Journal of Biotechnology 314–315 (2020) 25–33

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Journal of Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec

Research Article

Ultrasonic-assisted extraction of polyphenols and antioxidants from Picea T

abies bark
Daniela Chmelováa,*, Dominika Škulcováa, Barbora Legerskáa, Miroslav Horníkb,
Miroslav Ondrejoviča
a
University of Ss. Cyril and Methodius in Trnava, Faculty of Natural Sciences, Department of Biotechnology, Nam J. Herdu 2, SK-91701, Trnava, Slovak Republic
b
University of Ss. Cyril and Methodius in Trnava, Faculty of Natural Sciences, Department of Ecochemistry and Radioecology, Nám. J. Herdu 2, SK-917 01, Trnava,
Slovak Republic

ARTICLE INFO ABSTRACT

Keywords: Spruce bark represents a reservoir of bioactive compounds. The aim of this study was to investigate the effect of
bark independent variables (temperature, liquid to solid ratio, time and methanol content) and their interaction
spruce within the extraction process by the response surface methodology (RSM). The effect of conventional (solvent
antioxidants extraction; SE) and modern (ultrasound-assisted extraction; UAE) methods for the extraction of antioxidants
polyphenols
(antioxidant capacity; AC) and polyphenols (total polyphenol content; TPC) was compared. Maximum yields of
RSM
ultrasound-assisted extraction
AC and TPC by SE and UAE were obtained at modified optimal conditions of 63 °C, methanol content of 53 % (v/
v) and 38 mL of extraction solvent per gram of dry material. Two-step extraction process consisting of the fast
washing and slow diffusion steps was suitable described by Peleg and Patricelli mathematic models. The HPLC
fingerprints of both extracts did not show significant differences while the content of phenolic compounds ex-
tracted by UAE was 1.1- to 7.1-times higher than that obtained by SE, quantified by HPLC.

1. Introduction spruce species have proven effects on human health, for example, that
can work as antioxidants to terminate the oxidation reactions re-
Spruce species are one of the most widespread conifers in the sponsible for the tissue damage in living organisms (Rummukainen
European temperate forests (Spiecker, 2003). They belong to important et al., 2004; García-Pérez et al., 2012). Halliwell (1990) characterizes
raw materials generated by the forest industry in Nordic countries. The antioxidants as substances which are able to significantly delay or
bark of spruce represents one of the major biomass feedstocks that is prevent oxidation of the substrate at low concentrations. Polyphenols,
currently considered as an undervalued resource and the most of this such as stilbenes, phenolic acids, flavonoids, and lignans are the major
material is incinerated. However, bark is rich in phytochemicals which compounds with the antioxidant activity present in conifers (Kähkönen
could potentially be used for various industrial applications (in food, et al., 1999; Kreps et al., 2017). The main polyphenols of Picea abies can
pharmaceutical and chemical industries as bio-based products etc.) be classified into the groups of monoaryl compounds, stilbene gluco-
(Pietarinen et al., 2006; Balas and Popa, 2007; García-Pérez et al., sides, lignans, flavonoids, catechins or proanthocyanidins (Pan and
2012). Valorization of bark could lead to a more efficient use of re- Lundgren, 1995; Lacoste et al., 2015; Talmaciu et al., 2016).
sources and waste minimization (Rosell et al., 2014; Tofan et al., 2016). The recovery of plant polyphenols can be performed using nu-
Generally, the bark contains utilizable compounds, such as poly- merous extraction techniques. The extraction of polyphenols with a
saccharides, pectin substances, cross-linked polyesters (suberin and solvent (hot-water, low-molecular weight alcohols) belongs to con-
cutin), terpenoids and polyphenols in unbound (free) or polymer forms ventional technique used in many industrial processes (Ghitescu et al.,
(lignin and high-molecular-weight tannins) (Pan and Lundgren, 1995; 2015), but this technique has some disadvantages. Nowadays, there is
Co et al., 2012; Feng et al., 2013; Kemppainen et al., 2014; Jablonsky an attention to modern techniques of extraction supporting the con-
et al., 2017). Some of these compounds present in wood and bark of ventional extraction procedure using ultrasound or pulsed electric field

Abbreviations: SE, solvent extraction; UAE, ultrasound-assisted extraction; AC, antioxidant capacity; TPC, total polyphenol content; RSM, response surface meth-
odology; GAE, gallic acid equivalent; R2, coefficient of determination; adjR2, adjusted coefficient of determination; RMSE, root mean square error; %AARD, per-
centage average absolute relative deviation
⁎
Corresponding author.
E-mail address: [email protected] (D. Chmelová).

https://doi.org/10.1016/j.jbiotec.2020.04.003
Received 20 December 2019; Received in revised form 1 April 2020; Accepted 6 April 2020
Available online 12 April 2020
0168-1656/ © 2020 Elsevier B.V. All rights reserved.
D. Chmelová, et al. Journal of Biotechnology 314–315 (2020) 25–33

