Bark is an attractive renewable raw material, comprised of all types of silviculture vegetation. This renewable resource is a major alternative raw material for the food, chemical and pharmaceutical industry. Valorization is a key component of an economic lignocellulosic biorefinery. In this paper are included three extraction techniques and comparisons of total phenolic content. All extractions were done on milled spruce bark (Picea abies). Microwave assisted extraction; accelerated solvent extraction and extraction with deep eutectic solvents were used as a technique for extracts isolation. Choline chloride-based eutectic solvents with car-boxylic acids (maleic or malic acid) and glycerol were used as extractants. The extractions were performed for 1 h at 60 0 C with continuous stirring. Accelerated solvent extraction (extractant 96.6% ethanol; temperature (120, 140, 160 0 C) with steam pre-treatment (10, 20, 30 min.) was used as another type of extraction technique. The total phenolic content was determined spectrophotometrically at 764nm using the Folin-Ci-ocalteu method. This test is based on the oxidation of phenolic groups by phosphomolybdic and phospho-tungstic acids (FC reagent). Extraction technique using deep eutectic solvents brings results of phenolic contents in ranged from 900 to 2000 mg GAE per 100 g of dry bark. Samples with range of phenolic contents between 136.2 and 230.3 mg GAE per 100 g of dry bark were prepared by using acceleratet solvent extraction. Closed-system microwave assisted extraction (time 3 to 20 min.), and temperature (60; 80; 100 0 C) was applied to extract total phenolics from spruce bark, using 96.6% ethanol as an extractant. The total extracted phenolics, as assessed by Folin-Ciocalteu assay, varied between 90.3 and 321 mg gallic acid equivalence (GAE) per 100 g of dry bark for different temperatures. The results indicated that the highest amount of total phenolic compounds were found in extracts when using extraction by deep eutectic solvents

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2. Journal of Hygienic Engineering and Design
72
Abstract
Bark is an attractive renewable raw material, comprised
of all types of silviculture vegetation. This renewable
resource is a major alternative raw material for the
food, chemical and pharmaceutical industry. Valoriza-
tion is a key component of an economic lignocellulosic
biorefinery. In this paper are included three extraction
techniques and comparisons of total phenolic content.
All extractions were done on milled spruce bark (Picea
abies). Microwave assisted extraction; accelerated sol-
vent extraction and extraction with deep eutectic sol-
vents were used as a technique for extracts isolation.
Choline chloride-based eutectic solvents with car-
boxylic acids (maleic or malic acid) and glycerol were
used as extractants. The extractions were performed
for 1 h at 60 0
C with continuous stirring. Accelerated
solvent extraction (extractant 96.6% ethanol; tempera-
ture (120, 140, 160 0
C) with steam pre-treatment (10,
20, 30 min.) was used as another type of extraction
technique. The total phenolic content was determined
spectrophotometrically at 764nm using the Folin–Ci-
ocalteu method. This test is based on the oxidation of
phenolic groups by phosphomolybdic and phospho-
tungstic acids (FC reagent).
Extraction technique using deep eutectic solvents
brings results of phenolic contents in ranged from 900
to 2000 mg GAE per 100 g of dry bark. Samples with
range of phenolic contents between 136.2 and 230.3
mg GAE per 100 g of dry bark were prepared by using
acceleratet solvent extraction. Closed-system micro-
wave-assisted extraction (time 3 to 20 min.), and tem-
perature (60; 80; 100 0
C) was applied to extract total
phenolics from spruce bark, using 96.6% ethanol as an
extractant. The total extracted phenolics, as assessed
by Folin-Ciocalteu assay, varied between 90.3 and 321
mg gallic acid equivalence (GAE) per 100 g of dry bark
for different temperatures.
The results indicated that the highest amount of total
phenolic compounds were found in extracts when us-
ing extraction by deep eutectic solvents
Key words: Extractives, Antioxidants, Food supplement,
Spruce bark.
1. Introduction
In addition to the main components of wood - cel-
lulose, hemicellulose and lignin, a small amount of
extraction or related substances is found. Accesso-
ry substances are compounds that can be extracted
with various solvents (e.g., ether, alcohol, water) from
individual wood species without any change in wood
building (Perelygin, [1]).
The amount and composition of extracts among relat-
ed tree species is very variable. Significant differences
in composition can also be seen in different parts of
the same tree. In general, the content of extracts is
higher in the bark, leaves and roots than in the tree
core (wood).
