applied
sciences
Article
Optimization of the Ultrasound-Assisted Extraction of Bioactive
Compounds from Cannabis sativa L. Leaves and Inflorescences
Using Response Surface Methodology
Zbigniew Kobus 1 , Anna Pecyna 1, * , Agnieszka Buczaj 1 , Monika Krzywicka 1 , Artur Przywara 2
and Rafał Nadulski 3
Citation: Kobus, Z.; Pecyna, A.;
Buczaj, A.; Krzywicka, M.; Przywara,
A.; Nadulski, R. Optimization of the
Ultrasound-Assisted Extraction of
Bioactive Compounds from Cannabis
sativa L. Leaves and Inflorescences
Using Response Surface
Methodology. Appl. Sci. 2022, 12,
6747. https://doi.org/10.3390/
app12136747
Academic Editor: Chiara Cavaliere
Received: 30 May 2022
Accepted: 1 July 2022
Published: 3 July 2022
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Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
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Attribution (CC BY) license (https://
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4.0/).
Appl. Sci. 2022, 12, 6747. https://doi.org/10.3390/app12136747
1 Department of Technology Fundamentals, University of Life Sciences, Gł˛eboka 28, 20-612 Lublin, Poland;
zbigniew.kobus@up.lublin.pl (Z.K.); agnieszka.buczaj@up.lublin.pl (A.B.);
monika.krzywicka@up.lublin.pl (M.K.)
2 Department of Machinery Exploitation and Management of Production Processes, Gł˛eboka 28,
University of Life Sciences, 20-612 Lublin, Poland; artur.przywara@up.lublin.pl
3 Department of Food Engineering and Machines, University of Life Sciences, Gł˛eboka 28,
20-612 Lublin, Poland; rafal.nadulski@up.lublin.pl
* Correspondence: anna.pecyna@up.lublin.pl
Abstract: This study investigated the effects of particle size and ultrasonic parameters on the yields
of bioactive compounds extracted from the leaves and inflorescences of hemp. The total flavonoid
and anthocyanin contents were determined using the spectrophotometric method. The response
surface methodology (RMS) was employed to optimize the yield of bioactive substances. On the basis
of the developed model, the highest flavonoid yield was obtained under the following extraction
conditions: particle size, 0.59 mm; extraction time, 10.71 min; ultrasound intensity, 7.13 W·cm−2 ;
extraction yield, 9.28 mg QE·g−1 ; determination coefficient, R2 = 0.97. The optimal conditions for
extracting anthocyanins were as follows: particle size, 0.25 mm; extraction time, 15 min; ultrasound
intensity, 8.60 W·cm−2 ; extraction efficiency, 20.27 mg Cy-GE·100 g−1 ; determination coefficient,
R2 = 0.87. This study helped confirm the importance of pulsed ultrasound-assisted extraction in
obtaining bioactive compounds from hemp.
Keywords: Box–Behnken design; pulsed ultrasound-assisted extraction; hemp; flavonoids; anthocyanins;
response surface methodology
1. Introduction
Hemp (Cannabis sativa L.) is a herbaceous, wind-pollinated plant that is widespread
around the world and belongs to the Cannabaceae family. The species Cannabis sativa L. (true
hemp) includes fiber (Cannabis sativa L. var. sativa) and Indian (narcotic) (Cannabis sativa L.
var. indica) hemp. These differ in their levels of cannabinoids, mainly THC, the content of
which in fiber hemp is below 0.2% [1–4].
Poland has a long tradition of growing cannabis. Hemp is cultivated in areas with
valuable natural resources, including the Lublin region, Podlasie, and Greater Poland. The
Lublin region currently ranks fourth in terms of its cultivation area, but it is first in terms of
the number of farms.
C. saliva L. has recently received a lot of attention for its nutritional and pharmaceutical
value, although in the past, it was grown mainly for the fibers from hemp stalk and for
the oil from hemp seeds [5–7]. The medicinal use of cannabis has been known for over
5000 years, and the pharmacological properties of cannabinoids, dominant in cannabis, can
be useful in the treatment of various diseases [7,8]. Indeed, cannabinoids (phytocannabi-
noids) are one of the most important bioactive compounds in cannabis [9]. The best-known
cannabinoids found in Cannabis sativa L. are THC and CBD.
https://www.mdpi.com/journal/applsci
Appl. Sci. 2022, 12, 6747 2 of 14