(García-Pérez et al., 2012; Royer et al., 2013; Ghitescu et al., 2015; function (Equation 1):
Bouras et al., 2016; Lazar et al., 2016). These are more efficient, faster, k k k 1
and with minimum consumption of solvent (Altemimi et al., 2015), but Y = b0 + bi Xi + bii Xi2 + bij Xi Xj
for their effective application, the finding of optimal conditions of i 1 i 1 i 1 j 2
bioactive compound extraction is an essential step. i<j (1)
However, up to date, there is a lack of information regarding the where Xi is independent variable causing the Y response and bi is re-
optimization of the antioxidants extracted from the spruce bark. The gression coefficient describing the dependence between measured
aim of this study was to investigate the effect of different extraction properties and coded values of observed factors. Three-dimensional
parameters including temperature, methanol content and liquid to solid surface response plots were generated by varying of two variables
ratio on two response variables describing the extraction yields for within the experimental range and holding the third constant at the
antioxidants and polyphenols present in spruce bark (P. abies) (anti- optimized point. The coefficients of the response surface equation were
oxidant capacity; AC and total polyphenol content; TPC) by applying estimated.
the response surface methodology (RSM) and to compare the extracts
obtained by solvent extraction (SE) and ultrasound-assisted extraction
2.4. Chemical analysis
(UAE). The experimental data of kinetics of both realized extraction
processes at the optimal conditions were fitted with three empirical
AC was calculated using the DPPH method (Yen and Chen, 1995)
models, namely Page, Peleg and Patricelli models in order to determine
and was performed in the reaction containing extract (25 μL) and 100
the best model describing the extraction of antioxidants and poly-
μL of DPPH methanolic solution (0.012 %; w/v). After incubation at
phenols from spruce bark. Moreover, HPLC fingerprints of spruce bark
laboratory temperature, the absorbance was determined at a wave-
extracts obtained by SE and UAE under optimized conditions were
length of 540 nm using an ELx800 Absorbance Reader (BioTek, Wi-
compared.
nooski, Vermont, USA). Results were expressed in mg of gallic acid
equivalent (GAE) per gram of dry material (2 - 20 mg/L of GAE; y =
2. Materials and methods
-0.0165x - 0.4829; R2 = 0.999).
TPC in spruce extracts was measured by the method of Singleton
2.1. Materials and chemicals
and Rossi (1965) using Folin-Ciocalteu reagent. The sample (40 μL) was
mixed with 40 μL of Folin-Ciocalteu reagent and the mixture was in-
The bark from spruce (Picea abies) was purchased from a local
cubated for 10 min. Subsequently, 400 μL of sodium carbonate (20 %;
company focusing on wood processing (Slovakia). The dried samples of
w/v) was added. The mixture was kept for 10 min with shaking at la-
spruce bark were milled using a knife mill. The size of the particles was
boratory temperature and centrifuged at 10,000 RPM using Eppendorf
1.0 - 2.5 mm. Spruce bark was analyzed according to NREL/TP-510-
Mini Spin Microcentrifuge (Eppendorf, Hamburg, Germany) for 10 min.
42618 (Sluiter et al., 2012). Methanol, Folin-Ciocalteu reagent, sodium
Obtained supernatant (240 μL) was used for the determination of TPC
carbonate, formic acid, n-hexane, chloroform and ethyl acetate were
at a wavelength of 690 nm using an ELx800 Absorbance Reader. TPC
purchased from Mikrochem (Slovakia). 2,2-diphenyl-1-picrylhydrazyl