During the storage of wood, the content of extracts
decreases and their chemical composition chang-
es. There is a chain reaction generating free radicals,
which are particularly potent antioxidants. The influ-
ence of metal ions and light accelerates this process
of autoxidation. Nevertheless, only small quantities
of wood are excreted in the wood, have a great effect
Original scientific paper
UDC 582.475-114.43:542.61]:615.017
COMPARISON OF DIFFERENT EXTRACTION METHODS FOR THE
EXTRACTION OF TOTAL PHENOLIC COMPOUNDS FROM SPRUCE BARK
Ales Haz1*
, Michal Jablonsky1
, Veronika Majova1
, Andrea Skulcova1
, Petra Strizincova1
1
Department of Wood, Pulp, and Paper, Institute of Natural and Synthetic Polymers,
Slovak University of Technology, Radlinského 9, 81237 Bratislava, Slovak Republic
*
e-mail: ales.haz@stuba.sk

3. Journal of Hygienic Engineering and Design
73
on the properties and quality of the wood. They make:
smell, durability, permeability, insect resistance, or
lightfastness (Guangyu, [2]).
They are used as synthetic preservatives to increase the
life of wood or to protect it against molds and pests.
They act as a response to injury or act as a defensive
mechanism of wood. Many volatile extracts, in partic-
ular monoterpenes, camphor, α-pin, are causing the
characteristic smell of wood. While polyphenols and
tannins in hardwood hardly affect the color of wood.
This property is mainly used in the furniture industry
and in the production of floors. Tannins are extensive-
ly used in leather processing (Fengel, [3]). Nonpolar
low-oxygen extracts, such as: terpenes, oils, fats, resins,
affect the hygroscopicity and permeability of wood.
On the other hand, however, they have a high energy
value and increase the flammability of wood, espe-
cially the presence of resins, which is used in the pro-
duction of fuels. Some woody companion substances
are very valuable, for example, quinines, while others
are allergenic and toxic. The disadvantage is that the
group of valuable materials in the place of contact with
wood accelerates metal corrosion.
Extract substances are of great importance in medi-
cine. For example, substances derived from the bark of
a medical queen contain a quantity of quinine, which
has a beneficial effect in the treatment of malaria.Ther-
apeutic effects are also associated with asthma treating
agents. Mannitol, which can be extracted from woods
such as Fraxinus ornus or Olea europaea, has been used
in the past as a laxative (Harkin, [4]).
2. Materials and Methods
All extractions were made on milled spruce bark (Picea
abies). Microwave assisted extraction, accelerated sol-
vent extraction, and extraction with deep eutectic sol-
vents were used as a technique for extracts isolation.
Choline chloride-based eutectic solvents with carbox-
ylic acids (maleic or malic acid) and glycerol were used
as extractants. Choline chloride (≥ 98% mass fraction
purity), maleic acid (p.a.), malic acid (p.a.), glycerol
(86%), and ethanol (96.6%), was purchased from Sig-
ma-Aldrich.
2.1 Accelerated solvent extraction
Before extraction all samples were treated by steam
(temperature: 120, 140, 160 0
C, time of exposure: 10,
20, 30 min.) Accelerated solvent extraction (extract-
ant 96.6% ethanol) was then used as one type of ex-
traction technique. Extraction was performed with the
Accelerated Solvent Extractor model Dionex 350. The
extraction pressure (1500 psi) was imposed by the ASE
350 apparatus. Samples, typically 12 g, were placed
into stainless steel extraction chambers. Parameter of
static time (time for reaching final temperature) was 6
min. for 120 0
C, 140 0
C and 8 min. for 160 0
C. The sam-
ple in stainless steel cell was flushed with 50% volume
of used solvent and extract was collected in vial after
static time. Samples were sequentially extracted three
times. The extraction was carried out in duplicate. All
extracts were evaporated to dryness in rotary evapo-
rator at 40 0
C.
2.2 Microwave assisted extraction
Closed-system microwave-assisted extraction (MAE)
was performed using a MicroSYNTH Labstation (max-
imum output; 1.5 kW, 2.45 GHz, Milestone Inc., Shel-
ton, CT, USA, maximum temperature 250 0
C, maximum
pressure 100 bar) with an HPR 100 (high-pressure 100
ml) reactor. Sample (~2 g) was suspended in 20 mL of
96.6% ethanol, followed by microwave irradiation at
60, 80 and 100 0
C for: 1, 2, 3, 5, 10, 15 and 20 min. in
the reactor. To reach desired temperature, 3 min. heat-
up time was applied. After MAE, the extracted liquors
were cooled to room temperature (maximum cooling
time < 15 min.) and immediately filtered through No.
1 filter paper to separate the extract and the residue.
