ias, i, Romania. The
gallic acid used to obtain the calibration curve for the total phenol content assay, the 2,2diphenyl-1-picrylhydrazyl (DPPH) and 2,2′ -azino-bis(3-ethylbenzothiazoline-6-sulfonic
acid) (ABTS) reagents used for the antioxidant activity assays and hide powder used to
adsorb tannins and pyrogallol used as an external standard for the tannin content assay, were
all acquired from Sigma-Aldrich GmbH (Steinheim, Germany), while the Folin–Ciocâlteu
reagent used for the phytochemical characterization assays was obtained from Merck KGaA
(Darmstadt, Germany).
The standards used for the chromatographic assays (GC-FID and UPLC-PDA) were
acquired from Sigma-Aldrich GmbH, Steinheim, Germany. For the GC-FID analysis of the
extracts’ volatile compound content, nine standards were used: α- and β-pinene (99.0%),
3-carene (99.8%), α-phellandrene (98.9%), limonene (99.7%), sabinene (99.9%), myrcene
(99.8%), camphene (98.9%), and tricyclene (99.8%). For the UPLC-PDA analysis of the
extracts, standards from Sigma-Aldrich GmbH, Steinheim, Germany were utilized: gallic
acid—GAL (99%), sinapic acid—SINAP (99%), vanillic acid—VANIL (97%), chlorogenic
acid—CLR (99%), quercetin—QUER (95%), taxifolin—TAXI (85%), catechin—CAT (99%),
epicatechin—EPICAT (90%), eleutheroside B—ELEB (98%), and from Extrasyntese luteolin7-O-glucoside—LG (98%), luteolin-3′ , 7-diglucoside—LDG (97%).
3.2. Extraction
Ultrasound assisted (UAE) and microwave-assisted extractions (MAE) of the spruce
bark, were conducted in different concentration hydro-alcoholic mixtures. The extraction
protocols for both extraction methods were previously described in two studies conducted
on beech bark [34,65] and are briefly presented in Table 4.
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Table 4. Extraction parameters.
Extraction
Procedure
Extracted
Material
(Spruce Bark)
Solvent
(Ethanol)
Temperature
(◦ C)
Extraction
Time (min)
Ultrasound/Microwave
Power (W)
Ultrasound
Frequency
(kHz)
UAE
1g
70%
65
30
240
40
MAE
2g
50%
-
4
300
-
Two extractions were performed with each method for every bark sample, thus
resulting in two subvariants for every experimental variant. Triplicate measurements were
then performed on each one of these subvariants, leading to six measurements per variant,
for all of the used assays.
Eight experimental variants resulted after the extraction procedures, presented in
Table 5.
Table 5. Experimental variants for each collection site and extraction method used.
Collection Site
P. abies Bark Sample
Extraction Procedure
Extracts Attained
(Experimental Variant)
Maramures, ului Mountains National
Park site 1 (47◦ 47′ 05′′ N, 24◦ 34′ 20′′ E)
M1
UAE
MAE
M1 US
M1 M
Maramures, ului Mountains National
Park site 2 (47◦ 47′ 05′′ N, 24◦ 34′ 20′′ E)
M2
UAE
MAE
M2 US
M2 M
Tas, ca (46◦ 53′ 34′′ N, 26◦ 02′ 05′′ E)
MT
UAE
MAE
MT US
MT M
Dolhasca (47◦ 25′ 49′′ N, 26◦ 36′ 34′′ E)
MD
UAE
MAE
MD US
MD M
3.3. Assessment of the Content of Total Polyphenols (TPC)
For assessing the TPC, the Folin–Ciocâlteu method was used [66], slightly modified
as previously mentioned in one of our studies [61]. Briefly, 40 µL of diluted (1:5) extract
were brought together in a vial with distilled water, Folin–Ciocâlteu reagent and a 20%
Na2 CO3 solution. This mixture was then kept at room temperature for 30 min. Finally,
the absorbance was spectrophotometrically measured at a wavelength of 765 nm. For
every experimental variant six measurements were performed. A calibration curve was
used to express the TPC as gallic acid equivalents per gram of vegetal matrix (mg GAE/g
vegetal matrix).
3.4. Assessment of the Content of Total Tannins (TTC)
For the TTC determination of the tested extracts, a method utilized for the determination of tannins in herbal drugs was used [67]. This method uses the Folin–Ciocâlteu
assay to assess the TPC of the extracts spectrophotometrically, before and after treatment
with hide powder. This procedure uses the property of tannins to get adsorbed by the hide
powder, thus the difference in absorbance recorded for the tested samples before and after
treatment with hide powder, indirectly represents the absorbance of the tannins adsorbed.
