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An innovative ultrasound-assisted extraction (UAE) is rapid non-thermal extraction technique, and in comparison to conventional extraction (CE), offers high reproducibility in short time with simplified manipulation, reduced solvent consumption and lower energy. Optimization of ultrasonic conditions was conducted for devices with nominal output power of 100 and 400 W including influence of geometrical parameters of probes regarding ultrasound-assisted extraction. Results showed that the optimal parameters to extract total phenols and rosmarinic acid as a dominant compound in sage extracts were as follows: solvent: 30 % ethanol, extraction duration: 11 min, output power of ultrasonic device: 400 W. The antioxidant capacity of obtained extract correlated with concentration of total phenols and flavonoids, and among individual phenols the rosmarinic acid contributed the most to the antioxidant capacity. On the basis of achieved results and conducted statistical analysis (p0.05) it was shown how UAE resulted in shortening time of extraction, increased extraction capacity of phenolic compounds with utilisation of solvents with less amount of organic phase.
M. DENT et al., Comparison of Conventional and Ultrasound-assisted Extraction…, Chem. Biochem. Eng. Q., 29 (3) 475–484 (2015) 475
Sage (Salvia officinalis L.) is a popular herb
which is native to the Mediterranean region and
cultivated worldwide. Sage is an important source
of antioxidants used as preservatives and has wider
implications for the dietary intake of natural antiox-
idants.1,2 This antioxidant effect has been attributed
to the main phenolic components, caffeic acid di-
mer – rosmarinic acid1 and flavonoids, being mostly
present as flavones and their glycosides. Flavone
glycosides are apparently common in sage, and
most of them are flavones 7-glycosides represented
by apigenin and luteolin 7-glucoside and their cor-
responding 3- and 7-glucuronides.2 In the last few
decades, research regarding the extraction of pheno-
lic compounds found in plants have attracted spe-
cial interest regarding their application in the food
industry. Extraction is a very important step in the
isolation, identification, and use of polyphenols.3
The conventional extraction methods, which have
been employed for decades, require prolonged ex-
traction times and relatively larger quantities of sol-
vent.4,5 Therefore, various novel extraction tech-
niques have been developed for the extraction of
bioactive compounds from herbs, including ultra-
sound-assisted extraction,6–8 microwave-assisted ex-
traction,9 supercritical fluid extraction.10 Ultra-
sound-assisted extraction is an upcoming extraction
technique that can offer high reproducibility in
shorter time, higher yields of bioactive compounds,
simplified manipulation, decreased temperature
during processing, reduced solvent consumption,
and lower energy input.11–13 In our previous paper,5
the influence of solvent polarity and composition,
time and temperature of conventional extraction on
mass fraction of polyphenols from sage were re-
searched. The mixtures of ethanol and water are
possibly the most suitable solvents for the extraction
of sage due to different polarities of the active con-
stituents, and acceptability of this solvent system
for human consumption.4,5 Albu et al.3 and Sališova
et al.14 have investigated the difference in the appli-
cation of conventional extraction and ultrasonic-as-
sisted extraction on the concentration of biological-
ly active compounds in sage. They concluded that
the content of biologically active compounds is ap-
proximately 60 % higher under the influence of ul-
trasound. Rosmarinic acid is the more active of
these antioxidants, but it is relatively easily degrad-
ed in solvent and the rate of degradation is sol-
vent-dependant.6 The ultrasonic-assisted extraction
has been widely used for obtaining polyphenols
from plants using ethanol, mixture of ethanol/wa-
Comparison of Conventional and Ultrasound-assisted Extraction Techniques
on Mass Fraction of Phenolic Compounds from Sage (Salvia officinalis L.)
M. Dent, V. Dragović-Uzelac, I. Elez Garofulić, T. Bosiljkov,
D. Ježek, and M. Brnčić*
Faculty of Food Technology and Biotechnology, University of Zagreb,
Pierottijeva 6, Zagreb, Croatia
An innovative ultrasound-assisted extraction (UAE) is the rapid non-thermal ex-
traction technique, which in comparison to conventional extraction (CE), offers high re-
producibility in a short time with simplified manipulation, reduced solvent consumption
and lower energy. Optimization of ultrasonic conditions was conducted for devices with
nominal output power of 100 and 400 W, including the influence of geometrical param-
eters of probes regarding ultrasound-assisted extraction. The results showed that the op-
timal parameters for extraction of total phenols and rosmarinic acid as a dominant com-
pound in sage extracts were as follows: solvent: 30 % ethanol, extraction duration of 11
minutes, and ultrasonic device output power of 400 W. The antioxidant capacity of the
obtained extract correlated with the concentration of total phenols and flavonoids, and
among individual phenols the rosmarinic acid contributed the most to the antioxidant
capacity. The achieved results and statistical analysis (p 0.05) have shown how UAE
resulted in shorter extraction time, and increased extraction capacity of phenolic com-
pounds by using solvents with a less amount of organic phase.
Key words:
conventional extraction, flavone glycosides, polyphenols, rosmarinic acid, sage, ultra-
sound-assisted extraction
*Corresponding author:; phone: 0038514605223
doi: 10.15255/CABEQ.2015.2168
Original scientific paper
Received: January 12, 2015
Accepted: September 9, 2015
476 M. DENT et al., Comparison of Conventional and Ultrasound-assisted Extraction…, Chem. Biochem. Eng. Q., 29 (3) 475–484 (2015)
ter,3,15–19 water and acetone.17 Ultrasound-assisted
extraction can also provide the opportunity for en-
hanced extraction of heat-sensitive bioactive com-
ponents at lower processing temperatures15, and is a
more effective technique than conventional thermal
extraction with most of the plants extracted within
15 minutes.3 The mechanism of ultrasound in liq-
uids relies on the mechanical effect caused by the
implosion of cavitational bubbles. During implo-
sion of micro-sized cavitational bubbles, strong
shear forces are created, while both high pressures
and temperatures generated as a consequence of the
bursting bubbles, cause rapid plant tissue disruption
allowing cellular material release and improved
mass transfer as well. An important part of ultra-
sound-assisted processing is overall optimization of
the process. The frequency (kHz), amplitude (%),
applied cycle (%), nominal output power (W), and
geometrical parameters of the sonotrode (length and
diameter mm) should be carefully prepared and
taken into consideration.20–22 The choice of method
for the extraction of polyphenols from sage depends
on the type of compounds to be extracted, and is
found to be dependent on the solvent employed.
From literature data,3 extraction of polyphenols us-
ing polar solvents is significantly improved by son-
ication. It is important to note that extraction time is
significantly shortened with the use of ultrasound.
The aim of this study was to examine and com-
pare CE and UAE of sage leaves (Salvia officinalis
L.). The research was carried out in two steps: in
the first step, conventional extraction and ultra-
sound-assisted extraction with 30 % ethanol, and
setting of the optimum conditions for each method.