The panicle elements of the plant (inflorescences), along with the leaves, are used
in the production of industrial hemp extracts. They contain volatile terpenes and phy-
tocannabinoids, including cannabidiol, better known by the trade name CBD. The latter
is usually presented to consumers in the form of dietary supplements, cosmetics, and
even drugs, although its production in Poland is currently prohibited. The popularity of
cannabidiol is likely due to its pro-health and healing properties, some of which have been
scientifically proven and some of which are described within the so-called natural medicine
movement [10]. CBD oils, ointments, and capsules have a beneficial effect on general health
and can be used in the symptomatic treatment of eczema and other skin diseases, as well
as various types of pain, especially rheumatoid arthritis, joint diseases, cancer, and even
multiple sclerosis. The health properties of CBD also factor into the popularity of daily skin
care cosmetics that have been derived from it, the popularity of which increases every year.
Hemp is also rich in natural antioxidants and other bioactive ingredients, such as
bioactive peptides, phenolic compounds, tocopherols, carotenoids, and phytosterols. The
content of these ingredients is mostly influenced by environmental and agronomic factors
and, to a lesser extent, by genetic variability. Fibrous hemp inflorescences are a source
of polyphenolic compounds with proven health-promoting properties [11]. Bioactive
substances are biologically active compounds of a natural origin that can have a beneficial
and multidirectional effect on the body. As food ingredients, they can modify, strengthen,
or weaken various body functions, thus limiting the development of disease processes.
These compounds are characterized by antioxidant, anti-inflammatory, neuroprotective,
antihypertensive, antiproliferative, and hypocholesterolemic effects, which have mainly
been assessed with in vitro studies [2,8,12–15].
In recent years, the demand for bioactive substances has increased, which has also
resulted in a search for new, more efficient methods for their extraction. To this end,
various separation techniques are used, such as supercritical fluid extraction, microwave-
assisted extraction, accelerated solvent extraction, ultrasound-assisted extraction, and
pulsed electric-field-assisted extraction. The ultrasound-assisted extraction method enables
the extraction of bioactive ingredients in a very short time, at a low temperature, and with
lower energy and solvent requirements [16]. As a non-thermal extraction technique, it
better preserves the functionality of bioactive compounds; however, process variables,
such as frequency, power, duty cycle, temperature, time, type of solvent, and liquid–solid
ratio, must be individually selected for each raw material [17]. The advantage of ultrasonic
treatment is also the inactivation of microorganisms and enzymes, which extends the shelf
life of the obtained products [18].
Most of the work on cannabis is devoted to ultrasound-assisted continuous extrac-
tion, which, as noted, enables the extraction of bioactive ingredients in a very short time,
at a low temperature, and with lower energy and solvent requirements. Flores-Sanchez
and Verpoorte [19], as well as Choi et al. [20], conducted continuous ultrasound-assisted
extractions (for 10 min) in order to obtain cannabinoids and flavonoids from cannabis.
Nagy et al. [8] performed an ultrasonically assisted extraction (for 10 min) on sponta-
neous C. sativa, demonstrating the presence of several flavonoid derivatives. The total
flavonoid amounts in the leaves and the male and female inflorescences were 3.84, 6.09,
and 7.79 mg·g−1 , respectively.
Currently, the pulsed ultrasound field method is used more and more often in order
to support the process of extraction of bioactive compounds [21]. The advantages of this
solution are comparable or higher extraction yields, slower temperature increases during
the extraction process, and significantly lower energy consumption [22,23]. Thus far, no
research has been conducted on the effect of pulsed ultrasound-assisted extraction with
respect to obtaining bioactive substances from cannabis inflorescences. The aim of this study
was to determine the optimal conditions for the ultrasound-assisted extraction of bioactive
compounds from Cannabis sativa L. with the help of the Box–Behnken experimental design.
 bioactive compounds from Cannabis sativa L. with the help of the Box–B
experimental design.