The tannins absorbance was translated into TTC expressed as mg pyrogallol/g vegetal material by using pyrogallol as an external standard. The exact work protocol was previously
described [61], but this study used a 1:50 extract dilution and a 15% Na2 CO3 solution.
3.5. Terpenoids Content
In order to identify and quantify the potential volatile compounds present in the
bark extracts, gas chromatography with flame ionization detection (GC-FID) was used.
A GC with a TG-WAXMS capillary column, coupled with a FID detector was used for this
procedure. The linearity interval was determined using five calibration solutions prepared
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in duplicates. For the calculation of the concentrations, the densities of the compounds
were taken into account and a series of five dilutions was prepared from a stock solution
of terpenes. The concentrations of the solutions in the calibration curves were ranged
as follows: for Cα-pinene, β-pinene = 23.55–1177.5 mg/L, for Ccamphene = 11.85–150.3 mg/L,
for C3-carene = 12.25–148 mg/L, for Cα-phellandrene = 0.5–10 mg/L, for Climonene = 20–150.6 mg/L,
for Csabinene =5–50 mg/L, for Cmyrcene = 24.27–485.4 mg/L, for Ctricyclene = 5.15–122.1 mg/L,
respectively. If the signals given by some samples were out of linearity interval, the samples
were diluted in order to fit within linearity range of the method. The precision was
determined as relative standard deviation (% RSD) calculated for the three repeated measurements for the upper limit concentrations of the calibration curve (i.e., Cα-pinene, β-pinene
= 1177.5 mg/L, Ccamphene = 150.3 mg/L, C3-carene = 148 mg/L, Cα-phellandrene = 10 mg/L,
Climonene = 150.6 mg/L, Csabinene = 50 mg/L, Cmyrcene = 485.4 mg/L, Ctricyclene = 122.1 mg/L).
The extracts were directly injected into the GC and helium (He) was used as a carrier
gas. The identification process consisted in the comparison of the peak retention times
from the standard solution chromatogram with the retention times of the peaks registered
on the tested extracts chromatograms. The identified compounds were quantified based on
their peak height. The detailed analysis protocol was previously described [61].
3.6. Individual Polyphenol Content
The individual phenolic compounds from the tested extracts were identified and
quantified using ultra-performance liquid chromatography (UPLC). The detector used
for the UPLC analysis consisted in a photodiode array (PDA) detector. Chromatographic
separation was performed at 35 ◦ C on a Luna C18 (2) 150 × 4.6 mm, 3 µm column (Phenomenex). The mobile phase consisted of a mixture of formic acid 0.1% (A) and acetonitrile
(B). The gradient ellution was: 0–0.1 min: 90% A, 10% B; 0.1–20.1 min: 90%→ 20% A;
20.1–25.1 min: 20% A; 25.1–26.1 min: 20% A → 90% A; 26.1–30.1 min: 90% A. The flow
rate was 1 mL/min and the injection volume was 20 µL. The 210 nm, 280 nm, and 370 nm
wavelengths were monitored. The identification process consisted in the comparison of
the retention times of the peaks on the standard solution chromatogram with those on the
extracts’ chromatograms. The compounds associated with each peak were then quantified
based on the peak area and identified based on the similarity of UV spectra when compared
to standard solutions. This method was also described in our previous study [61].
3.7. Antioxidant Capacity (AC)
3.7.1. DPPH Assay
The AC was assessed using the DPPH method [68]. A 50-fold dilution was applied
before the assay for each experimental variant. The diluted extracts were then treated with
a DPPH solution and after 30 min the absorbance was spectrophotometrically measured at
517 nm. The exact method was previously described [61]; however, in this study 150 µL of
sample were used.
3.7.2. ABTS Assay
In order to assess the AC by the ABTS method [69] the experimental variants were
treated with the ABTS reagent and after 5–6 min the absorbance was spectrophotometrically
measured at 734 nm, in a microplate reader. The exact method was also previously
described [61].
3.8. Antibacterial Activity
The minimum inhibitory concentration (MIC) was used as an indicator of the antibacterial activity. For the MIC evaluation, the microdilution method that was previously
outlined [70], was used. In addition to this method, we used resazurin to ease the visualization of bacterial growth in the wells of the microplate [71]. For the accuracy of the results,
the extracts were first subjected to an evaporation step, to remove any trace of ethanol that
could modify antibacterial activity.