In second step, the extraction was carried out with
different solvents (30 % acetone and water) under
optimal conditions for each method set in the first
step. The effects of the extraction parameters: sol-
vents polarity (water, 30 % ethanol and acetone) on
extraction methods, extraction time (20, 30, 40 min)
and temperature (40, 50, 60 °C) for CE, and the in-
fluence of different ultrasonic devices (100 and 400
W) and extraction time (8 to 12 min) for UAE, with
respect to the amount of extracted polyphenols were
investigated. The influence and relevance of the op-
erating parameters required during the extractions
were checked and evaluated using a response sur-
face methodology studying phenolic content. The
analysis of individual polyphenols of the extracts
was made by HPLC with UV/PDA detection.
Material and methods
Chemicals and reagents
Ethanol, sodium carbonate, and sodium nitrite
were purchased from Gram–mol Company (Zagreb,
Croatia). Folin-Ciocalteu reagent, apigenin-7-gluco-
side, luteolin-3-glucoside, rosmarinic, caffeic, gal-
lic, vanillic and syringic acid were purchased from
Sigma-Aldrich company (Steinheim, Germany).
Acetone was purchased from Kemika Company
(Zagreb, Croatia). Methanol (HPLC grade) was
purchased from J.T. Baker Company (Deventer, the
The plant material (sage) was collected on the
Island of Pag (Croatia) in July 2008. The leaves of
sage (Salvia officinalis L.) were dried immediately
after harvesting in a shady and well-aired place for
two weeks. Thereupon, the dried leaves were packed
in paper bags, sealed, and kept in a dark, dry and
cool place. Before use, the dry leaves were crushed
using a house blender (Mixy, Zepter International).
Extraction of polyphenols
Conventional extraction was performed in a
water bath shaker (Memmert WB14, SV1422,
Schwabach, Germany). The crushed dried sage
leaves (1 g) were weighed into a 100 mL glass cup,
dissolved in 30 % ethanol (20 mL) and extracted in
a water bath at 40, 50 and 60 °C for 20, 30 and 40
minutes. Further extraction experiments were per-
formed with 30 % acetone and pure distilled water
under optimal extraction conditions (60 °C, 30 min).
After the extraction, the flask was removed from
the bath and cooled to room temperature with cool-
ing water. The extracts were filtered through What-
man no. 40 filter paper (Whatman International
Ltd., Kent, UK) using a Büchner funnel, and the
filtrates were adjusted to 25 mL in volumetric flasks
with appropriate organic solvent or distilled water.
The extracts were stored at –18 °C until analyses
(no more than 7 days).
Ultrasound-assisted extraction experiments
were carried out with 2 different ultrasonic devices
with maximal power of 100 W and 400 W under
maximal working amplitude equal to 100 % of
maximum nominal output power of the device. The
ultrasonic devices were manufactured by “Dr
Hielscher”, Teltow, Germany). The frequency was
constant at 24 kHz for 100 W, and 30 kHz for 400
W ultrasonic devices, and enabled transient cavita-
tions with bubbles implosion effect. The device was
working throughout the experiment in continuous
mode, i.e. cycle = 1, which means how the ultra-
sound was propagated in 100 % of the time through-
out the medium. As previously mentioned for pro-
cess optimization, two different ultrasonic devices
were used to determine appropriate sonotrode for
the used volume of the sample. The setup was not
cooled down. The reason for this was to obtain the
M. DENT et al., Comparison of Conventional and Ultrasound-assisted Extraction…, Chem. Biochem. Eng. Q., 29 (3) 475–484 (2015) 477
best yield in a shorter processing time without addi-
tional energy consumption for cooling using ultra-
sonic in comparison with conventional extraction.
The crushed dried sage leaves (1 g) were weighed
into a 100 mL glass cup, dissolved in 30 % ethanol
(20 mL), and directly sonicated. The extrinsic pa-
rameters of extraction time (8, 10, 11, 12 min) and
the ultrasonic device (100 and 400 W) were varied.
The ultrasonic probe was immersed into the mixture
directly. Further extraction experiments were per-
formed with 30 % acetone and pure distilled water
under optimal extraction time of 11 minutes. The
temperature was in the range of 21.8 to 88.0 °C.
After extraction, the extracts were filtered through
Whatman no. 40 filter paper (Whatman Internation-
al Ltd., Kent, UK) using a Büchner funnel, and the
filtrates were adjusted to 25 mL in a volumetric flask
with appropriate organic solvent or distilled water.
The obtained polyphenolic extracts (CE and
UAE extraction) were used for determination of to-
tal phenols, flavonoids, antioxidant capacity spec-
trophotometrically, and individual phenols using
HPLC coupled with UV/PDA detector. All treat-
ments were carried out in triplicate.
Determination of total phenols (TP)
The total phenols content of the extracts ob-
tained by both conventional extraction and ultra-
sound-assisted extraction were determined by a
modified spectrophotometric method using Fo-
lin-Ciocalteu reagent, calibrated against rosmarinic
acid as the reference standard.23 Quantification of
total phenols was made by using the calibration
curve of rosmarinic acid, which was prepared by di-
luting the stock standard with the extraction sol-
vents to yield 50 to 500 mg per L of TP. The results
were calculated according to the calibration curve
for rosmarinic acid and the mass fraction of total
phenols, derived from triplicate analyses and ex-
pressed as mg of rosmarinic acid equivalents (RA)
per 100 g dry matter (dm).
Determination of flavonoids (FL)
Flavonoid compounds were determined from
the same extracted samples that were used for de-
termination of total phenolic content. The flavo-
noids were determined by a modified photometric
method using AlCl3,
24 calibrated against rutin as the
reference standard. Quantification of flavonoids
was made by using the calibration curve of rutin,
which was prepared by diluting the stock standard
with the extraction solvents to yield 50 to 250 mg
per 100 mL of flavonoids. The results were calcu-
lated according to the calibration curve for rutin and
the mass fraction of flavonoids, derived from tripli-
cate analyses and expressed as mg of rutin equiva-
lents per 100 g dry matter (dm).
DPPH radical-scavenging activity
The DPPH radical-scavenging activity was de-
termined using the method proposed by Brand-Wil-
liams et al.25 Briefly, 1 mL of phenolic extracts was
added to 1 mL of DPPH methanolic solution (0.5
mmol L–1 ) with 3 mL of methanol. The mixture
was shaken vigorously and allowed to stand at room
temperature in the dark for 20 minutes. Absorbance
was measured at 517 nm. Methanol was used to ad-
just zero and DPPH methanol solution as a refer-
ence sample. The results were corrected for dilution
and expressed in mmol trolox equivalents per kg.
All determinations were performed in triplicate.
HPLC analysis
Separation of sage phenols was performed by
HPLC analysis, using a Varian Pro Star System
(Agilent Technologies, Inc., Santa Clara, CA, US)
equipped with a ProStar 230 solvent delivery sys-
tem, Rheodyne® 7125 injector and Pro Star 330
UV-photo diode array detector. Chromatographic
separation was performed on a Zorbax ODS column
(250 x 4.6 mm i.d. 5 mm; Agilent Technologies).