Appl. Sci. 2022, 12, 6747 2. Materials and Methods 3 of 14

2.1. Raw Material and Regents

Driedand
2. Materials true hemp was obtained from an organic farm located in Biała P
Methods
County,
2.1. Lubelskie
Raw Material Voivodeship. The material was crushed in the Zelmer MM 1200
and Regents
thenDried
sieved
trueand
hempdivided into fractions.
was obtained For further
from an organic research,
farm located in Biała 3Podlaska
fractions with the fo
County,
Lubelskie Voivodeship. The material was crushed in
mean particle weights were used: 0.25, 0.75, and 1.25 mm. the Zelmer MM 1200 device, then
sieved and divided into fractions. For further research, 3 fractions with the following mean
Potassium chloride, sodium acetate and hydrochloric acid (Stanlab, Lublin,
particle weights were used: 0.25, 0.75, and 1.25 mm.
werePotassium
used for the analysis
chloride, of anthocyanins.
sodium acetate and hydrochloricForaciddetermining
(Stanlab, Lublin,flavonoids,
Poland) alu
chloride
were and
used for thequercetin (Sigma-Aldrich—Merck,
analysis of anthocyanins. Taufkirchen,
For determining flavonoids, Germany)
aluminum chloridewere u
reagents
and used
quercetin for analytical procedures
(Sigma-Aldrich—Merck, wereGermany)
Taufkirchen, of analytical
were grade.
used. All reagents
used for analytical procedures were of analytical grade.
2.2.Methods
2.2. Methods
Theresearch
The research
waswas carried
carried out according
out according to the
to the scheme schemeinpresented
presented Figure 1. in Figure 1.

Figure 1. Scheme of the conducted research.

Figure 1. Scheme of the conducted research.
2.2.1. Ultrasound-Assisted Extraction
Raw
2.2.1. material in the amount Extraction
Ultrasound-Assisted of 1.5 g from each fraction was placed into a conical flask,
and an aqueous solution of ethyl alcohol with a concentration of 60% was poured over it.
Raw
The flask wasmaterial in the
sealed with amount
a 25.4 of 1.5diameter
mm (1 inch) g fromultrasonic
each fraction
probe,was
then placed
placed ininto
the a conic
and an
water aqueous
bath solution
to stabilize of ethyl(30
the temperature ◦
alcohol with
C). The a concentration
experimental of 60%
samples were was poured
sonicated
with a VC750 Sonics processor (Sonics and Materials Inc., Newtown,
The flask was sealed with a 25.4 mm (1 inch) diameter ultrasonic probe, then CT, USA) operating at place
a frequency of 20 kHz. The sonication was performed at three amplitudes—30%, 50%, and
water bath to stabilize the temperature (30 °C). The experimental samples were so
70%—corresponding to the ultrasound intensity—1.6, 5.1, and 8.6 W·cm−2 , respectively.
with a VC750
The samples wereSonics processor
sonicated (Sonicsprocessor
in the following and Materials Inc., Newtown,
arrangement: CT,
5 s on–10 s off. TheUSA) op
at a frequency
effective operationof 20 kHz.
times were 5,The sonication
10, and was
15 min, and theperformed at three
total extraction amplitudes—30
times were 15, 30,
and4570%—corresponding
and to the
min, respectively. The extracts ultrasound
obtained in this wayintensity—1.6, 5.1, and 8.6
were stored in a refrigerator
(2 ◦ C) and collected for further chemical analysis.
respectively. The samples were sonicated in the following processor arrangement
10 s off. The effective operation times were 5, 10, and 15 min, and the total extractio
were 15, 30, and 45 min, respectively. The extracts obtained in this way were sto
refrigerator (2 °C) and collected for further chemical analysis.
Appl. Sci. 2022, 12, 6747 4 of 14

2.2.2. The Total Flavonoid Content (TFC)

TFC was determined with spectrophotometry using quercetin as the reference stan-
dard. First, the sample extract (1.0 mL) was mixed with 1 mL of a 2% AlCl3 × 6H2 O
solution (in methanol), and the volume of the mixture was made up to 10 ml with distilled
water. After incubating the mixture for 10 min at room temperature in the dark, absorbance
was measured at 430 nm. The results were calculated as mg quercetin equivalent per 1 g
dry weight (mg QE·g−1 dw).

2.2.3. The Total Anthocyanin Content (TAC)

The TAC was determined with the spectrophotometric method from absorbance
measurements taken at different pH levels. First, the sample extract (0.5 mL) was mixed
with 4 mL of potassium chloride and sodium acetate buffers at pH 1.0 and 4.5. After leaving
the mixture for 15 min at room temperature, the absorbance was measured at 520 and
700 nm. The correct absorbance was determined from Formula (1):

A = (A520 − A700 )pH1.0 − (A520 − A700 )pH4.5 (1)

The total anthocyanin content was expressed as cyanidin 3-glucoside equivalent (Cy-
GE) in mg/g dry weight using Formula (2):

A
TAC = Mw · N (2)
Lε
A—correct absorbance;
L—cuvette thickness;
N—dilution factor;
Mw —molar mass of cyanidin 3-glucoside = 26,900;
ε—molecular absorbance of cyanidin 3-glucoside = 449.2.