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3.9. Statistical Interpretation of the Data
The differences that occurred between the variants’ TPC, TTC, AC, and volatile
compounds content were statistically interpreted using a one-way ANOVA test to detect
potential differences in the means of the data sets. Afterward, Tukey’s post hoc test was
performed if any mean difference occurred, to identify the specific variants that significantly
differ (significance level α = 0.05, p ≤ 0.05) from each other. A Pearson correlation test was
also performed to assess the linear correlation between the TPC and the AC of the extracts.
These tests were performed in the GraphPad Prism 8 statistical software.
4. Conclusions
The results of this study indicated that differences occur between the barks collected
from different locations and between the extracts obtained by different methods, regarding
the phytochemical composition, antioxidant, and antibacterial activities. It has been shown
that the UAE is more efficient for extracting phenolic compounds and tannins (excepting
the case of the M1 variants), while the MAE generally resulted in higher yields of terpenoids
(mainly α-pinene and β-pinene). Minor differences were registered between the collecting
sites of the barks; however, these differences were not significant.
The antioxidant activity of the extracts attained by UAE was also higher compared to
their MAE counterparts, a strong positive correlation being identified between the TPC and
AC of the extracts, indicating that phenolic compounds are the main antioxidants present
in the extracts and thus the primary antioxidant agents in the bark of P. abies.
The antibacterial effects of the spruce bark extracts were more evident against Grampositive bacteria, all of the experimental variants having similar efficiencies against S. aureus
(methicillin sensitive and resistant), with M1 M and MD M variants being slightly more
efficient against these strains. Although, the extracts’ activity against Gram-negative
bacteria was clearly weaker, the growth inhibition of E. coli, K. pneumonia, and P. aeruginosa
still occurred for some of the experimental variants at higher MICs.
Considering the presented data, the spruce bark may represent an important source
of phytochemicals with antioxidant and antibacterial activities. The valorization of this
by-product and all of its bioactive constituents may lead to new natural products with
important therapeutic uses. However, more data regarding the toxicological profile, doses
and detailed mechanism of action are required in order to achieve this goal.
Author Contributions: Conceptualization, C.T.; methodology, A.N., R.S, ., A.D.M., D.I.S. and L.F.;
resources, C.T. and D.I.S.; data curation, A.N., R.S, ., L.F., A.D.M. and D.I.S.; writing—original draft
preparation, A.N. and R.S, .; writing—review and editing, C.T.; funding acquisition, C.T. and D.I.S.;
All authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by a grant of the Romanian Ministry of Education and Research,
Project PN 19110302 “Research on the variation trends specific to stable isotopes in different tree
species: deepening the fractionation mechanisms and the chemical processes interconnected on the
soil–water–plant chain”.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data sharing not applicable.
Acknowledgments: The authors would like to thank the Center for Advanced Medical and Pharmaceutical Research (George Emil Palade University of Medicine, Pharmacy, Science, and Technology of
Târgu Mures, ) and its staff for the support offered during sample analysis.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Elansary, H.O.; Szopa, A.; Kubica, P.; Al-Mana, F.; Mahmoud, E.A.; Zin El-Abedin, T.K.A.; Mattar, M.A.; Ekiert, H. Phenolic
Compounds of Catalpa speciosa, Taxus cuspidata, and Magnolia acuminata Have Antioxidant and Anticancer Activity. Molecules
2019, 24, 412. [CrossRef]
Plants 2021, 10, 870
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
13 of 15
Mármol, I.; Quero, J.; Jiménez-Moreno, N.; Rodríguez-Yoldi, M.J.; Ancín-Azpilicueta, C. A Systematic Review of the Potential
Uses of Pine Bark in Food Industry and Health Care. Trends Food Sci. Technol. 2019, 88, 558–566. [CrossRef]
Busquets-Ferrer, M.; Czabany, I.; Vay, O.; Gindl-Altmutter, W.; Hansmann, C. Alkali-Extracted Tree Bark for Efficient Bio-Based
Thermal Insulation. Constr. Build. Mater. 2021, 271, 121577. [CrossRef]
Hamad, A.M.A.; Ates, S.; Olgun, Ç.; Gür, M. Chemical Composition and Antioxidant Properties of Some Industrial Tree Bark
Extracts. BioResources 2019, 14, 5657–5671.