The composition of solvents and gradient elution
conditions had previously been described by Fecka
et al.26 with modification of formic acid in mobile
phases. The mobile phase was composed of solvent
A (3 % formic acid in acetonitrile) and solvent B
(3 % formic acid in water). The following gradient
was carried out: 10 % A in B, rising to 40 % A after
25 minutes, to 70 % A after 30 minutes, and then to
10 % A after 35 minutes. The flow rate was kept
constant at 0.9 mL per minute for a total run time of
35 minutes. The injection volume for all the sam-
ples was 20 µL. The extracts were filtered through
a 0.45 µL diameter membrane filter prior to analy-
sis. The detection wavelength of 278 nm was used
for the detection of phenolic acids. Flavones glyco-
sides were detected at 360 nm. Identification of the
compounds was achieved by comparing their reten-
tion times and UV-VIS spectra with those of au-
thenticated standards. Phenolic compounds were
separated in a series of descending polarity, based
on the comparison of their retention time and reten-
tion time of the standards, and based on a compari-
son of typical UV spectra. Quantitative determina-
tions were carried out using the calibration curves
of the standards. Phenolic acids (rosmarinic, caffe-
ic, vanillic and syringic) and flavonoids (luteo-
lin-3-glucoside and apigenin-7-glucoside) were
used as standards. Calibration curves of the pheno-
lic acids and flavone glycosides standards were
made by diluting stock standards (concentration of
478 M. DENT et al., Comparison of Conventional and Ultrasound-assisted Extraction…, Chem. Biochem. Eng. Q., 29 (3) 475–484 (2015)
0.5 to 2.0 mg per mL) in extraction solvents (etha-
nol, acetone or water) to yield 0.001– 0.020 mg per
mL. Mass fractions of phenolic compounds were
calculated from the calibration curves of phenolic
acids and flavones glycosides, and expressed as mg
of phenolic acid or flavones glycosides equivalent
per 100 g dry matter. The values were expressed as
means (N = 3) ± S.D. Salvianolic K and salvianolic
I acids and methyl rosmarinate were quantified as
equivalents of rosmarinic acid. Flavone glycosides
6-hydroxyluteolin-7-glucoside, luteolin-7-glucuron-
ide and luteolin-3-glucuronide were quantified as
equivalents of luteolin-3-glucoside and apigen-
in-7-glucuronide as the equivalent of apigen-
Statistical analysis
All data were expressed as means (N = 3) ±
standard deviations (S.D.) of triplicate measure-
ments and analysed by software (Statistica 12.0).
One-way analysis of variance (ANOVA)27,28 was
carried out on the significant differences between
the extraction methods and the solvents used. Two
different extraction methods were examined. The
effects of solvent extraction conditions (extraction
solvent, extraction time and temperature, power of
ultrasonic devices) were investigated. Differences
were considered significant at p0.05.
Results and discussion
In the first step of this study, two methods
(conventional and ultrasound-assisted extraction)
were used to extract polyphenols from sage under
the previously described conditions. Table 1 shows
the extraction capacity of conventional extraction
for total phenols, flavonoids, antioxidant capacity,
and quantification results of phenolic acids and fla-
vonoids. Two variables influencing the extraction
were investigated by means of the conventional
process, which are extraction temperature (40, 50,
60 °C) and time (20, 30, 40 min). In the further ex-
periments, chosen were temperatures below 60 °C
and shorter extraction times up to 40 minutes, be-
cause higher temperatures may be attributed to ther-
mal degradation of polyphenols.5 Studying the ex-
traction time (20, 30, 40 min), it was observed that
the mass fraction of the extracted phenols slightly
Table 1 – Quantification of individual phenolic compounds in the sage extracts obtained in ultrasonic bath at 40, 50 and 60 °C for
20, 30 and 40 minutes with 30 % ethanol
(min) Total phenols
Phenolic acids, mg/100 g
Flavo noids
Flavone glycosides, mg/100 g DPPH
TE kg–1)
Σ hydroxy-
cinnamic acids
Σ hydroxy-
benzoic acids
Σ luteolin
Σ apigenin
20 3698.08±20.12 1226.22±33.34 74.81±11.85 39.41±4.45 1340.38±9.21 611.66±12.12 118.31±9.15 1.69±0.75
30 5547.71±10.56 1983.25±34.51 102.65±12.21 54.07±5.13 1199.97±8.96 893.51±14.70 221.17±5.47 1.69±0.83
40 4539.49±12.33 2254.59±11.71 100.69±10.99 58.84±7.66 1186.18±11.72 792.24±15.94 197.12±8.42 1.64±0.95
20 3874.10±23.17 2241.71±23.55 100.39±11.09 71.40±10.97 1313.89±7.89 816.10±16.45 205.31±3.49 1.68±1.03
30 4276.08±12.75 2288.32±32.92 108.45±12.53 67.78±8.85 1443.29±14.63 885.48±12.99 189.32±2.96 1.67±1.04
40 4221.07±11.45 1906.12±20.68 95.43± 6.69 65.64±9.52 1364.41±9.72 797.31±13.75 180.03±4.69 1.69±0.99
20 5203.15±18.55 3032.19±10.92 96.33± 7.88 85.85±10.95 1322.66±14.44 1343.40±21.50 247.31±5.78 1.70±0.87
30 6399.79±21.85 3499.32±22.78 101.32±12.10 94.16±10.82 1924.60±9.85 1426.14±20.76 267.43±8.45 1.72±0.94
40 4744.35±13.55 3455.18±13.75 95.03± 9.24 88.61±9.09 2083.36±10.14 1392.77±19.16 248.29±6.75 1.69±0.74
time NS NS NS NS 0.028019 NS NS NS
temperature NS 0.003539 NS 0.000227 NS 0.003452 0.036498 NS
Correlation, r 0.7170 0.5070
p0.05 significant statistically; NS, not significant statistically.
Content of phenolic acids and flavone glycosides are expressed as mg per 100 g of dry sage; the sum of hydroxycinnamic acids
(caffeic acid, salvianolic K and I acids, methyl rosmarinat); the sum of hydroxybenzoic acids (vanillic and syringic acids); Luteolin
glycosides (6-hydroxyluteolin-7-glucoside, luteolin-7-glucuronide, luteolin-7-glucoside, luteolin-3-glucuronide); Apigenin glycosides
(apigenin-7-glucuronide, apigenin-7-glucoside).