was expressed in mg of GAE per gram of dry material (10 - 100 mg/L of
(DPPH) radical, gallic acid, 6-hydroxy-2,5,7,8-tetramethylchromane-2-
GAE; y = 0.0048x + 0.0165; R2 = 0.991).
carboxylic acid (TROLOX), butylated hydroxyanisole (BHA) and buty-
lated hydroxytoluene (BHT) were procured from Sigma-Aldrich (Ger-
many) and TLC sheets ALLUGRAM ® SIL G (Silica gel 60; 0.20 mm) 2.5. TLC analysis
were obtained from Macherey-Nagel (Germany).
The polyphenol profiles of spruce bark extracts prepared using
2.2. Extraction procedure solvents with different polarity (n-hexane, chloroform, ethyl acetate,
methanol, 50 % (v/v) methanol and distilled water) were determined by
In a preliminary study, the effect of solvent extraction (SE) without TLC analysis using a multiple development method. The extract (4 μL)
application of ultrasound on antioxidant capacity (AC) and total poly- was spotted onto activated Silica gel plate (10 × 10 cm; TLC Silica gel
phenol content (TPC) was investigated. Dried spruce bark (1 g) was 60 F254) air-dried and then stored in a developing chamber.
extracted at different liquid-solid ratio (10:1, 20:1, 50:1, and 100:1 [mL Development of the TLC plate involved a three-step elution: first with
of methanol to 1 g of dry material]) with aqueous-methanolic solutions methanol:formic acid mixture (10:1; v/v) to 1/3 of the plate height,
(0 - 100 %; v/v). The extraction procedure was performed at 150 RPM second with chloroform:methanol (9:1; v/v) to another 2/3 and the last
and at different temperatures (20, 30, 40, 50, and 60 °C) for 24 hours. In with n-hexane:ethyl acetate (6:4; v/v) to 3/3 of TLC plate height. Each
the selected time intervals (5, 10, 15, 30, 45, 60, 120, 240, 360, 480, cycle included the process of drying. Subsequently, the air-dried TLC
600, 840, 960, 1080, 1260 and 1440 min), the samples of extracts were plate was sprayed with Folin-Ciocalteu reagent for the detection of
taken and AC and TPC were determined by methods described in Sub- polyphenols. The polyphenol profiles of different spruce bark extracts
chapter 2.4. present on the chromatograms were evaluated using ImageJ program
ver. 1.46r (National Institute of Health, USA).
2.3. Experimental design
2.6. HPLC analysis
RSM was used to investigate the effect of temperature, methanol
content and liquid to solid ratio on the dependent variables (AC and The HPLC fingerprints of spruce bark extracts prepared under op-
TPC) obtained for 200 min of spruce bark extraction. The comparison of timal conditions were analyzed using HPLC System Agilent 1200 Series
the efficiency of SE and ultrasound-assisted extraction (UAE) from the (Agilent Technologies, Santa Clara, California, USA) equipped with
point of view of AC and TPC values obtained was also realized. UAE was degasser, binary pump, column temperature controller and DAD SL
carried out using an ultrasonic bath PS10 000 A detector. System control and data analysis were processed using the
(NOTUS-POWERSONIC, Slovakia; bath frequency 35 kHz, power 320 Agilent ChemStation software Rev. B.04.03 (Agilent Technologies). The
W). The set-up allowed the control of time and temperature of the ex- injection volume of each sample and the standard solution was 10 μL.
traction. The chromatographic separation was performed at 30 °C in the Eclipse
The relationships between the responses and the three selected XD8-C18 column (3.5 μm, 3.0 × 100 mm) using 1 % (v/v) water so-
variables were approximated by the following second-order polynomial lution of acetic acid (A) and methanol (B) as the mobile phase at a flow