2.3. Box–Behnken Experimental Design and Statistical Analyses

The experiment was performed on the basis of the Box–Behnken experimental design
in the Design-Expert v13, Stat-Ease, Minneapolis, MI, USA). A three-level, three-factor
BBD plan was used to determine the best combination of cannabis inflorescence extraction
variables. Particle size (X1 ), extraction time (X2 ), and ultrasound intensity (X3 ) were
independent variables, whereas the dependent variables were the total flavonoid content
and the total anthocyanin content.
The experiment comprised a total of 15 combinations, including 3 center points to
estimate the pure error, and was carried out in randomized order. All experiments were
performed in triplicate. The levels of the three factors evaluated in the design are listed in
Table 1.
A generalized, second-order polynomial model was used to explain the effect of the
independent variables on each response of interest according to the following equation:

Y = β0 + β1 X1 + β2 X2 + β3 X3 + β11 X21 + β22 X22 + β33 X23 + β12 X1 X2 + β13 X1 X3 + β23 X2 X3 (3)
where Y is the response variables (the total flavonoid content or the total anthocyanin
content); X1 , X2 , and X3 are the independent variables; β0 represents the constant; and β1,2,3 .
B11,22,33 , and β12,13,23 are the linear, quadratic, and interactive coefficients, respectively.
The experimental data were assessed via analysis of variance (ANOVA). The statistical
significances of the regression coefficients were checked with an F-test, and p-values less
than 0.05 were considered significant.
The optimal extraction conditions were estimated through Derringer’s desirability
prediction tool, aiming at a maximum attainable response for each independent factor. The
validity of the developed model was assessed by comparing the experimental values and
the predicted values. Two additional independent experiments were conducted using the
 A generalized, second-order polynomial model was used to explain the e
independent variables on each response of interest according to the following e
Y = β + β X + β X + β X + β X + β X + β X + β X X + β X X + β 5 ofX14X
Appl. Sci. 2022, 12, 6747

where Y is the response variables (the total flavonoid content or the total an
content); X1, X2, and X3 are the independent variables; β0 represents the constan
optimal conditions estimated with the models for each dependent variable (separately), as
Β11,22,33
well, as
andoneβexperiment
12,13,23 are for
theboth
linear, quadratic, and interactive coefficients, respecti
variables.
The experimental data were assessed via analysis of variance (ANO
Table 1. The Box–Behnken response surface design.
statistical significances of the regression coefficients were checked with an F-t
values lessRun than 0.05 were considered X1 significant.
X2 X3
1.
The optimal extraction0.75conditions were 5estimated through 8.6 Derringer’s d
2. 1.25 10 1.6
prediction tool,3. aiming at a0.25 maximum attainable 10 response for8.6each independ
The validity4. of the developed 0.25model was assessed
10 by comparing 1.6 the experimen
5. 0.75 10 5.1
and the predicted
6. values. Two 0.25additional independent
5 experiments 5.1 were condu
7. 0.75 10
the optimal conditions estimated with the models for each dependen 5.1
8. 0.75 10 5.1
(separately),9. as well as one experiment
0.25 for both
15 variables. 5.1
10. 0.75 5 1.6
11. 0.75 15 1.6
3. Results and
12. Discussion 1.25 10 8.6
13. 1.25 10 5.1
3.1. Total Flavonoid
14. Content 1.25 5 5.1
15. 0.75 15 8.6
The total flavonoid content extracted from the hemp ranged from 3.02 to 9
∙g−1,3.varying according to the experimental conditions (Figure 2). Similar resu
Results and Discussion
total3.1.flavonoid content,
Total Flavonoid Content ranging from 1.83 to 11.20 mg QE∙g dw, were obtaine
−1

studiesThe ontotal
hemp parts
flavonoid fromextracted
content the Cannabis sativa
from the hemp L.from
ranged variety, as carried
3.02 to 9.35 mg QE ·g−out1, by D
varying according to the experimental
[15]. In the extracts obtained with 50% ethanol, conditions (Figure 2). Similar results for the
the highest flavonoid contenttotal
flavonoid content, ranging from 1.83 to 11.20 mg QE·g−1 dw, were obtained in aerial studies
mg on QE∙g −1 dw for young plants and 5.21 mg QE∙g−1 dw mature plants. Studies
hemp parts from the Cannabis sativa L. variety, as carried out by Drinić et al. [15]. In the
by extracts
Izzo etobtained
al. [24] showed
with thatthe
50% ethanol, the average
highest flavonoid
flavonoid content
content was 11.20 mginQEthe
·g−1 inflore
dw for young plants and 5.21was
mg QE − 1
·g dw mature plants.
−1 forStudies conducted
cannabis cv. Carmagnola about 0.62 mg∙g samples withbymoistu
Izzo et al. [24] showed that the average flavonoid content in the inflorescences of cannabis
ranging from 8% to 12%.
cv. Carmagnola was about 0.62 mg·g−1 for samples with moisture content ranging from
8% to 12%.