Fedorova, T.E.; Fedorov, S.V.; Babkin, V.A. Phenolic Compounds of Picea obovata Ledeb. Bark. Russ. J. Bioorganic Chem. 2019, 45,
927–932. [CrossRef]
Burlacu, E.; Nisca, A.; Tanase, C. A Comprehensive Review of Phytochemistry and Biological Activities of Quercus Species.
Forests 2020, 11, 904. [CrossRef]
Jablonsky, M.; Nosalova, J.; Sladkova, A.; Haz, A.; Kreps, F.; Valka, J.; Miertus, S.; Frecer, V.; Ondrejovic, M.; Sima, J.; et al. Valorisation
of Softwood Bark through Extraction of Utilizable Chemicals. A Review. Biotechnol. Adv. 2017, 35, 726–750. [CrossRef] [PubMed]
Ignat, I.; Volf, I.; Popa, V.I. A Critical Review of Methods for Characterisation of Polyphenolic Compounds in Fruits and Vegetables.
Food Chem. 2011, 126, 1821–1835. [CrossRef] [PubMed]
Cos, arcă, S.-L.; Moacă, E.-A.; Tanase, C.; Muntean, D.L.; Pavel, I.Z.; Dehelean, C.A. Spruce and Beech Bark Aqueous Extracts:
Source of Polyphenols, Tannins and Antioxidants Correlated to In Vitro Antitumor Potential on Two Different Cell Lines. Wood
Sci. Technol. 2019, 53, 313–333. [CrossRef]
Tanase, C.; Cos, arcă, S.; Muntean, D.-L. A Critical Review of Phenolic Compounds Extracted from the Bark of Woody Vascular
Plants and Their Potential Biological Activity. Molecules 2019, 24, 1182. [CrossRef]
Edreva, A.; Velikova, V.; Tsonev, T.; Dagnon, S.; Gürel, A.L.; Aktas, L. Stress-Protective Role of Secondary Metabolites: Diversity
of Functions and Mechanisms. Gen. Appl. Plant Physiol. 2007, 34, 67–78.
Bhattacharya, A.; Sood, P.; Citovsky, V. The Roles of Plant Phenolics in Defence and Communication during Agrobacterium and
Rhizobium Infection. Mol. Plant Pathol. 2010, 11, 705–719. [CrossRef]
Neiva, D.M.; Luís, Â.; Gominho, J.; Domingues, F.; Duarte, A.P.; Pereira, H. Bark Residues Valorization Potential Regarding
Antioxidant and Antimicrobial Extracts. Wood Sci. Technol. 2020, 54, 559–585. [CrossRef]
Silva, V.; Igrejas, G.; Falco, V.; Santos, T.P.; Torres, C.; Oliveira, A.M.P.; Pereira, J.E.; Amaral, J.S.; Poeta, P. Chemical Composition,
Antioxidant and Antimicrobial Activity of Phenolic Compounds Extracted from Wine Industry by-Products. Food Control 2018,
92, 516–522. [CrossRef]
Grzesik, M.; Naparło, K.; Bartosz, G.; Sadowska-Bartosz, I. Antioxidant Properties of Catechins: Comparison with Other
Antioxidants. Food Chem. 2018, 241, 480–492. [CrossRef] [PubMed]
Kuntzler, S.G.; Costa, J.A.V.; de Morais, M.G. Development of Electrospun Nanofibers Containing Chitosan/PEO Blend and
Phenolic Compounds with Antibacterial Activity. Int. J. Biol. Macromol. 2018, 117, 800–806. [CrossRef]
Li, R.; Narita, R.; Nishimura, H.; Marumoto, S.; Yamamoto, S.P.; Ouda, R.; Yatagai, M.; Fujita, T.; Watanabe, T. Antiviral Activity
of Phenolic Derivatives in Pyroligneous Acid from Hardwood, Softwood, and Bamboo. ACS Sustain. Chem. Eng. 2017, 6, 119–126.
[CrossRef]
Medini, F.; Megdiche, W.; Mshvildadze, V.; Pichette, A.; Legault, J.; St-Gelais, A.; Ksouri, R. Antiviral-Guided Fractionation and
Isolation of Phenolic Compounds from Limonium densiflorum Hydroalcoholic Extract. Comptes Rendus Chim. 2016, 19, 726–732.