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D. Chmelová, et al. Journal of Biotechnology 314–315 (2020) 25–33

Fig. 1. Comparison of phenolic profiles in spruce bark samples extracted with S1 – distilled water, S2 – 50 % (v/v) methanol, S3 – methanol, S4 – ethyl acetate, S5 –
chloroform or S6 – n-hexane.

rate of 0.5 mL/min. The HPLC mobile phase was prepared daily, filtered analysis was used to identify the relationship between AC and TPC.
through the membrane filter (0.45 μm) and degassed before injection
into the column. The linear gradient program was set as follows: 0 - 40 3. Results and discussion
min, 10 - 100 % B; 40 - 45 min, 100 % B. The separated compounds
were monitored at a wavelength of 254 nm. 3.1. Preliminary extraction experiments
For the identification and quantification of selected phenolic com-
pounds, the curves of analytes were obtained using the standards The content of holocellulose (51.5 ± 0.7 %), lignin (25.2 ± 1.1 %),
(syringic, vanillic, p-coumaric, ferulic, gallic and sinapinic acids, ca- ash (3.8 ± 0.2 %), extractives (14.3 ± 0.3 %) and the moisture content
techin and quercetin). The final concentration of phenolic compounds (8.8 ± 0.1 %) were determined in spruce bark. Moreover, the spruce
was expressed in mg per 100 g of dry material (spruce bark). bark contains a wide spectrum of phenols (Pan and Lundgren, 1995;
Lacoste et al., 2015; Talmaciu et al., 2016), which have various biolo-
2.7. Kinetics of solvent extraction and ultrasound-assisted extraction gical effects including anti-inflammatory, antibacterial, antiviral, anti-
allergic, and hepatoprotective effects (Haggag et al., 2011), but anti-
The kinetics of antioxidants and polyphenols extraction from spruce oxidant activity belongs to the most well-known biological activities of
bark expressed in AC and TPC values and realized under optimal con- polyphenols (Talmaciu et al., 2016). The selection of suitable extraction
ditions calculated by RSM were fitted with three mathematic models. conditions is linked to the amount of extracted compounds, affecting
Namely Page model (Jokić et al., 2010) (Equation 2), Peleg model the rate and yield of the extraction process. This is also associated with
(Peleg, 1988) (Equation 3) and Patricelli model (Patricelli et al., 1979) the physical and chemical properties of the target compounds including
(Equation 4) were used in order to determine the best-fitting model. the other extractable compounds and the components of matrix. Plant
phenolic compounds can be present in their free aglycone or glycosi-
ct = exp( k. t n) (2)
cally bound forms which determines their polarity. Therefore, the se-
where k and n are Page model constants; and ct represents AC or TPC lection of a suitable extraction solvent is the first step of optimization.
value [mg GAE/g of dry material] at the time t [min]. The influence of extraction solvents with different polarity was in-
t vestigated by TLC analysis coupled with image analysis of the devel-
ct = c0 + oped TLC plates (Fig. 1).
K1 + K2. t (3)
The result suggests that 50 % (v/v) aqueous-methanolic solvent
where ct is AC or TPC value [mg GAE/g of dry material] at the time t; c0 belongs to the most effective extractants for the polyphenols extraction
represents the initial AC or TPC value [mg GAE/g of dry material] at from spruce bark (Fig. 1). These results agree with Şahin and Samli
extraction time t = 0; K1 is the Peleg rate constant [min.g/mg]; and K2 (2013), which found that water used as extraction solvent can change
is the Peleg capacity constant [g/mg]. the plant material structure due to swelling, so the solvent can pene-
ct = c1 (1 exp( k1. t )) + c2 (1 exp( k2. t )) (4) trate effectively into a material. Thus, water acts as a swelling agent and
alcohol increases the solubility of polyphenols. The extraction of
where ct is AC or TPC value [mg GAE/g of dry material] at the time t polyphenols from spruce bark was performed by different solvents, al-
[min]; k1 represents a constant for the washing step and k2 is a constant though, aqueous-alcoholic solutions were almost exclusively used in
for the diffusion step. c1 and c2 are AC or TPC values [mg GAE/g of dry this way (Co et al., 2012; Ghitescu et al., 2015; Talmaciu et al., 2015;
material] at equilibrium for the washing and diffusion steps, respec- Talmaciu et al., 2016; Spinelli et al., 2019).
tively. From preliminary experiments (data not shown), maximum amount
Coefficient of determination (R2), adjusted coefficient of determi- of antioxidants and polyphenols were obtained by solvents containing
nation (adjR2), root mean square error (RMSE) and percentage average 50 - 80 % and 50 - 70 % (v/v) methanol, respectively. It was found that
absolute relative deviation (%AARD) were calculated for applied the values of monitored parameters quickly increased and reached the
models to assess the model adequacy. maximum level at 60 °C. Talmaciu et al. (2016) confirmed that ex-
posure to higher temperatures for a long time may cause loss of poly-
2.8. Statistical analysis phenols, so they recommend to make the extraction at temperature not
higher than 50 °C. However, our results showed that the extraction
The Statgraphic Centurion XV (Statpoint Technologies, Warrenton, temperature of 60 °C is propitious to obtain the maximum values of
Virginia, USA) was used for the statistical analysis of experimental data. TPC. Ghitescu et al. (2015) reported that the increase of extraction
All determinations were carried out in triplicates. Pearson correlation temperature from 40 °C to 60 °C had a positive effect on TPC in spruce

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D. Chmelová, et al. Journal of Biotechnology 314–315 (2020) 25–33