(a)
Figure 2. Cont.
. 2022, 12, x FOR PEER REVIEW
Appl. Sci. 2022, 12, 6747 6 of 14

(b)

(c)
Figure 2. Response
Figure 2. Response surfaces obtained with
surfaces the Box–Behnken
obtained with the experimental design for the
Box–Behnken total
experimen
flavonoid content (mg QE·g−1 ) in hemp depending on the (a) intensity and time, (b) particle size and
flavonoid content (mg QE∙g−1) in hemp depending on the (a) intensity and ti
time, and (c) particle size and intensity.
time, and (c) particle size and intensity.
Based on the obtained results, the effects of the independent variables on the depen-
dent variables are presented in Table 2.
Based on the obtained results, the effects of the independ
dependent variables are presented in Table 2.

Table 2. Effects of the independent variables on the dependent variab

significance with respect to the extraction of flavonoid compounds from he
Appl. Sci. 2022, 12, 6747 7 of 14

Table 2. Effects of the independent variables on the dependent variables and their statistical signifi-
cance with respect to the extraction of flavonoid compounds from hemp.

Variables p Value Coefficient

X1 0.0054 −1.15
X2 0.0153 +0.8872
X3 0.0016 +1.51
X1 X2 0.4980 −0.2534
X1 X3 0.5001 −0.2521
X2 X3 0.0202 −1.17
X21 0.0002 −3.58
X22 0.0457 −0.9557
X23 0.0445 −0.8214

It was observed that the linear and quadratic terms of the particle size, extraction
time, and ultrasound intensity significantly affected (p < 0.05) the extraction of flavonoid
compounds. However, the linear and quadratic term of the particle size, and the quadratic
terms of the extraction time and ultrasound intensity demonstrated negative correlations,
indicating that an increase in the magnitude of these variables may favor the extraction of
flavonoid compounds only up to a certain value. This effect is very visible in the case of
the disintegration degree, as the total flavonoid content initially increased with the rising
particle size, from 0.25 to 0.75 mm, and then decreased as the particle size increased from
0.75 to 1.25 mm. The degree of fragmentation was the variable that most significantly
influenced the extraction of flavonoid compounds.
In general, the yield of flavonoids increased with an increased time of extraction.
Above 14 min, a slight decrease in the total flavonoid content was visible, so a further
extension of the extraction time may reduce the extraction of the target compound. Being
exposed to ultrasound for too long causes structural damage in the solute and reduces the
extraction efficiency, which was confirmed during the extraction of phenolic compounds
from waste coffee grounds [25], phenolic compounds from black chokeberry waste [26],
and flavonoids from hawthorn seeds [27]. The extraction time had the least influence on
the total flavonoid content in the obtained extracts. The total flavonoid content increased
with the intensity of the ultrasound. The growth in the intensity of the ultrasound from
1.6 to 8.6 W·m−2 increased the total flavonoid content by 47.7%. However, applying
ultrasound intensity above the test range may reduce the extraction yield of the target
compound. The different effects of ultrasound power on the content of phenolic compounds
were observed by Al-Dhabi et al. [25]. They demonstrated an increase in the efficiency
of phenolic compound extraction from coffee-ground waste when the ultrasound power
increased from 100 to 244 W, as well as a decrease in efficiency when the ultrasound power
exceeded 250 W. In the work of Al-Dhabi et al. [25], a statistically significant negative
interaction was found between the extraction time and the intensity of the ultrasound,
which means that the effect of the combined action of the two predictors is less than the
sum of the individual effects.
The parameters presented in Table 2 were re-estimated considering only the significant
terms (p < 0.05). From the regression analysis, the model was adjusted to the experimental
data, as presented in Equation (4):

TFC = −10.61 + 19.19X1 + 1.28X2 + 1.78X3 − 0.07X2 X3 − 14.32X21 − 0.04X22 − 0.07X23 (4)

The predictive equation was verified with analysis of variance (ANOVA), as can be
seen in Table 3, and it provided a satisfactory fit to the experimental data.
Appl. Sci. 2022, 12, 6747 8 of 14

Table 3. ANOVA results for extraction efficiency and the total flavonoid content.