M. DENT et al., Comparison of Conventional and Ultrasound-assisted Extraction…, Chem. Biochem. Eng. Q., 29 (3) 475–484 (2015) 479
increased at 30 minutes, and a decrease at longer
times of extraction (40 min) at all temperatures. The
results of quantification are shown individually for
rosmarinic acid as it was the most abundant com-
pound in the sage extracts, while the results for oth-
er identified phenolic acids are shown as the sum of
hydroxycinammic acids (caffeic, salvianolic K and
I acids, methyl rosmarinate) and hydroxybenzoic
acids (vanillic and syringic acids) because they
were present in very low mass fractions. Also, the
results of quantification of flavonoids are expressed
as the sum of luteolin glycosides (6-hydroxyluteo-
lin-7-glucoside, luteolin-7-glucuronide, luteolin-7 -
glucoside, luteolin-3-glucuronide) and api ge nin gly-
cosides (apigenin-7-glucuronide, api ge nin-7-glu coside),
because some of them were present in low mass
fractions. As can be observed from the results ob-
tained by conventional extraction, temperature also
has a great impact on the extraction of rosmarinic,
hydroxybenzoic acids, and flavone glycosides. A
temperature increase from 40 to 60 °C had signifi-
cantly increased the extraction efficiency in conven-
tional extraction (Table 1). When extraction tem-
perature rose to 60 °C, the mass fraction of phenolic
acids and flavone glycosides decreased with in-
creased extraction time (30 to 40 min). We suggest
that the extraction temperature in the extraction of
sage phenols by conventional extraction in a water
bath should not exceed 60 °C. As already discussed,
the obtained results and the results previously de-
scribed5 lead us to conclude that the extraction ca-
pacity decreases at higher temperatures. The reduc-
tion in yields of extracted phenolics at times longer
than 30 minutes and higher temperatures may prob-
ably be attributed to the thermal degradation and
polymerization reaction occurring due to the combi-
nation of various phenols among themselves, hav-
ing an effect on the analytical quantification.29 Tak-
ing into account these facts, temperature (60 °C)
and extraction time (30 min) were selected as the
For the ultrasound-assisted extraction, the nom-
inal output power of ultrasonic device with different
frequencies (24 and 30 kHz) and the nominal output
powers (100 and 400 W) with duration of extraction
(8, 10, 11, 12 min) parameters were optimized. The
influence of sonication time ranging from 8, 10, 11
to 12 minutes on total phenols and flavonoids mass
fraction is shown in Table 2. Our obtained results
showed that phenols recovery increased with the
time of sonication, reaching a maximum at 11 min-
utes for the sage total phenols and flavonoids anal-
ysed. The extraction efficiencies were lower during
first 8 minutes of sonication, indicating that more
time of ultrasound propagation was required for cell
walls disruption releasing phenols from the cell
constituents. Longer sonication times of 10 and 11
minutes improved extraction efficiencies and hence
increased the rate of extraction, while prolonged ap-
plication of 12 minutes did not increase the mass
fraction of total phenols and flavonoids to a larger
extent. The highest extraction capacity was reached
by extraction time of 11 minutes, regardless of the
ultrasonic devices (100 and 400 W). The highest
mass fraction of TP (7813.69 mg RA/100 g) with
Folin-Ciocalteu method was obtained with the fol-
lowing parameters of ultrasonic extraction: device
output power of 100 W, sonication times of 11 min-
utes, while the highest value was not confirmed
with conducted HPLC analysis. Considering the
above-mentioned, this may have been due to the
non-phenolic components in the extract reacting
with the Folin-Ciocalteu reagent. Longer sonication
times of 10 and 20 minutes improved extraction ef-
ficiencies, and hence increased the rate of ex-
traction, while prolonged application of 30 minutes
did not benefit greatly in phenol yields. When com-
pared to 30 minutes of extraction, more than 60 %
phenolics were extracted in the first 10 minutes.
The time increases up to 10 minutes provoked an
almost linear rise in total phenols yields, suggesting
that maximum recovery was already attained before
10 minutes.7,13 Statistical evaluation (ANOVA) con-
firmed that phenol extraction yields were not highly
time-dependent (p 0.05) (Table 2). The positive
effect on the extraction yield of phenolic acids and
flavone glycosides was observed with the increase
in ultrasonic power. The extraction yield of pheno-
lic acids and flavone glycosides increased, but not
significantly (p0.05). The mass fraction of ros-
marinic acid was much higher than the others, and
had an obvious increasing trend from 100 to 400 W.
The total value of phenolic acids and flavone glyco-
sides of ethanol extract to the extraction time of 11
minutes with 400 W ultrasonic devices was ob-
tained in higher mass fraction than using the 100 W
devices. The achieved results and the conducted sta-
tistical analysis suggested that the extraction time
influenced statistically significantly the extraction
of phenolic compounds from sage up to 11 minutes,
while after that time, a decrease occurred (p ≥ 0.05)
(Table 2). Prolonged extraction time (8, 10 and 11
min) resulted in increased extraction capacity of
rosmarinic acid isolation but also of other phenolic
acids and flavone glycosides.
When compared to 30 minutes of conventional
extraction at 60 °C, most of the polyphenols were
extracted in the first 11 minutes. Direct sonication
with a probe was more efficient than conventional
extraction for sage. Generally, in a directly sonicated
system, the energy of the probe unit is directly fo-
cused on a localized sample zone, thereby providing
more efficient cavitation into the treated solution.30–32
It is important to note that by using ultrasound, the
480 M. DENT et al., Comparison of Conventional and Ultrasound-assisted Extraction…, Chem. Biochem. Eng. Q., 29 (3) 475–484 (2015)
extraction time was significantly shortened compared
to conventional extraction. Based on the results from
the first step of this research, optimal extraction con-
ditions were defined for optimal extraction condi-
tions for both extraction methods. Optimisation of
the extraction procedure was based on total and indi-
vidual phenols. The optimal extraction conditions se-
lected were as follows: 11 minutes of extraction time
with 400 W device, and 30 minutes in water bath at
60 °C with conventional extraction.
In the second step, the extraction was carried
out at optimal processing conditions with the other
two solvents (30 % acetone and water). Ultra-
sound-assisted extraction (with 400 W device) in
other solvents, 30 % acetone and water, in the same
extraction conditions, obtained a somewhat lower
mass fraction of TP, 30 % acetone (5202.88 mg
RA/100 g), water (4284.64 mg RA/100 g). Table 3
shows the total phenolic and flavonoid content of
the three solvent extractions from sage. The differ-
ence in polarities of the extracting solvents might
influence the solubility of the chemical constituents
in a sample and its extraction yield. Therefore, in
the selection of a solvent for optimizing the recov-
ery of total phenols and flavonoids from the sample,
30 % ethanol exhibited the highest value of total
phenols and flavonoids, hence was is considered as
the most efficient solvent system for extracting phe-
nolic compounds from sage. The 30 % acetone ex-
tracts of sage leaves ranked next with no significant
difference from the 30 % ethanol and water extracts
(p 0.05, table 3). The highest extraction capacity
were achieved by extraction with 30 % ethanol, and
significantly less with 30 % acetone and water in
the same extraction conditions. In the conventional
extraction method, the choice of solvent extraction
and extraction method had no significant impact on
the mass fraction of total phenols and flavonoids
(Table 3). After the photometric determination of
total phenols, the extracted samples were subjected
to HPLC UV/PDA analysis. The effect of these sol-
vent systems in extracting phenolic compounds
from sage were quantitatively measured and com-
pared. Higher mass fractions of rosmarinic acid as
the most represented compound in sage, were ex-
tracted by implementing 30 % ethanol than with (30
% acetone and water) under same extraction condi-
tions. The statistical analysis showed how the sol-
vent used had no significant influence on the ex-
traction of rosmarinic acid (p0.05) (Table 3).