bark extract. But, the increase of extraction temperature over 64 °C is influence (p < 0.05). Moreover, the increase of extraction temperature
not possible due to physicochemical characteristics of used solvents from 51 to 63 °C affected the value of AC (Fig. 2A). Methanol content
(mainly methanol). Longer extraction time than 200 minutes is also not showed significant positive linear (p < 0.05) and negative quadratic (p
necessary to obtain the higher values of AC and TPC, respectively. The < 0.05) effects. The increase of methanol content from 40 to 54 % (v/v)
increase of liquid to solid ratio from 10:1 to 20:1 (mL of solvent to gram caused the increase of AC extracted from spruce bark (Fig. 2A), al-
of dry material) caused the increase of both evaluated parameters. The though, methanol content higher than 54 % (v/v) resulted in lower AC
highest yield of AC was observed for the ratio from 20:1 to 100:1 (1.35 - yields. Also, the liquid to solid ratio had a positive linear (p < 0.05) and
1.46 mg GAE/g of dry material). The increase of liquid to solid ratio negative quadratic (p < 0.05) effects on AC yields. For AC of extracts
was also manifested in higher values of TPC (data not shown), al- obtained by UAE, all linear effects were significant at the level of p <
though, Lazar et al. (2016) noted that the higher liquid to solid ratio 0.05 and the parameters of quadratic effect (BB, CC) were also sig-
(20:1) did not significantly affect TPC in the case of spruce bark. The nificant (p < 0.05). Moreover, interaction effect of methanol con-
independent variables for spruce bark extraction without application of tent*liquid to solid ratio had a negative effect (p < 0.05) (Table 2). The
ultrasound were selected from these preliminary experiments. From the results of UAE are quite similar to the results of spruce bark extraction
results, the ranges of each parameter (temperature, methanol content without ultrasound assistance. It is evident that the increase of ex-
and liquid to solid ratio) were chosen, and the optimization by RSM was traction temperature from 51 to 61 °C and methanol content from 40 to
carried out. In this study, two extraction techniques, namely SE and 48 % (v/v) significantly affected AC (Fig. 2C). In comparison with the
UAE, were compared in order to obtain spruce bark extract with high extraction without application of ultrasound the higher value of AC was
antioxidant activity. Ultrasound frequency is an important factor in the observed for UAE.
extraction of polyphenols from plants. Liu et al. (2013) and Altemimi The extraction temperature had a positive linear effect on TPC (p <
et al. (2016) reported that the lower frequency (37 and 45 kHz, re- 0.005) in extracts without application of ultrasound (Table 2). Similarly
spectively) is more appropriate than the higher frequency due to the to AC, the increase of extraction temperature from 51 to 63 °C affected
collapse of bubbles in the samples. For this reason, UAE in this work the values of TPC (Fig. 2-B). Methanol content had a significant positive
was realized at 35 kHz. linear (p < 0.05) and negative quadratic (p < 0.05) effects. As shown
in Fig. 2B, the value of TPC was increased and reached the maximum
with increasing methanol content from 40 to 56 % (v/v). However, the
3.2. Effect of extraction parameters on antioxidant capacity and total following increase of methanol content from 56 to 80 % (v/v) caused
polyphenol content of spruce bark and analysis of response surfaces the significant decrease in the value of TPC. This means that the high
methanol content can negatively affect TPC. The third variable, liquid
AC and TPC yields for all spruce bark extracts prepared by extrac- to solid ratio, had a positive linear effect on TPC (p < 0.05). All tested
tion without or with ultrasound are listed in Table 1. UAE of spruce parameters of quadratic effect had a negative effect on the value of TPC
bark increased the values of AC and TPC from 1.1- to 1.2-times. Ob- (p < 0.05). For the values of TPC in extracts obtained by UAE, the
tained experimental data were used to calculate the coefficients of the linear (extraction temperature, methanol content) and all quadratic
second-order polynomial equations. For extraction without application parameters were significant while interaction parameters of extraction
of ultrasound, coefficients R2 reached the values 98.9 % and 99.4 % for temperature*methanol content as well as methanol content*liquid to
AC and TPC, respectively. Calculated values of R2 of the models for AC solid ratio had a negative effect (p < 0.05). The increase of extraction
and TPC and for extracts obtained by UAE were 99.7 % and 99.1 %, temperature up to 61 °C and methanol content up to 51 % (v/v) had a
respectively. 3D plots for better understanding interactions between positive effect on TPC (Fig. 2D). Further, UAE increased the values of
selected variables are shown in Fig. 2. The third variable, liquid to solid TPC. The above-mentioned results agree with the findings of Ghitescu
ratio (30 mL of solvent to gram of dry material), represents the constant et al. (2015) and Lazar et al. (2016) and suggest the positive effect of
in the 3D plots. ultrasound on the values of TPC for spruce bark. All these results con-
Table 2 summarizes the regression coefficients and analysis of var- firm that selected independent parameters had the comparable effects
iances calculated for AC and TPC in extracts. For AC in non-ultra- on AC and TPC in extracts obtained with or without ultrasound.
sonicated extracts, temperature had a significant positive linear

Table 1
Combination of extraction temperature, methanol content and liquid to solid ratio with their coded levels obtained from RSM and the measured values of AC [mg
GAE/g dry material] and TPC [mg GAE/g; dry material] after SE and UAE.
Extraction variables SE UAE

X1 X2 X3 AC TPC AC TPC
Extraction temperature [°C] Methanol content [%] Liquid to solid ratio [mL/g]