Sum of Mean
Source DF F-Value p-Value
Squares Square
Yield (%)
Model 90.57 7 12.94 31.01 <0.0001 significant
X1 10.55 1 10.55 25.28 0.0015
X2 6.30 1 6.30 15.09 0.0060
X3 18.31 1 18.31 43.89 0.0003
X2 X3 5.43 1 5.43 13.02 0.0086
X21 47.34 1 47.34 113.48 <0.0001
X22 3.37 1 3.37 8.08 0.0249
X23 2.49 1 2.49 5.97 0.0445
Residual 2.92 7 0.4172
not
Lack of Fit 2.65 5 0.5298 3.91 0.2162
significant
Pure Error 0.2711 2 0.1355
Total 93.49 14
R2 = 0.9688; adj-R2 = 0.9375; CV = 11.13; Adeq Precision = 15.004

The model is statistically significant (p < 0.0001), and the lack of model fit is statistically
insignificant (p > 0.2162), which indicates that the model has been validated correctly. The
high value of the R2 coefficient (0.9688) and the corrected R2 (0.9375) indicates the existence
of a large correlation between the input variables and the total flavonoid content. A low CV
value (11.13%) means that the deviations between the experimental and predicted values
are low, and the reliability of the experiment and its precision is high. Adequate precision
greater than four is desirable, and the ratio was found to be 15.004, which indicates an
adequate signal and confirms that this model is significant for this extraction process.

3.2. The Total Anthocyanin Content

The results of the extraction of anthocyanin using an ultrasonic extractor are presented
in Figure 3.
The total anthocyanin content extracted from the hemp ranged from 10.66 to 17.16 mg
Cy-GE·100 g−1 , varying according to the experimental conditions. None of the available re-
ports have analyzed TAC in cannabis. Based on these results, the effects of the independent
variables on the dependent variables are presented in Table 4.
It was observed that only the linear terms of the particle size, extraction time, and
ultrasound intensity significantly affected (p < 0.05) the extraction of anthocyanins. How-
ever, the linear term of the particle size demonstrated negative correlations. As the particle
size increased from 0.25 to 1.25, there was a clear decrease in the total anthocyanin content
in the obtained extracts, from 15.30 to 12.52 mg Cy-GE·100 g−1 (Figure 3). This is most
likely due to the area in which anthocyanins are stored in the plant material. Anthocyanins
occur in plant vacuoles [28]; therefore, a greater degree of fragmentation facilitates their
washing out and contributes to a higher extraction efficiency. Wang et al. [29] showed that
a material particle size of 0.9 mm was sufficient to achieve an optimal pectin yield. The
smaller particle sizes of raw materials with larger specific surface areas have a tendency to
increase the heat and mass transfer between solvents and matrices [29]. Particle size had
the least influence on the total anthocyanin content in the obtained extracts, and its increase
caused the anthocyanin value to drop by 22.2%.
 found to be 15.004, which indicates an adequate signal and confirms that this model is
significant for this extraction process.

3.2. The Total Anthocyanin Content

Appl. Sci. 2022, 12, 6747 The results of the extraction of anthocyanin using an ultrasonic extractor
9 of 14 are
presented in Figure 3.

(a)

Appl. Sci. 2022, 12, x FOR PEER REVIEW 9 of 13

(b)

(c)
Figure
Figure3.3. Response
Response surfaces obtainedwith
surfaces obtained withthethe Box–Behnken
Box–Behnken experimental
experimental designdesign
for thefor the total
total
anthocyanin content(mg
anthocyanin content (mg Cy-GE∙100
Cy-GE ·100 g−1 ) g
−1) in hemp depending on the (a) intensity and time, (b)
in hemp depending on the (a) intensity and time, (b) particle
particle size
size and and
time, time,
and and (c)size
(c) particle particle size and intensity.
and intensity.

The total anthocyanin content extracted from the hemp ranged from 10.66 to 17.16
mg Cy-GE∙100 g−1, varying according to the experimental conditions. None of the available
reports have analyzed TAC in cannabis. Based on these results, the effects of the
independent variables on the dependent variables are presented in Table 4.
Appl. Sci. 2022, 12, 6747 10 of 14

Table 4. Effects of the independent variables on the dependent variables and their statistical signifi-
cance with respect to the extraction of anthocyanins from hemp.