Table 2 – Effect of different ultrasonic devices and sonication times on mass fractions of phenolic acids and flavone glycosides.
Extraction was performed with 30 % ethanol under ultrasound working conditions (100 % amplitude/duty cycle) and
sonication time (8, 10, 11 and 12 min).
Phenolic acids, mg/100 g Flavone glycosides, mg/100 g DPPH
(mmol TE
Σ hydroxy-
cinnamic acids
Σ hydroxy-
benzoic acids Flavonoids Σ luteolin
Σ apigenin
8 6892.67±19.58 3622.96±10.83 38.58±2.36 25.36±3.20 1569.75±8.16 1433.59±11.12 193.47±6.47 1.64±0.98
10 7146.44±20.47 3915.68±11.14 34.21±0.98 37.55±3.51 2072.21±10.51 1719.19±12.19 232.26±4.83 1.64±0.84
11 7813.69±14.36 3991.59±12.18 45.14±1.57 43.80±5.35 2122.96±11.84 1689.59±9.45 237.32±4.63 1.64±0.75
12 7224.21±15.48 3549.36±13.75 42.25±1.89 39.39±4.74 1412.32±14.79 1549.59±8.66 218.41±2.59 1.62±0.97
85833.88 ±23.45 2461.98±14.58 90.24±14.98 110.63±4.82 1552.11±10.14 1046.60±42.35 144.34±12.11 1.59±1.01
10 5393.91±24.56 3545.89±14.58 89.60±13.54 115.14±3.89 1884.66±12.42 1562.28±37.79 195.46±14.23 1.59±1.16
11 6775.52±21.12 4160.31±20.01 117.30±8.47 120.22±4.59 1928.79±14.21 1961.03±49.90 248.20±14.14 1.57±1.25
12 5595.69±21.76 3699.59±16.88 115.20±13.44 100.49±2.96 1913.77±13.83 1620.05±44.53 216.05±15.34 1.46±1.37
device 0.015128 NS NS NS NS NS NS 0.030183
time NS NS 0.000204 0.000060 NS NS NS NS
Correlation, r0.6742 0.0980
p0.05 significant statistically; NS, not significant statistically.
Content of phenolic acids and flavone glycosides are expressed as mg per 100 g of dry sage; the sum of hydroxycinnamic acids
(caffeic acid, salvianolic K and I acids, methyl rosmarinate); the sum of hydroxybenzoic acids (vanillic and syringic acids); Luteolin
glycosides (6-hydroxyluteolin-7-glucoside, luteolin-7-glucuronide, luteolin-7-glucoside, luteolin-3-glucuronide); Apigenin glycosides
(apigenin-7-glucuronide, apigenin-7-glucoside).
M. DENT et al., Comparison of Conventional and Ultrasound-assisted Extraction…, Chem. Biochem. Eng. Q., 29 (3) 475–484 (2015) 481
In the extracts obtained by the probe system of
400 W under optimal conditions, the mass fractions
of all identified polyphenols were higher than in ex-
tracts obtained with the probe system of 100 W, and
especially with regard to conventional extraction.
Improvement was obtained using a 400 W probe sys-
tem. Here, additional stirring was not required since
the probe itself provided sufficient mixing of the het-
erogeneous mixture. The mass fraction of rosmarinic
acid was the highest in ethanol extracts (4160.31
mg/100 g), followed by acetone (3937.13 mg/100 g),
while it was the lowest in the water extracts (2654.14
mg/100 g). Other phenolic acids were determined in
higher mass fractions in ethanol extracts with regard
to acetone and water extracts. Luteolin and apigenin
glycosides were the highest in the ethanol extract,
followed by acetone, and the lowest in the water ex-
tracts. The mass fractions of all identified phenols in
the water extracts were low compared to ethanol and
acetone extracts, although ultrasound enhanced the
extraction, so the mass fractions of all identified phe-
nols were higher in ultrasound-assisted extraction
than conventional extraction (Table 3). Statistical
analysis showed a significant influence of the ex-
traction method and solvent on the amount of hy-
droxybenzoic acids and luteolin glycosides, while
only the extraction solvent had a significant influence
on the amount of hydroxycinamic acids (p ≤ 0.05,
Table 3). Several studies have also revealed the effi-
ciency of ethanol and acetone water mixtures in ex-
tracting polyphenols from samples of plant materials.
Ethanol is possibly a preferable solvent because of its
nontoxic, environmentally safe and inexpensive fea-
tures, while acetone may lead to an unacceptable lev-
el of residue in the extracts.3,6,10 Therefore, ethanol
and water are possibly the most suitable solvent sys-
tem for the extraction of sage due to the different po-
larities of the active constituents, and the acceptabil-
ity of this solvent system for human consumption.
By ultrasound-assisted extraction for 11 min-
utes, using 30 % ethanol, the mass fraction of TP
(6775.52 mg RA/100 g) was 20 % higher than with
conventional extraction for 30 minutes with the same
solvent, TP (6399.79 mg RA/100 g). By using other
extraction solvents (acetone and water), the mass
fraction of TP was lower. Under ultrasonic extraction,
ethanol showed a greater enhancement in the mass
fraction of total phenols and flavonoids compared
with acetone and water, which suggested it could be
a viable solvent for extraction under these conditions.
Sonication also appeared to reduce the dependence
on the extraction solvent itself, with yields greatly
enhanced when employing ethanol.3 From the results
of individual phenols obtained, it appeared that 30 %
ethanol was a better extraction solvent for rosmarinic
acid under the conditions studied (Table 3). This is
probably due to the more polar nature of rosmarinic
acid favouring the more polar ethanol solvent. Wang
et al.17 investigated the effect of extraction time on
the content of rosmarinic acid using 30 % ethanol as
the solvent. They found that 10 minutes of sonication
was sufficient to extract the phenols. It was found
that there was little difference using 30 % ethanol or
Table 3 – Effect of extraction solvent type on mass fractions of total phenols and phenolic acids under optimal extraction conditions
of both extraction methods. Ultrasound extraction was performed under ultrasound working conditions (100 % amplitude/duty cycle,
ultrasonic device of 400 W) and sonication time of 11 min. Conventional extraction was performed at 60 °C for 30 min.