56 (0) 60 (0) 30 (0) 2.76 ± 0.04 9.80 ± 0.14 3.00 ± 0.16 10.43 ± 0.97
61 (1) 40 (-1) 40 (1) 2.83 ± 0.08 9.83 ± 0.93 3.35 ± 0.33 10.57 ± 0.33
61 (1) 80 (1) 20 (-1) 1.89 ± 0.06 7.89 ± 0.44 2.37 ± 0.07 8.07 ± 0.61
51 (-1) 80 (1) 40 (1) 2.26 ± 0.08 8.42 ± 0.59 2.12 ± 0.08 7.41 ± 0.58
51 (-1) 40 (-1) 20 (-1) 1.87 ± 0.06 7.89 ± 0.95 2.11 ± 0.09 7.88 ± 0.41
61 (1) 80 (1) 40 (1) 2.40 ± 0.02 8.72 ± 0.92 2.40 ± 0.17 8.29 ± 0.19
51 (-1) 80 (1) 20 (-1) 1.51 ± 0.03 6.17 ± 0.44 2.03 ± 0.08 7.48 ± 0.67
61 (1) 40 (-1) 20 (-1) 2.28 ± 0.06 8.31 ± 0.27 2.71 ± 0.03 9.12 ± 0.06
51 (-1) 40 (-1) 40 (1) 2.22 ± 0.13 8.31 ± 0.76 2.95 ± 0.35 8.69 ± 0.42
56 (0) 60 (0) 30 (0) 2.59 ± 0.08 9.42 ± 0.46 3.15 ± 0.18 10.45 ± 0.33
56 (0) 60 (0) 30 (0) 2.59 ± 0.05 9.56 ± 0.48 3.01 ± 0.22 10.18 ± 0.14
56 (0) 26.5 (-1.682) 30 (0) 1.80 ± 0.00 8.03 ± 0.37 2.41 ± 0.10 7.44 ± 0.57
56 (0) 60 (0) 46.7 (1.682) 2.78 ± 0.04 9.83 ± 0.35 2.92 ± 0.55 9.92 ± 0.19
47.6 (-1.682) 60 (0) 30 (0) 2.16 ± 0.12 8.18 ± 0.96 2.86 ± 0.38 8.91 ± 0.79
56 (0) 93.5 (1.682) 30 (0) 1.51 ± 0.03 6.35 ± 0.62 1.46 ± 0.20 5.56 ± 0.82
56 (0) 60 (0) 13.3 (-1.682) 1.58 ± 0.01 7.29 ± 0.99 2.33 ± 0.08 8.40 ± 0.38
64.4 (1.682) 60 (0) 30 (0) 2.78 ± 0.10 9.92 ± 0.08 3.48 ± 0.07 10.71 ± 0.67

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D. Chmelová, et al. Journal of Biotechnology 314–315 (2020) 25–33

Fig. 2. Response surface model plots showing the effects of independent variables on AC and TPC at constant value of liquid to solid ratio (30 mL solvent to gram dry
material). Effect of extraction temperature and methanol content on AC in non-ultrasonicated extracts (A) and ultrasonicated extracts (B) and on TPC in extracts after
SE (C) and UAE (D).

3.3. Optimization and verification of the model extraction.

Modified optimal extraction conditions were verified and there was
The optimal extraction conditions to obtain maximum AC and TPC no significant difference between predicted and experimental values of
for spruce bark are summarized in Table 3. To verify the effect of UAE all tested dependent variables (p < 0.05). Thus, this model can be
on AC and TPC values for spruce bark, optimal extraction conditions potentially used for the extraction of antioxidants and polyphenols (AC
were identified (see Table 3). The optimal extraction conditions to and TPC) from the spruce bark. In fact, the UAE had the ability to in-
obtain maximum values of AC and TPC were modified as follows: ex- crease the values of AC and TPC for spruce bark. Under modified op-
traction temperature - 63 °C, methanol content - 53 % (v/v) and liquid timal conditions of extraction with ultrasound, the experimental values
to solid ratio - 38 mL to gram of dry material. Ghitescu et al. (2015) of AC and TPC were 3.57±0.05 and 11.06±0.00 mg GAE/g of dry
reported that the optimal conditions to obtain the maximum values of material, respectively. TPC in spruce bark extracts varied from 13.23 to
TPC for spruce bark were as follows: extraction temperature - 54 °C, 43.10 mg GAE/g of dry material showing the differences between used
time - 60 min and ethanol concentration - 70 % (v/v). But they found extracted materials (Ignat et al., 2013; Ghitescu et al., 2015; Lazar
that the increase of extraction temperature from 40 to 60 °C led to the et al., 2016). The comparison among the experimental results obtained
increase of TPC. This finding agrees with Lazar et al. (2016), who ob- in this study and other studies is difficult due to differences in the
served that the increase of extraction temperature from 45 to 60 °C methodology used for the determination of polyphenols.
caused the higher values of TPC for spruce bark. Moreover, they found Conventional methods of extraction commonly require the longer
that the maximum value of TPC was measured after 70 min of time to reach the maximum yields of extractable compounds than

Table 2
Results of analysis of variance and obtained regression coefficients of the predicted second order polynomial models for antioxidants and polyphenols (AC and TPC)
extracted from spruce bark by SE and UAE.
SE UAE

Effect Source AC TPC AC TPC

Regression coefficients Regression coefficients Regression coefficients Regression coefficients

Constant −11.4128 −31.2392 −4.05779 −38.4908

Linear A: Extraction temperature 0.264586* 0.967416* −0.0612834* 0.951794*
B: Methanol content 0.122064* 0.207241* 0.160554* 0.532355*
C: Liquid to solid ratio 0.109825* 0.288834* 0.20334* 0.229901*
Quadratic AA −0.00166234 −0.00757467* 0.00132468 −0.00701786*
BB −0.000831575* −0.00213413* −0.00101989* −0.00339397*
CC −0.0014513* −0.00364367* −0.00161526* −0.00407589*
Interaction AB −0.000625 0.0001 −0.000475 −0.0020625*
AC −0.0001 −0.0008 −0.00065 0.002325
BC 0.000225 0.0007125 −0.00085* −0.00131875*