2.3 Extraction with deep eutectic solvents
Milled spruce bark (Picea abies) was added into flask
in ratio 1 : 1 (choline chloride: carboxylic acids) and in
ratio 1 : 2 (choline chloride : glycerol). Choline chloride
(≥ 98%, Sigma Aldrich, Bratislava, Slovakia) was mixed
with malic and maleic acid and glycerol (86%). Eight
carboxylic acids were tested: tartaric acid (99.5%), lac-
tic acid (9%, WVR®
), malonic acid (p.a.), malic acid (p.a.),
maleic acid (p.a.), glycolic acid (p.a.), oxalic acid × 2H2
O
(p.a.), and citric acid × H2
O (p.a.). The mixtures were
stirred in an oil bath to form a homogeneous liquid at
60 °C to 80 °C, depending on the carboxylic acid.
The dried and weighed ground bark was added to the
DESs at a 1 : 20 (wt/wt) ratio. The extraction was per-
formed for 1 h at 60 0
C with continuous stirring in a
closed flask.
2.4 Folin-Ciocalteu method/Gallic acid equivalent
method (GAE)
The total phenolic content was determined spectro-
photometrically (VWR UV-VIS 1600PC) at 764nm using
the Folin-Ciocalteu method. A spectrophotometric Fo-
lin-Ciocalteu (FC) method was used to determine the
proportion of phenol components in the extract. This
method is commonly used for the determination of
phenols from woody extracts, but the procedures vary
considerably. One part of this method is a FC agent in
which phosphotungstic acid and phosphomolybdic

4. Journal of Hygienic Engineering and Design
74
acid complexes are present. Reaction of complexes
with phenolic compounds results in a blue discolor-
ation. The principle is the oxidation of phenols in al-
kaline solution of Na2
CO3
. Gallic acid is used as a stan-
dard. The resulting value is subsequently converted to
an equivalent amount of gallic acid. (Mrázová, [5]). All
analyses were carried out in duplicate and the results
were expressed in mg GAE (gallic acid equivalent)/g
dry residue.
3. Results and Discussion
3.1 Accelerated solvent extraction
The application of ASE is an important method for ob-
taining extractive compounds from lignicellulosic ma-
terials. Opportunities and conditions of extraction by
accelerated solvent extraction were confirmed in this
study. Extraction yield of phenolics is affected by the
addition to the time and temperature of extraction
and the type of solvent. From the liquid fraction was
­determined the concentration of the ­extractives.
All extracts were prepared with ethanol at: 120,140
and 160 0
C.
The concentration of extractives was determined from
the obtained extract by solvent evaporation (Table 1).
3.2 Microwave assisted extraction
Total extracted phenolics, assessed by Folin-Ciocalteau
assay, varied between 42.7 and 265.0 mg GAE/100 g
dry bark for different particles at temperature of ex-
traction 60 0
C (Fig. 3). The yield reached and 90.3 and
321.1 mg GAE/100 g dry bark at 60 0
C to 100 0
C, respec-
tively when extracting 1 mm particles. Other studies
on European softwood bark extracts (Jerez et al. [6];Ye-
sil-Celiktas etal., [7]), reported values in the same range
as determined in our work. In determining the propor-
tion of polyphenols in a liquid extract a positive impact
on the efficiency of the extraction time also could be
seen, but we do not see a reduction in the yields of
polyphenols at 15 minutes. From Table 2 higher yields
of polyphenols are achieved at higher temperatures.
Table 2. Yield of extracts and content of phenolics (ASE, steam pre-treatment)
Parameters
Temperature [0
C]
60 80 100
Time of steam
exposure [min.]
1 10 20 1 10 20 1 10 20
Yield of extract [%] 5.18 5.78 5.6 5.24 5.93 5.76 5.7 7.04 6.63
Phenolic content
[mg GAE/100 g]
90.3 120.1 153.1 139.7 250.7 265.7 155.3 302.3 321.1
Table 1. Yield of extracts and content of phenolics (ASE, steam pre-treatment)
Parameters
Temperature [0
C]
120 140 160
Time of steam
exposure [min.]
10 20 30 10 20 30 10 20 30
Yield of extract [%] 16.41 17.44 18.08 19.54 20.77 21.45 26.38 27.37 28.44
Phenolic content
[mg GAE/100 g]
136.2 144.7 150.1 162.2 172.4 178.0 218.9 227.2 236.3
Table 3. Yield of extracts and content of phenolics (DES, 60 min.)
Parameters
Temperature [0
C]
60
Time of steam
exposure [min.]
ChChl : malic acid ChChl : maleic acid ChChl : glycerol
Yield of extract [%] 14.68 11.87 11.40
Phenolic content
[mg GAE/100 g]
900 2000 1700

5. Journal of Hygienic Engineering and Design
75
3.3 Extraction with deep eutectic solvents
The effect of DESs on extraction and fractionation is
not fully understood. The application of DESs to indi-
vidual components (e.g., cellulose, lignin) may lead to
their solubilisation (Kroon et al., [8]). When applied to
lignocellulosic biomass, its complexity and composi-
tion can complicate extraction, fractionation, or del-
ignification. Researches focused on the verification of
selectivity in the fractionation process reflect the com-
plexity of DESs selectivity (de Dios, [9]; Jablonský et
al., [10]; Kumar et al., [11]; Grande, [12]; Škulcová et al.,
[13]). Yield of extracts and content of phenolics (DES,
60 min.) is presented in Table 3.