method Solvent type
Phenolic acids, mg/100 g Flavone glycosides, mg/100 g
total phenols rosmarinic
cinnamic acids
benzoic acids Flavonoids luteolin
30 % ethanol 6399.79±21.85 3499.32±22.78 101.32±7.42 43.80±5.35 1924.60±9.85 1426.14±45.32 267.43±12.11
30 % acetone 6192.09±20.18 3820.17±18.54 92.10±11.27 39.81±4.01 1597.13±12.41 1177.36±34.25 282.46±19.40
water 3493.34±15.83 625.22±19.98 80.35±6.52 26.59±3.08 866.05±6.42 902.53±39.12 205.16±20.27
30 % ethanol 6775.52±21.12 4160.31±20.01 117.30±8.47 94.16±4.54 1928.79±14.21 1961.03±49.90 248.20±14.14
30 % acetone 5202.88±21.48 3937.13±15.32 99.57±14.37 65.41±4.22 1712.00±10.45 1736.48±44.94 217.84±22.40
water 4284.64±25.76 2654.14±14.88 83.88±8.82 46.21±5.11 1459.01±12.52 1323.42±49.52 210.33±21.37
Extraction method NS NS NS 0.030195 NS 0.010377 NS
Extraction solvent NS NS 0.011312 0.049781 NS 0.013783 NS
p 0.05 significant statistically; NS, not significant statistically.
Content of phenolic acids and flavone glycosides are expressed as mg per 100 g of dry sage; the sum of hydroxycinnamic acids
(caffeic acid, salvianolic K and I acids, methyl rosmarinate); the sum of hydroxybenzoic acids (vanillic and syringic acids); Luteolin
glycosides (6-hydroxyluteolin-7-glucoside, luteolin-7-glucuronide, luteolin-7-glucoside, luteolin-3-glucuronide); Apigenin glycosides
(apigenin-7-glucuronide, apigenin-7-glucoside).
482 M. DENT et al., Comparison of Conventional and Ultrasound-assisted Extraction…, Chem. Biochem. Eng. Q., 29 (3) 475–484 (2015)
acetone as an extraction solvent, while the content of
rosmarinic acid was about 20 % higher than with wa-
ter. With water alone as an extraction solvent, the
content of rosmarinic acid was about 30 % lower
than with the other solvents. From our results, water
is not a good solvent for extracting phenols from
sage, but it has been observed that the addition of
small percentages of water to the extraction solvent
helps increase the effectiveness of extraction of the
analytes of interest from the sample.5 All the factors
had positive effects on the rosmarinic acid content.
Research results are in accordance with literature
data stating that caffeic acid and their derivates,33 sal-
vianolic K and 5,9,34 sagerinic,1 caffeic acid,34–36 meth-
yl rosmarinate,26,37 play a central role in the composi-
tion of Lamiaceae. The content of free caffeic acid in
our extracts was lower compared to syringic and es-
pecially rosmarinic acid. The results of our study
showed that rosmarinic acid was present in all these
sage extracts. Rosmarinic acid was the dominant
compound, while other phenolics were present in
significantly lower mass fractions. HPLC analysis
showed higher mass fractions of phenolic acids and
flavone glycosides in samples obtained by ultrasound
than by conventional extraction under the same po-
larity of solvents with significant shortening of treat-
ment time (threefold).
In recent years, there have been several reports
on the application of UAE in the isolation of various
phenolic compounds from plant materials.3,13,14,16 The
mass fractions of rosmarinic acid and other identified
phenols in ethanol extracts obtained by applying ul-
trasonic-assisted extraction with directly immersed
probe (output power of 400 W, 11 minutes) achieved
about 20 % higher extraction capacity compared to
conventional extraction in a water bath (60 °C, 30
minutes). These results are consistent with studies of
other authors.3,14 It is important to emphasise that ul-
trasound-assisted extraction shortened the extraction
time threefold. During sonication, the cavitations
process causes the swelling of cells or the breakdown
of cell walls, which allows high diffusion rates across
the cell wall in the first case, or a simple washing out
of the cell contents in the second.15 Besides the sol-
vent, temperature and pressure, better recovery of
cell contents can be obtained by optimising ultra-
sound application factors, including frequency, soni-
cation power and time, as well as ultrasonic wave
disruption.11 Ultrasound as a technology and ultra-
sound-assisted extraction can be called an ″environ-
ment-friendly″ or ″green″ technique.19 Overall, ultra-
sound-assisted extraction of polyphenols by using
food grade solvents has strong potential for its indus-
trial development as an efficient and environ-
ment-friendly process for preparation of extracts rich
in natural antioxidants aimed at replacing synthetic
antioxidants.16 UAE has been recognised for applica-
tion in industry to improve efficiency and reduce ex-
traction time.15
A good correlation was obtained between antiox-
idant properties of sage extracts and their total phenols
content, while the sage extracts possessed higher radi-
cal scavenging ability for both extraction methods.
The results obtained for flavonoids and DPPH assays
showed a low degree of correlation. Tables 1 and 2
show the linear correlation between the mass fraction
of total phenols, flavonoids and antioxidant capacities
of sage extracts obtained by different extraction tem-
perature and time. It can be concluded that increased
temperature increased the antioxidant capacity of the
sage extracts. This parallel relationship proves that the
total phenol content directly affects the antioxidant ca-
pacity of sage extracts.
By applying different extraction methods, the ul-
trasound-assisted extraction employing an ultrasonic
device with direct agitation, resulted in the highest
recovery of total and individual polyphenols coupled
with lower solvent consumption compared to con-
ventional extraction. Direct sonication with a probe
system was more efficient than conventional ex-
traction. The energy of the probe unit was directly
focused on a localised sample zone thereby provid-
ing efficient cavitations into the extracting solution.
The values of polyphenols extracted with ultra-
sound-assisted extraction under optimal conditions
(output power of 400 W, 11 minutes) using 30 %
ethanol were 20 % higher than with conventional ex-
traction (60 °C, 30 min) with a meaningful (up to
threefold) shortening of processing time. Improve-
ment of all identified polyphenols was obtained us-
ing a 400 W probe system under optimal conditions,
especially relative to the probe system of 100 W. The
results showed that rosmarinic acid was the dominant
compound in the sage extracts, while other phenolics
were present in significantly lower mass fractions.
Total and individual polyphenols were determined in
higher mass fractions in ethanol extracts relative to
acetone and water extracts.
This work was financially supported by the
following projects granted by Ministry of Science,
Education and Sports of the Republic of Croatia:
“Ultrasound-assisted extraction of bioactive com-
pounds” (910–08/11–01/00207) coordinated by
Professor Mladen Brnčić, and “Biologically active
compounds in some wild and cultivated plants”
(058–00000–3488) coordinated by Professor Verica
Dragović Uzelac.
M. DENT et al., Comparison of Conventional and Ultrasound-assisted Extraction…, Chem. Biochem. Eng. Q., 29 (3) 475–484 (2015) 483
CE Conventional extraction
dm Dry matter
FL – Flavonoids
TP – Total phenols
RA Rosmarinic acid
UAE – Ultrasound-assisted extraction
1. Lu, Y., Yeap Foo, L., Rosmarinic acid derivates from Salvia
officinalis, Phytochem. 51 (1999) 91.