*
Statistically significant at p-value <0.05

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D. Chmelová, et al. Journal of Biotechnology 314–315 (2020) 25–33

Table 3
Predicted and experimental values of AC and TPC [mg GAE/g; dry material] – antioxidants and polyphenols extracted from spruce bark under optimized extraction
conditions.
Type of extraction Dependent variable Extraction variables Predicated value Experimental value

X1 X2 X3
Extraction temperature [°C] Methanol content [%] Liquid to solid ratio [mL/g]

SE AC 63.40 54.60 39.90 3.02 3.03 ± 0.07

TPC 62.20 56.40 38.30 10.20 10.04 ± 0.12
UAE AC 63.60 48.20 37.30 3.62 3.57 ± 0.05
TPC 61.30 51.40 38.20 11.23 11.06 ± 0.00

Fig. 3. Effect of extraction time on AC (A) and TPC (B) during SE and UAE under optimal extraction conditions.

modern extraction techniques (Altemimi et al., 2016). From our pre- equilibrium yields of TPC were 10.14 and 11.02 mg GAE/g of dry
liminary experiments, it was found that the extraction time of 200 min material for SE and UAE, respectively. Patricelli and Peleg models
is propitious to obtain the maximum extraction of antioxidants and showed a positive effect of ultrasound assistance in the context of
polyphenols from spruce bark by SE (data not shown). Obtained results amount of extracted antioxidants and polyphenols. Ultrasound helps
show that the extraction time to reach the maximum AC or TPC in disrupt plant cell walls and enhances the release of cell content
extracts exposed to ultrasound waves under optimal conditions was 2- (Altemimi et al., 2016; Chemat et al., 2017). It was found that the
times lower than the extraction time required for non-ultrasonicated calculated mass transfer coefficients for both evaluated variables (AC,
extracts (Fig. 3). TPC) from Patricelli model were higher for washing step than for dif-
AC of optimized extracts was relatively compared to standard an- fusion step. Thus, the application of ultrasound accelerates mainly the
tioxidants, namely TROLOX, BHA and BHT. For non-ultrasonicated washing step of extraction process.
extract, the lower activity of the extract was observed in comparison It has been reported that TPC and AC can be linked by high corre-
with TROLOX (2.7 ± 0.5-times) and BHA (2.2 ± 0.4-times) and quite lations (García-Pérez et al., 2010; Rosales-Castro et al., 2015; Chmelová
similar was reached with BHT (0.3 ± 0.0-times) and for ultrasonicated et al., 2018). This finding was also confirmed in our study (Fig. 4). The
extract of spruce bark, the activity was lower than TROLOX (2.2 ± 0.1- significant correlation was observed between the values of AC and TPC
times) and BHA (1.9 ± 0.1-times) activities, but almost comparable (R2 = 0.969; p < 0.005) for optimized extract of spruce bark. The
activity was observed for BHT (0.2 ± 0.0-times). The amount of similar correlation was identified for optimized extract of spruce bark
polyphenols can be increased with decreasing of particle size. obtained by UAE (R2 = 0.952; p < 0.005). It suggests that mainly
Pӑtrӑuţanu et al. (2019) found that the highest TPC in spruce bark polyphenols are responsible for antioxidant activity in spruce bark.
extract was determined for the smallest particle size (< 0.25 mm).
Thus, it can be concluded that the application of ultrasound is suitable
for increasing the effectiveness and rate of the extraction process. 3.4. HPLC analysis
To explain the extraction of antioxidants and polyphenols from
spruce bark samples and to compare the effect of conventional SE and In order to evaluate the main differences between optimized ex-
modern UAE, the experimental data were fitted with models of Page, tracts of spruce bark obtained with or without ultrasound, HPLC fin-
Peleg and Patricelli (Table 4). gerprints were compared (Fig. 5). It seems that only UAE increases the
Page model describing the extraction of antioxidants and poly- amount of extractable compounds because the presence of similar peaks
phenols from spruce bark showed the lowest R2 value (Table 4). From was observed in both extracts. The phenolic acid type compounds were
this result, it can be concluded that Page model is not appropriate to detected in both extracts, mainly ferulic, syringic, vanillic, p-coumaric
explain the extraction process. If the constant c0 in Peleg model is not and sinapinic acid (Table 5). Ultrasonication of spruce bark sample
neglected, this model is adequate to fit the extraction process. The re- increased the amount of selected phenolic acids in the range of 1.1- to
sults of both SE and UAE are best described by Peleg and Patricelli 7.1-times. Talmaciu et al. (2016) determined the similar concentration
models with the highest calculated values of R2 and adjR2 and the of ferulic and p-coumaric acids in dry extracts of spruce bark obtained
lowest values of RMSE and %AARD (Table 4). In Patricelli model, the by supercritical fluid extraction. The composition of selected phenols is
extraction process is divided into two steps involving rapid washing partially consistent with the study of Lazar et al. (2016), although, the
(solvation) of bioactive substances from plant biomass and slow diffu- presence of gallic acid, catechin and syringic acid was observed in bark
sion of compounds through the plant material (Karacabey et al., 2011). extract. Ignat et al. (2013) identified some phenolic acids, such as gallic
The equilibrium yields of AC were 3.07 and 3.59 mg GAE/g of dry acid, catechin, vanillic acid and quercetin (1.39 mg/100 g of dried
material for SE and UAE, respectively (Table 4). Additionally, the material). The presence of this flavonoid was not confirmed in our
work. It must be noted that the composition of secondary metabolites in