[CrossRef]
Karimi, A.; Mohammadi-Kamalabadi, M.; Rafieian-Kopaei, M.; Amjad, L.; Salimzadeh, L. Determination of Antioxidant Activity,
Phenolic Contents and Antiviral Potential of Methanol Extract of Euphorbia spinidens Bornm (Euphorbiaceae). Trop. J. Pharm. Res.
2016, 15, 759–764. [CrossRef]
Martins, N.; Barros, L.; Santos-Buelga, C.; Henriques, M.; Silva, S.; Ferreira, I.C.F.R. Evaluation of Bioactive Properties and
Phenolic Compounds in Different Extracts Prepared from Salvia Officinalis L. Food Chem. 2015, 170, 378–385. [CrossRef] [PubMed]
Martins, N.; Barros, L.; Henriques, M.; Silva, S.; Ferreira, I.C.F.R. Activity of Phenolic Compounds from Plant Origin against
Candida Species. Ind. Crops Prod. 2015, 74, 648–670. [CrossRef]
Elansary, H.O.; Salem, M.Z.M.; Ashmawy, N.A.; Yessoufou, K.; El-Settawy, A.A.A. In Vitro Antibacterial, Antifungal and Antioxidant
Activities of Eucalyptus Spp. Leaf Extracts Related to Phenolic Composition. Nat. Prod. Res. 2017, 31, 2927–2930. [CrossRef]
Cao, J.; Han, J.; Xiao, H.; Qiao, J.; Han, M. Effect of Tea Polyphenol Compounds on Anticancer Drugs in Terms of Anti-Tumor
Activity, Toxicology, and Pharmacokinetics. Nutrients 2016, 8, 762. [CrossRef]
Mahboubi, A.; Asgarpanah, J.; Sadaghiyani, P.N.; Faizi, M. Total Phenolic and Flavonoid Content and Antibacterial Activity of
Punica Granatum L. Var. pleniflora Flowers (Golnar) against Bacterial Strains Causing Foodborne Diseases. BMC Complement.
Altern. Med. 2015, 15, 366. [CrossRef]
de Camargo, A.C.; Regitano-d’Arce, M.A.B.; Rasera, G.B.; Canniatti-Brazaca, S.G.; do Prado-Silva, L.; Alvarenga, V.O.; Sant’Ana,
A.S.; Shahidi, F. Phenolic Acids and Flavonoids of Peanut By-Products: Antioxidant Capacity and Antimicrobial Effects. Food
Chem. 2017, 237, 538–544. [CrossRef]
Lima, V.N.; Oliveira-Tintino, C.D.M.; Santos, E.S.; Morais, L.P.; Tintino, S.R.; Freitas, T.S.; Geraldo, Y.S.; Pereira, R.L.S.; Cruz, R.P.;
Menezes, I.R.A.; et al. Antimicrobial and Enhancement of the Antibiotic Activity by Phenolic Compounds: Gallic Acid, Caffeic
Acid and Pyrogallol. Microb. Pathog. 2016, 99, 56–61. [CrossRef] [PubMed]
Plants 2021, 10, 870
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
14 of 15
Martins, N.; Barros, L.; Santos-Buelga, C.; Silva, S.; Henriques, M.; Ferreira, I.C.F.R. Decoction, Infusion and Hydroalcoholic
Extract of Cultivated Thyme: Antioxidant and Antibacterial Activities, and Phenolic Characterisation. Food Chem. 2015, 167,
131–137. [CrossRef] [PubMed]
Guendouze-Bouchefa, N.; Madani, K.; Chibane, M.; Boulekbache-Makhlouf, L.; Hauchard, D.; Kiendrebeogo, M.; Stévigny, C.;
Okusa, P.N.; Duez, P. Phenolic Compounds, Antioxidant and Antibacterial Activities of Three Ericaceae from Algeria. Ind. Crops
Prod. 2015, 70, 459–466. [CrossRef]
Lagadinou, M.; Onisor, M.O.; Rigas, A.; Musetescu, D.-V.; Gkentzi, D.; Assimakopoulos, S.F.; Panos, G.; Marangos, M. Antimicrobial
Properties on Non-Antibiotic Drugs in the Era of Increased Bacterial Resistance. Antibiotics 2020, 9, 107. [CrossRef] [PubMed]
Elshamy, A.A.; Aboshanab, K.M. A Review on Bacterial Resistance to Carbapenems: Epidemiology, Detection and Treatment
Options. Future Sci. OA 2020, 6, FSO438. [CrossRef] [PubMed]
Meullemiestre, A.; Petitcolas, E.; Maache-Rezzoug, Z.; Chemat, F.; Rezzoug, S.A. Impact of Ultrasound on Solid–Liquid Extraction
of Phenolic Compounds from Maritime Pine Sawdust Waste. Kinetics, Optimization and Large Scale Experiments. Ultrason.