2. Lu, Y., Yeap Foo, L., Flavonoid and phenolic glycosides
from Salvia officinalis, Phytochem. 55 (2000) 263.
3. Albu, S., Joyce, E., Paniwnyk, L., Lorimer, J. P., Mason, T. J.,
Potential for the use of ultrasound in the extraction of antiox-
idants from Rosmarinus officinalis for the food and pharma-
ceutical industry, Ultrason. Sonochem. 11 (2004) 261.
4. Durling, N. E., Catchpole, O. J., Grey, J. B., Webby, R. F.,
Mitchell, K. A., Foo, L. Y., Perry, N. B., Extraction of pheno-
lics and essential oil from dried sage (Salvia officinalis) us-
ing ethanol-water mixtures, Food. Chem. 101 (2007) 1417.
5. Dent, M., Dragović-Uzelac, V., Penić, M., Brnčić, M.,
Bosiljkov, T., Levaj, B., The effect of Extraction Solvents,
Temperature and Time on the Composition and Mass Frac-
tion of Polyphenols in Dalmatian Wild Sage (Salvia offici-
nalis L.) Extracts, Food. Technol. Biotech. 51(1) (2013) 84.
6. Paniwnyk, L., Cai, H., Albu, S., Mason, T. J., Cole, R., The
enhancement and scale up of the extraction of anti-oxidants
from Rosmarinus officinalis using ultrasound, Ultrason.
Sonochem. 16 (2009) 287.
7. Jerman, T., Trebše, P., Vodopivec, B. M., Ultrasound-assist-
ed solid liquid extraction (USLE) of olive fruit (Olea euro-
pea) phenolic compounds, Food. Chem. 123 (2010) 175.
8. Hossain, M. B., Brunton, N. P., Patras, A., Tiwari, B., ODon-
nell, C. P., Martin-Diana, A. B., Barry-Ryan, C., Optimization
of ultrasound assisted extraction of antioxidant compounds
from marjoram (Origanum majorana L.) using response sur-
face methodology, Ultrason. Sonochem. 19 (2012) 582.
9. Dragović-Uzelac, V., Elez Garofulić, I., Jukić, M., Penić,
M., Dent, M., The Influence of Microwave-Assisted Ex-
traction on the Isolation of Sage (Salvia officinalis L.),
Food. Technol. Biotech. 50(3) (2012) 377.
10. Lu, Y., Yeap Foo, L., Wong, H., Sagecumarin, a novel caffe-
ic acid trimer from Salvia officinalis, Phytochem. 52 (1999)
11. Wang, L., Weller, C. L., Recent advances in extraction of
nutraceuticals from plants, Trends, Food. Sci. Technol. 17
(2006) 300.
12. Virot, M., Tomao, V., Le Bourvellec, C., Renard, C. M. C. G.,
Chemat, F., Towards the industrial production of antioxidants
from food processing by-products with ultrasound-assisted
extraction, Ultrason. Sonochem. 17(6) (2010) 1066.
13. Klen, T. J., Vodopivec, B. M., Optimisation of olive oil phe-
nol extraction conditions using a high-power probe ultra-
sonication, Food. Chem. 134 (2012) 2481.
14. Sališová, M., Toma, Š., Mason, T. J., Comparison of con-
ventional and ultrasonically assisted extractions of pharma-
ceutically active compounds from Salvia officinalis, Ultra-
son. Sonochem. 4 (1997) 131.
15. Vilkhu, K., Mawson, R., Simons, L., Bates, D., Applications
and opportunities for ultrasound assisted extraction in the
food industry-A review, Inno. Food. Sci. Emerg. Technol. 9
(2008) 161.
16. Khan, M. K., Albert-Vian, M., Fabiano-Tixier, A.-S., Dan-
gles, O., Chemat, F., Ultrasound assisted extraction of poly-
phenols (flavanone glycosides) from orange (Citrus sinen-
sis L.) peel, Food. Chem. 119 (2010) 851.
17. Wang, H., Provan, G. J., Helliwel, K., Determination of
rosmarinic acid and caffeic acid in aromatic herbs by
HPLC, Food. Chem. 87 (2004) 307.
18. Šic Žlabur, J., Voća, S., Dobričević, N., Brnčić, M., Dujmić,
F., Rimac Brnčić, S., Optimization of Ultrasound assisted
extraction of functional ingredients from Stevia rebaudiana
Bertoni leaves, Int. Agrophys. 29 (2015) 231.
19. Roselló-Soto, E., Galanakis, C. M., Brnčić, M., Orlien V.,
Trujillo, F. J., Mawson, R., Knoerzer, K., Tiwari, B. K., Bar-
ba, F. J., Clean Recovery of Antioxidant Compounds from
Plant Foods, By-Products and Algae Assisted by Ultra-
sounds Processing: Modeling approaches to optimize pro-
cessing conditions, Trends Food Sci. Technol. 42 (2015)
20. Dujmić, F., Brnčić, M., Karlović, S., Bosiljkov, T., Ježek, D.,
Tripalo, B., Mofardin, I., Ultrasound-Assisted Infrared Dry-
ing of Pear Slices: Textural Issues, J. Food. Process. Eng.
36 (2013) 397.
21. Herceg, Z., Brnčić M., Jambrak Režek, A., Rimac Brnčić,
S., Badanjak, M., Sokolić, I., Possibility of application high
intensity ultrasound in milk industry, Mljekarstvo 59 (2009)
22. Brnčić, M., Karlović, S., Rimac Brnčić, S., Penava, A.,
Bosiljkov, T., Ježek, D., Tripalo, B., Textural properties of
infra-red dried apple slices as affected by high power ultra-
sound pre-treatment, Afr. J. Biotech. 9(41) (2010) 6907.
23. Singleton, V. L., Rossi, J. A., Colorimetry of total phenolics
with phosphomolybdic- phosphotungstic acid reagents,
Am. J. Enol. Viticult. 16 (1965) 144.
24. Zhishen, J., Mengcheng, T., Jianming, W., The determina-
tion of flavonoid contents in mulberry and their scavenging
effects on superoxide radicals, Food. Chem. 64 (1999) 555.
25. Brand-Williams, W., Cuvelier, M. E., Berset, C., Use of free
radical method to evaluate antioxidant activity, Lebensm.
Wiss. Technol. 28 (1995) 25.
26. Fecka, I., Turek, S., Determination of polyphenolic com-
pounds in commercial herbal drugs and spices from Lami-
aceae: thyme, wild thyme and sweet marjoram by chro-
matographic techniques, Food. Chem. 108 (2008) 1039.