Variables p Value Coefficient

X1 0.0141 −1.39
X2 0.0060 +1.72
X3 0.0003 +3.25
X1 X2 0.6191 +0.2818
X1 X3 0.4789 −0.4070
X2 X3 0.0700 +1.22
X21 0.7883 −0.1570
X22 0.0901 −1.16
X23 0.7702 +0.1709

The ultrasound intensity was the variable that most significantly influenced the ex-
traction of anthocyanins. The total anthocyanin content increased with the growth of
the ultrasound intensity. An incremental increase in the ultrasound intensity, from 1.6
to 8.6 W·m−2 , raised the total anthocyanin content by 61%. As the extraction time rose
from 5 to 15 min, the total anthocyanin content increased by 28.2%. The linear term of the
extraction time has also been observed during the extraction of anthocyanins and phenolic
compounds from jabuticaba skin [30], as well as phenolic compounds from grape marc [31]
and grape seeds [32]. In a previous experiment on the extraction of anthocyanins from
hawthorn berries, we showed a growth in TAC with a commensurate increase in time when
using the pulse mode of the ultrasound [22]. However, in the case of the continuous mode,
a slight but statistically insignificant decrease in the total anthocyanin content was observed
at a time of 15 min and an amplitude of 36 µm [22]. Zou et al. [33] indicated that the
anthocyanin yield of mulberry quickly increased with the time of extraction, reaching the
highest value at 40 min. From 40 to 100 min, the yield was almost constant. Mane et al. [34]
investigated the effect of ultrasound-assisted extraction on the amount of anthocyanin ex-
tracted from Purple Majesty potatoes and pointed out that shorter times lead to the growth
of anthocyanins in the extracts, with 5 min being optimal. Longer extraction times have
shown a linear decrease in TAC obtained over a period of 120 min [34]. Tiwari et al. [35]
showed that higher levels of ultrasonic amplitude and time have an adverse effect on the
total anthocyanin content in grape juice.
The parameters presented in Table 4 were re-estimated, considering only the significant
terms (p < 0.05). From the regression analysis, the model was adjusted to the experimental
data, as presented in Equation (5):

TAC = 7.82 − 2.78X1 + 0.34X2 + 0.93X3 (5)

The predictive equation was verified with analysis of variance (ANOVA), as can be
seen in Table 5, and it provides a satisfactory fit to the experimental data.
The model is statistically significant (p < 0.0001), and the lack of model fit is statistically
insignificant (p > 0.1476), which indicates that the model has been validated correctly. High
values of the R2 coefficient (0.8735) and corrected R2 (0.8390) indicate the existence of a
large correlation between the input variables and the total anthocyanin content. A low CV
value (9.17%) means that the deviations between the experimental and predicted values
are low, and the reliability of the experiment and its precision is high. Adequate precision
was found to be 15.09, which indicates an adequate signal and confirms that this model is
significant for this extraction process.
Appl. Sci. 2022, 12, 6747 11 of 14

Table 5. ANOVA results for extraction efficiency and total anthocyanin content.

Sum of Mean
Source DF F-Value p-Value
Squares Square
Yield (%)
Model 123.55 3 41.18 25.33 <0.0001 significant
X1 15.47 1 15.47 9.52 0.0104
X2 23.68 1 23.68 14.56 0.0029
X3 84.40 1 84.40 51.90 <0.0001
Residual 17.89 11 1.63
not
Lack of Fit 17.26 9 1.92 6.15 0.1476
significant
Pure Error 0.6236 2 0.3118
Total 141.44 14
R2 = 0.8735; adj-R2 = 0.8390; CV = 9.17; Adeq Precision = 15.09

3.3. Optimization of the Extraction Conditions

Derringer’s desirability prediction tool was used to calculate the maximum yield of
flavonoids and anthocyanins. Based on this analysis, the optimal values of extraction for
individual components were obtained (Table 6).

Table 6. Comparison between the experimental yields and predicted yields of total flavonoid content
(TFC), total anthocyanin content (TAC), and the simultaneous extraction of both bioactive compounds
(TFC and TAC) determined in the optimized conditions.