Sonochem. 2016, 28, 230–239. [CrossRef]
Chemat, F.; Khan, M.K. Applications of Ultrasound in Food Technology: Processing, Preservation and Extraction. Ultrason.
Sonochem. 2011, 18, 813–835. [CrossRef] [PubMed]
Vinatoru, M.; Mason, T.J.; Calinescu, I. Ultrasonically Assisted Extraction (UAE) and Microwave Assisted Extraction (MAE) of
Functional Compounds from Plant Materials. TrAC Trends Anal. Chem. 2017, 97, 159–178. [CrossRef]
Tanase, C.; Domokos, E.; Cos, arcă, S.; Miklos, A.; Imre, S.; Domokos, J.; Dehelean, C.A. Study of the Ultrasound-Assisted
Extraction of Polyphenols from Beech (Fagus sylvatica L.) Bark. BioResources 2018, 13, 2247–2267. [CrossRef]
Azwanida, N.N. A Review on the Extraction Methods Use in Medicinal Plants, Principle, Strength and Limitation. Med. Aromat.
Plants 2015, 4. [CrossRef]
Talmaciu, A.I.; Volf, I.; Popa, V.I. Supercritical Fluids and Ultrasound Assisted Extractions Applied to Spruce Bark Conversion.
Environ. Eng. Manag. J. 2015, 14, 615–623. [CrossRef]
Chmelová, D.; Škulcová, D.; Legerská, B.; Horník, M.; Ondrejovič, M. Ultrasonic-Assisted Extraction of Polyphenols and
Antioxidants from Picea Abies Bark. J. Biotechnol. 2020, 314–315, 25–33. [CrossRef]
Burčová, Z.; Kreps, F.; Grivnová, P.; Strižincová, P.; Ház, A.; Jablonský, M.; Šurina, I.; Schmidt, Š. Spruce Bark as a Source of
Antioxidant Active Substances. BioResources 2019, 14, 5980–5987.
Zhao, T.; Solheim, H.; Långström, B.; Borg-Karlson, A.-K. Storm-Induced Tree Resistance and Chemical Differences in Norway
Spruce (Picea abies). Ann. For. Sci. 2011, 68, 657–665. [CrossRef]
Salem, M.Z.M.; Elansary, H.O.; Elkelish, A.A.; Zeidler, A.; Ali, H.M.; EL-Hefny, M.; Yessoufou, K. In Vitro Bioactivity and
Antimicrobial Activity of Picea abies and Larix decidua Wood and Bark Extracts. BioResources 2016, 11, 9421–9437. [CrossRef]
Metsämuuronen, S.; Sirén, H. Bioactive Phenolic Compounds, Metabolism and Properties: A Review on Valuable Chemical
Compounds in Scots Pine and Norway Spruce. Phytochem. Rev. 2019, 18, 623–664. [CrossRef]
Diouf, P.N.; Stevanovic, T.; Cloutier, A. Study on Chemical Composition, Antioxidant and Anti-Inflammatory Activities of Hot
Water Extract from Picea mariana Bark and Its Proanthocyanidin-Rich Fractions. Food Chem. 2009, 113, 897–902. [CrossRef]
Halliwell, B. Are Polyphenols Antioxidants or Pro-Oxidants? What Do We Learn from Cell Culture and In Vivo Studies? Arch.
Biochem. Biophys. 2008, 476, 107–112. [CrossRef] [PubMed]
Lazar, L.; Talmaciu, A.I.; Volf, I.; Popa, V.I. Kinetic Modeling of the Ultrasound-Assisted Extraction of Polyphenols from Picea abies
Bark. Ultrason. Sonochem. 2016, 32, 191–197. [CrossRef] [PubMed]
Tanase, C.; Cosarca, S.; Toma, F.; Mare, A.; Cosarca, A.; Man, A.; Miklos, A.; Imre, S. Antibacterial Activities of Spruce Bark
(Picea abies L.) Extract and Its Components Against Human Pathogens. Rev. Chim. 2018, 69, 1462–1467. [CrossRef]
Sládková, A.; Benedeková, M.; Stopka, J.; Šurina, I.; Ház, A.; Strižincová, P.; Čižová, K.; Škulcová, A.; Burčová, Z.; Kreps, F.; et al.