4. Conclusions
- The composition of bark Picea abies in terms of
monoterpenes, sesquiterpene, diterpenes and fatty
acid fractions was very complex. Antioxidant activity is
mainly connected with phenolic compounds which is
important parameter for further application.
- Different techniques show strong dependence with
this parameter. In case of extraction with deep eutectic
solvents, results can be explained as process which is
linked with solubility of lignin. Lignin consist of phen-
ylpropanoid units which can be determined with FC
method also. Wide range of phenolic content (90.3 -
2,000 mg gallic acid equivalence (GAE) per 100 g of dry
bark) shows strong dependence on used technique.
- All results lead to better knowledge of exact extract
composition and their potential risks during further
application in chemicals, pharmaceuticals, food, plas-
tics etc.
Acknowledgement
This work was supported by the Slovak Research and
Development Agency under the contracts Nos. APVV-
15-0052 and APVV-0393-14, andVEGA grant 1/0848/17.
This article was realized also thanks to the support for
infrastructure equipment by the Operation Program
Research and Development for the project “National
Center for Research and Application of renewable en-
ergy sources” (ITMS 26240120016, ITMS 26240120028)
for the project “Competence center for new ma-
terials, advanced technologies and energy“ (ITMS
26240220073) and for the project “University science
park STU Bratislava“ (ITMS 26240220084), co-financed
by the European Regional Development Fund. The au-
thors would like to thank for financial assistance from
the STU Grant scheme for Support of excellent Teams
of Young Researchers under the contract no. 1663.
5. References
[1] Perelygin L. M. (1965). Woodworking (in Slovakian).
Slovenské vydavateľstvo technickej literatúry,
Bratislava, Slovakia.
[2] Guangyu Y. Jaakkola P. (2011). Wood chemistry and
isolation of extractives from wood.
URL: http://biotuli-hanke.fi/files/download/Biotuli_Yangj
Jaakkola2011.pdf. Accessed 16 October 2017.
[3] Fengel D., Wegener G. (1989). Wood: Chemistry,
ultrastructure, reactions. Walter de Gruyter, Berlin,
Germany.
[4] Harkin J. M. Rowe W. J. (1971). Bark and its possible uses.
U.S.D: A. Forest service, Madison, USA.
[5] Mrázová E. (2011). Determination of phenolic substances
and antioxidant activity in cereals (in Czech). Diploma
thesis, Tomas Bata University in Zlín, Faculty of
Technology, Czech Republic.
[6] Jerez M., Selga A., Sineiro J., Torres J. L., Nunez M. J.
(2007). A comparison between bark extracts from Pinus
pinaster and Pinus radiata: Antioxidant activity and
procyanidin composition. Food Chem., 100, pp. 439-444.
[7] Yesil-Celiktas O., Ganzera M., Akgun I., Sevimli C.,
Korkmaz K. S., Bedir E. (2009). Determination of
polyphenolic constituents and biological activities of
bark extracts from different Pinus species. Journal of the
Science of Food and Agriculture, 89, (8), pp. 1339-1345.
[8] Kroon M. C., Casal M. F., van den Bruinhorst A. (2012).
Pretreatment of lignocellulosic biomass and recovery
of substituents using natural deep eutectic solvents/
compound mixtures with low transition temperatures.
International patent: WO2013/153203 A1.
[9] de Dios, S. L. G. (2013). Phase equilibria for extraction
processes with designer solvents. Dissertation Thesis,
University of Santiago de Compostela, Santiago, Spain.
[10] Jablonský M., Škulcová A., Kamenská L., Vrška M., Šima
J. (2015). Deep eutectic solvents: Fractionation of wheat
straw. BioResources, 10, (4), pp. 8039-8047.
[11] Kumar A. K., Parikh B. S., Pravakar M. (2016). Natural
deep eutectic solvent mediated pretreatment of rice
straw: Bioanalytical characterization of lignin extract
and enzymatic hydrolysis of pretreated biomass residue.
Environmental Science and Pollution Research, Volume
23, 10, pp. 9265-9275.
[12] Grande, P. M. (2014). Novel bio-based catalytic strategies
for the fractionation and valorization of lignocellulose.
Sc.D. Thesis, RWTH Aachen University, Aachen,
Germany.
[13] Škulcová A., Jablonský M., Ház A., Vrška M. (2016).
Pretreatment of wheat straw using deep eutectic solvents
andultrasound. Przeglad Papierniczy, 72, (4), pp. 243-247.
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