Extraction Variables Yield of Extraction

Optimized Response Predictive
Condition X1 X2 X3 Predicted Experimental a
Capacity (%)
TFC 0.59 10.71 7.13 TFC 9.28 8.98 ± 0.21 96.77 ± 2.27
TAC 0.25 15 8.60 TAC 20.27 17.83 ± 1.1 87.96 ± 5.43
TFC 8.99 8.52 ± 0.2 94.77 ± 2.22
TFC and TAC 0.60 12.51 8.60
TAC 18.43 16.74 ± 0.98 90.83 ± 5.32
X1 —particle size (mm); X2 —extraction time (min); X3 —ultrasound intensity (W·cm−2 ); a —mean ± standard
deviation of the three experiments.

The predictive capacity of the models was evaluated by comparing the predicted and
experimental values that were obtained from the tests, applying the optimized conditions
for each response. In all cases, the extraction yield of bioactive substances was slightly lower
than the values calculated on the basis of the developed models. Better agreement between
the predicted and experimental responses was obtained for the flavonoids, possibly due to
the greater number of parameters included in the model describing flavonoid extraction.
However, considering the high complexity of the matrix, the proposed models showed a
satisfactory predictive capacity for all evaluated compounds.

3.4. Energy Consumption during Pulsed Ultrasound-Assisted Extraction

Energy consumption during sonication is mainly dependent on the time and ampli-
tude of the ultrasonic vibrations and, to a lesser extent, on factors related to the extraction
environment, such as the viscosity and temperature of the solvent and the size and concen-
tration of the immersed solids. Some of these parameters can be changed during treatment.
Because of this, it is important to determine the total quantity of energy emitted into the
solid–liquid system [21]. The energy emitted by the ultrasonic device during extraction is
presented in Table 7.
Appl. Sci. 2022, 12, 6747 12 of 14

Table 7. The energy emitted by the ultrasonic device during extraction.

Ultrasound Intensity Time Energy Consumption

(W·cm−2 ) (min) (kJ)
1.6 5 2720
1.6 10 4843
1.6 15 7041
5.1 5 7631
5.1 10 15,410
5.1 15 21,821
8.6 5 14,011
8.6 10 22,945
8.6 15 32,012

The analysis of the data contained in Table 7 shows a directly proportional relationship
between the time of ultrasonic treatment and energy consumption; thus, it can be assumed
that, during sonication, the physical properties of the solvent were constant.

4. Conclusions
This study investigated the effects of extraction conditions on the yield of flavonoids
and anthocyanins using the Box–Behnken response surface methodology. The influence of
process variables (particle size, extraction time, and ultrasound intensity) on the extraction
efficiency depended on the tested bioactive substance.
For flavonoids, in the entire tested range, a significant positive effect was found with
respect to extraction time and ultrasound intensity on the efficiency of the process, whereas
in the case of particle size, the highest efficiency was obtained for a particle size of 0.75 mm.
The optimal conditions (on the basis of the model) for the extraction of flavonoids from
cannabis were as follows: particle size, 0.59 mm; extraction time, 10.71 min; and ultrasound
intensity, 7.13 W·cm−2 .
For anthocyanins, we observed a negative influence from the particle size distribu-
tion on the extraction efficiency and a positive effect from the ultrasound intensity and
time. The optimal conditions (on the basis of the model) for the extraction of antho-
cyanins were as follows: particle size, 0.25 mm; extraction time, 15 min; and ultrasound
intensity, 8.60 W·cm−2 .
The statistical data showed that the developed models were precise and adequate
compared with the experimental data. For both models (anthocyanins and flavonoids),
high values for the coefficient of determination (0.87–0.97) and the corrected coefficient of
determination (0.84–0.94) were obtained. The developed extraction procedure, with the
application of a pulsed ultrasound field, proved to be efficient for obtaining an enriched
fraction of bioactive compounds with very high flavonoid and anthocyanin contents from
Cannabis sativa L.

Author Contributions: Conceptualization, Z.K.; methodology, Z.K. and R.N.; validation, A.P. (Anna
Pecyna) and M.K.; formal analysis, M.K.; investigation, Z.K.; A.P. (Anna Pecyna) and A.B.; data
curation, A.P. (Artur Przywara); writing—original draft preparation, Z.K., A.P. (Anna Pecyna),
M.K. and R.N.; writing—review and editing, A.P. (Anna Pecyna), M.K. and A.P. (Artur Przywara);
visualization, A.B. and A.P. (Artur Przywara); supervision, Z.K.; funding acquisition, Z.K. All authors
have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
Appl. Sci. 2022, 12, 6747 13 of 14

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