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D. Chmelová, et al. Journal of Biotechnology 314–315 (2020) 25–33

Table 4
Calculated parameters of fitted models describing the antioxidants and polyphenols (AC and TPC) extraction from spruce bark by SE and UAE under optimized
extraction conditions.
Model Variable Type of extraction Constant Statistic parameters

- - k n RMSE R2 adjR2 %AARD

Page AC SE −0.2296 0.3005 0.12208 0.9561 0.9513 0.1350
UAE −0.8152 0.0878 0.05613 0.9765 0.9739 0.0620
TPC SE −1.3846 0.0981 0.18478 0.9886 0.9873 0.2043
UAE −1.8707 0.0504 0.33679 0.9365 0.9294 0.3723
c0 - K1 K2 RMSE R2 adjR2 %AARD
Peleg AC SE 0.9957 18.9869 0.4240 0.05488 0.9911 0.9889 0.0644
UAE 2.3595 25.4928 0.6773 0.03113 0.9928 0.9910 0.0365
TPC SE 4.4180 7.8591 0.1400 0.11417 0.9956 0.9945 0.1339
UAE 5.5072 2.4163 0.1657 0.09862 0.9946 0.9932 0.1157
c1 k1 c2 k2 RMSE R2 adjR2 %AARD
Patricelli AC SE 1.3853 0.2892 1.6879 0.0128 0.06380 0.9880 0.9829 0.0800
UAE 2.4773 0.7662 1.1171 0.0206 0.02310 0.9960 0.9943 0.0290
TPC SE 4.7508 0.7954 5.3900 0.0160 0.15173 0.9923 0.9890 0.1902
UAE 6.9110 0.5307 4.1066 0.0330 0.09122 0.9953 0.9933 0.1144

k and n – Page model constants; c0 – initial AC or TPC value [mg GAE/g of dry material] at extraction time t = 0; K1 – Peleg rate constant [min.g/mg]; K2 – Peleg
capacity constant [g/mg]; k1 – constant for the washing step; k2 – constant for the diffusion step; c1 and c2 – AC or TPC values [mg GAE/g of dry material] at
equilibrium for the washing and diffusion steps, respectively; R2 – coefficient of determination; adjR2 – adjusted coefficient of determination; RMSE – root mean
squared error; %AARD – percentage average absolute relative deviation.

spruce bark can be affected by various abiotic and biotic factors re- polyphenols under optimized conditions, Peleg and Patricelli models
sulting in different phenolic content and antioxidant activity. were the most appropriate. UAE increased the amount of antioxidants
and polyphenols within the washing step of extraction process.
Although, the phenolic profiles in both extracts obtained under mod-
4. Conclusions ified optimized conditions were comparable to each other, the con-
centrations of some identified phenolic compounds in spruce bark ex-
The results of the study indicated that UAE of spruce bark has the tracts obtained with application of ultrasound were higher in
ability to increase the values of AC and TPC. From the methodology comparison with extracts obtained without ultrasound, except for
point of view, it was found that TLC-densitometric method can be ferulic acid.
useful for rapid qualitative screening of polyphenols present in plant
extracts. The optimal conditions for simultaneous extraction of anti-
oxidants and polyphenols from spruce bark were comparable for both Declaration of Competing Interest
types of selected extractions, namely SE and UAE. The optimal condi-
tions were as follows: extraction temperature of 63 °C, methanol con- The authors declare that they have no known competing financial
tent of 53 % (v/v) and liquid to solid ratio 38 mL to gram of dry ma- interests or personal relationships that could have appeared to influ-
terial. The ultrasound increased the maximum values of AC and TPC ence the work reported in this paper.
and decreased the extraction time. From all applied mathematical
models describing the kinetics of extraction of antioxidants and

Fig. 4. Correlations between AC and TPC for optimized extraction of antioxidants and polyphenols from spruce bark.

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D. Chmelová, et al. Journal of Biotechnology 314–315 (2020) 25–33

Fig. 5. HPLC fingerprints of spruce bark extracts obtained by SE and UAE under optimized extraction conditions. 1 – vanillic acid; 2 – syringic acid; 3 – p-coumaric
acid; 4 – ferulic acid; 5 – sinapinic acid.

Table 5 1016/j.rser.2013.06.024.
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