Yield of Polyphenolic Substances Extracted From Spruce (Picea abies) Bark by Microwave-Assisted Extraction. BioResources 2016,
11, 9912–9921. [CrossRef]
Lacoste, C.; Čop, M.; Kemppainen, K.; Giovando, S.; Pizzi, A.; Laborie, M.-P.; Sernek, M.; Celzard, A. Biobased Foams from
Condensed Tannin Extracts from Norway Spruce (Picea abies) Bark. Ind. Crops Prod. 2015, 73, 144–153. [CrossRef]
Kemppainen, K.; Siika-aho, M.; Pattathil, S.; Giovando, S.; Kruus, K. Spruce Bark as an Industrial Source of Condensed Tannins
and Non-Cellulosic Sugars. Ind. Crops Prod. 2014, 52, 158–168. [CrossRef]
Bukhanko, N.; Attard, T.; Arshadi, M.; Eriksson, D.; Budarin, V.; Hunt, A.J.; Geladi, P.; Bergsten, U.; Clark, J. Extraction of
Cones, Branches, Needles and Bark from Norway Spruce (Picea abies) by Supercritical Carbon Dioxide and Soxhlet Extractions
Techniques. Ind. Crops Prod. 2020, 145, 112096. [CrossRef]
Kain, G.; Stratev, D.; Tudor, E.; Lienbacher, B.; Weigl, M.; Barbu, M.-C.; Petutschnigg, A. Qualitative Investigation on VOCEmissions from Spruce (Picea abies) and Larch (Larix decidua) Loose Bark and Bark Panels. Eur. J. Wood Wood Prod. 2020, 78, 403–412.
[CrossRef]
Ghimire, R.P.; Kivimäenpää, M.; Blomqvist, M.; Holopainen, T.; Lyytikäinen-Saarenmaa, P.; Holopainen, J.K. Effect of Bark Beetle
(Ips typographus L.) Attack on Bark VOC Emissions of Norway Spruce (Picea abies Karst.) Trees. Atmos. Environ. 2016, 126, 145–152.
[CrossRef]
Plants 2021, 10, 870
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
15 of 15
Albuquerque, B.R.; Prieto, M.A.; Barreiro, M.F.; Rodrigues, A.; Curran, T.P.; Barros, L.; Ferreira, I.C.F.R. Catechin-Based Extract
Optimization Obtained from Arbutus unedo L. Fruits Using Maceration/Microwave/Ultrasound Extraction Techniques. Ind.
Crops Prod. 2017, 95, 404–415. [CrossRef]
Liazid, A.; Palma, M.; Brigui, J.; Barroso, C.G. Investigation on Phenolic Compounds Stability during Microwave-Assisted
Extraction. J. Chromatogr. A 2007, 1140, 29–34. [CrossRef] [PubMed]
Spinelli, S.; Costa, C.; Conte, A.; La Porta, N.; Padalino, L.; Del Nobile, M.A. Bioactive Compounds from Norway Spruce Bark:
Comparison Among Sustainable Extraction Techniques for Potential Food Applications. Foods 2019, 8, 524. [CrossRef] [PubMed]
Jablonsky, M.; Majova, V.; Strizincova, P.; Sima, J.; Jablonsky, J. Investigation of Total Phenolic Content and Antioxidant Activities
of Spruce Bark Extracts Isolated by Deep Eutectic Solvents. Crystals 2020, 10, 402. [CrossRef]
Škulcová, A.; Haš, Z.; Hrdli, L.; Šima, J.; Jablonský, M. Green Solvents Based on Choline Chloride for the Extraction of Spruce
Bark (Picea abies). Cellulose Chem. Technol. 2018, 52, 171–179.
Kasparavičienė, G.; Ramanauskienė, K.; Savickas, A.; Velžienė, S.; Kalvėnienė, Z.; Kazlauskienė, D.; Ragažinskienė, O.; Ivanauskas,
K. Evaluation of Total Phenolic Content and Antioxidant Activity of Different Rosmarinus officinalis L. Ethanolic Extracts. Biologija
2013, 59. [CrossRef]
Nayak, B.; Dahmoune, F.; Moussi, K.; Remini, H.; Dairi, S.; Aoun, O.; Khodir, M. Comparison of Microwave, Ultrasound and
Accelerated-Assisted Solvent Extraction for Recovery of Polyphenols from Citrus sinensis Peels. Food Chem. 2015, 187, 507–516.
[CrossRef]
Dhanani, T.; Shah, S.; Gajbhiye, N.A.; Kumar, S. Effect of Extraction Methods on Yield, Phytochemical Constituents and
Antioxidant Activity of Withania Somnifera. Arab. J. Chem. 2017, 10, S1193–S1199. [CrossRef]
Karabegović, I.T.; Stojičević, S.S.; Veličković, D.T.; Todorović, Z.B.; Nikolić, N.Č.; Lazić, M.L. The Effect of Different Extraction
Techniques on the Composition and Antioxidant Activity of Cherry Laurel (Prunus laurocerasus) Leaf and Fruit Extracts. Ind.
Crops Prod. 2014, 54, 142–148. [CrossRef]
Nisca, A.; S, tefănescu, R.; Stegărus, , D.I.; Mare, A.D.; Farczadi, L.; Tanase, C. Comparative Study Regarding the Chemical
Composition and Biological Activity of Pine (Pinus nigra and P. sylvestris) Bark Extracts. Antioxidants 2021, 10, 327. [CrossRef]
Ferrentino, G.; Haman, N.; Morozova, K.; Tonon, G.; Scampicchio, M. Phenolic Compounds Extracted from Spruce (Picea abies) by
Supercritical Carbon Dioxide as Antimicrobial Agents against Gram-Positive Bacteria Assessed by Isothermal Calorimetry. J.
Therm. Anal. Calorim. 2020, 1–11. [CrossRef]
Li, F.; Jin, H.; Xiao, J.; Yin, X.; Liu, X.; Li, D.; Huang, Q. The Simultaneous Loading of Catechin and Quercetin on Chitosan-Based
Nanoparticles as Effective Antioxidant and Antibacterial Agent. Food Res. Int. 2018, 111, 351–360. [CrossRef] [PubMed]
Zhang, H.; Jung, J.; Zhao, Y. Preparation, Characterization and Evaluation of Antibacterial Activity of Catechins and Catechins–Zn
Complex Loaded β-Chitosan Nanoparticles of Different Particle Sizes. Carbohydr. Polym. 2016, 137, 82–91. [CrossRef]
Tanase, C.; Mocan, A.; Cos, arcă, S.; Gavan, A.; Nicolescu, A.; Gheldiu, A.-M.; Vodnar, D.C.; Muntean, D.-L.; Cris, an, O. Biological and Chemical Insights of Beech (Fagus sylvatica L.) Bark: A Source of Bioactive Compounds with Functional Properties.
Antioxidants 2019, 8, 417. [CrossRef]
Sánchez-Rangel, J.C.; Benavides, J.; Heredia, J.B.; Cisneros-Zevallos, L.; Jacobo-Velázquez, D.A. The Folin–Ciocalteu Assay
Revisited: Improvement of Its Specificity for Total Phenolic Content Determination. Anal. Methods 2013, 5, 5990–5999. [CrossRef]
Council of Europe. European Pharmacopoeia, 7th ed.; Council of Europe: Strasbourg, France, 2010.
Bondet, V.; Brand-Williams, W.; Berset, C. Kinetics and Mechanisms of Antioxidant Activity Using the DPPH.Free Radical Method.
LWT Food Sci. Technol. 1997, 30, 609–615. [CrossRef]
Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; Rice-Evans, C. Antioxidant Activity Applying an Improved ABTS
Radical Cation Decolorization Assay. Free Radic. Biol. Med. 1999, 26, 1231–1237. [CrossRef]
Tanase, C.; Cosarca, S.; Toma, F.; Mare, A.; Man, A.; Miklos, A.; Imre, S.; Boz, I. Antibacterial Activities of Beech Bark
(Fagus sylvatica L.) Polyphenolic Extract. Environ. Eng. Manag. J. 2018, 17, 877–884. [CrossRef]
Tanase, C.; Berta, L.; Coman, N.A.; Ros, ca, I.; Man, A.; Toma, F.; Mocan, A.; Nicolescu, A.; Jakab-Farkas, L.; Biró, D.; et al.
Antibacterial and Antioxidant Potential of Silver Nanoparticles Biosynthesized Using the Spruce Bark Extract. Nanomaterials
2019, 9, 1541. [CrossRef] [PubMed]