484 M. DENT et al., Comparison of Conventional and Ultrasound-assisted Extraction…, Chem. Biochem. Eng. Q., 29 (3) 475–484 (2015)
27. Hegedušić, V., Herceg, Z., Rimac, S., Rheological properties
of carboxymethylcellulose and whey model solutions before
and after freezing, Food. Technol. Biotech. 38(1) (2000) 19.
28. Brnčić, M., Ježek, D., Rimac Brnčić, S., Bosiljkov, T., Tripa-
lo, B., Influence of whey protein concentrate addition on
textural properties of corn flour extrudates, Mljekarstvo
58(2) (2008) 131.
29. Herrera, M. C., Luque de Castro, M. D., Ultrasound assist-
ed extraction of phenolic compounds from strawberries to
liquid chromatographic separation and photodiode array
ultraviolet detection, J. Chromatogr. A. 1100 (2005) 1.
30. Herceg, I. L., Jambrak, A. R., Šubarić, D., Brnčić, M., Brn-
čić, S. R., Badanjak, M., Tripalo, B., Ježek, D., Novotni, D.,
Herceg, Z., Texture and Pasting Properties of Ultrasonically
treated Corn Starch, Czech J. Food Sci. 28 (2010) 83.
31. Luque-Garcia, J. L., Luque de Castro, M. D., Ultrasound: A
powerful tool for leaching, Trends. Anal. Chem. 22 (2003)
32. Bosiljkov, T., Tripalo, B., Brnčić, M., Ježek, D., Karlović,
S., Jagušt, I., Influence of High Intensity Ultrasound With
Different Probe Diameter on the Degree of Homogeniza-
tion (variance) and Physical Properties of cow Milk, Afr. J.
Biotechnol. 10(1) (2010) 34.
33. Generalić, I., Skroza, D., Ljubenkov, I., Katalinić, A.,
Burčul, F., Katalinić, V., Influence of the phenophase on the
phenolic profile and antioxidant properties of Dalmatian
sage, Food. Chem. 127 (2011) 427.
34. Lu, Y., Yeap Foo, L., Antioxidant activities of polypolyphe-
nols from sage (Salvia officinalis), Food. Chem. 75 (2001)
35. Zgórka, G., Głowniak, K., Determination of rosmarinic acid
and caffeic acid in aromatic herbs by HPLC, J. Pharm.
Biomed. Anal. 26 (2001) 79.
36. Koşar, M., Dorman, H. J. D., Hiltunen, R., Effect on an
acid treatment on the phytochemical and antioxidant char-
acteristics of extracts from selected Lamiaceae species,
Food. Chem. 91 (2005) 525.
37. Lu, Y., Yeap Foo, L., Polyphenolics of Salvia-A review,
Phytochem. 59 (2002) 117.
... For plant maceration, 1 g of dry sample, ground and weighed on an analytical balance to 4 decimal places, was immersed in 10 ml solvent (pharmaceutical ethyl alcohol:distilled water). Maceration was carried out at room temperature, shielded from sunlight, for 7 days; the first 4 days with continuous stirring for 6 hours at 30 RPM on the Biosan mini-rotator, and the next 3 days without stirring (Dent 2015). ...
... Initially the plant material was hydrated in the solvent for 1 hour and then subjected to microwave irradiation for 3, 5 and 10 minutes at a maximum power of 250 W. Microwave-assisted extraction was performed using the NEOS-GR equipment, Milestone. The final temperature range of the samples was between 54-78°C (Dent 2015). ...
... UAE was carried out using a Hielscher UP200St ultrasonic extraction system under a working amplitude equal to 80% of the maximum rated output power of the device. In order to avoid overheating of the experimental samples and possible destruction of phytocompounds we used a cooling system, extracts were obtained at temperatures below 45°C (Dent 2015;Žlabur et al. 2016). ...
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In this study we targeted the noble metal nanoparticles (MNPs) biogenic synthesis capacity of two medicinal species with therapeutic potential, namely Melissa officinalis L. (lemon balm) and Salvia officinalis L. (sage), cultivated in Romania. Plant material was extracted by maceration, microwave assisted extraction (MAE) and ultrasound assisted extraction (UAE). Bright field scanning transmission electron microscopy and energy dispersive X-ray spectroscopy (BFSTEM-EDS) techniques were used in order to investigate particles shape, dispersion and chemical elemental analysis. The total polyphenol content for both simple extracts and nanostructured mixtures was determined using the Folin-Ciocalteu method and antioxidant activity using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. Identification and quantification of secondary metabolites of M. officinalis and S. officinalis were performed by ultra-high performance liquid chromatography (UHPLC). The Allium assay was used to evaluate the potential cytogenotoxic activity, for both simple and nanostructured phytochemical complexes, in the case of S. officinalis L. species being performed for the first time. Spherical shaped MNPs with diameters of about 20 nm were biosynthesised in lemon balm extracts. Larger AuNPs were phytosynthesized in sage extract obtained by UAE. Compared to the simple extracts, the antioxidant capacity as well as the amount of total polyphenols in the nanostructured extracts decreased, substantiating the involvement of bioorganic material in the reduction of metal ions. Low frequency of chromosomal aberrations corresponding to crude extracts and extracts supplemented with MNPs, suggest the cytoprotective, antigenotoxic, and safe use of these plant species as potential therapeutic forms in various diseases.
... We used the ultrasound-assisted extraction because it is a technique that generally achieves high reproducibility in a short period of time and a high yield of bioactive compounds. Additionally, this technique is characterized by its simplicity, a low temperature during processing, and a reduced consumption of solvents and energy [29]. On the other hand, the disadvantage of this extraction technique is the risk of possible free radicals formation when the ultrasound energy exceeds 20 kHz [29,30]. ...
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... Juntachote et al., showed that lower UAE amplitude application and increased time raised the plant's TPC, which is similar to the current study results [32]. Another research reveals that TPC was raised until a particular time and temperature level, and then the TPC was limitedly decreased as the extraction time and temperature increased [33]. Upadhyay et al., reported that increasing the extraction time increased the phenolic content of the extract, but most of the phenolics were lost as the extraction time increased [34]. ...
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The study aimed to optimize the yield and ultrasonic extraction (UAE) conditions of phenolics from citral genotype basil leaves using response surface methodology (RSM) and to produce a functional beverage using the optimized extract (OE). To develop a functional drink formulation (FDM), OE was enriched with chlorophyll, ascorbic acid, and riboflavin. The model was found to be suitable for accurately predicting experimental results. The optimum conditions were 100% for amplitude, 45 min for extraction time, and 1.06 g/100 mL for plant/solvent ratio. The TPC (mg GAE/g extract), radical scavenging (IC 50 mg/mL), and antidiabetic activities (IC 50 mg/mL) were 262.40, 0.14, and 2.30 for the OE and, 292,176, 0.156, and 1.67 for FDM, respectively. After the enrichment process, the total chlorophyll of FDM was 3 times higher than OE, whereas total carotenoid content was negligible for both. Additionally, FDM was accepted by panelists in relation to overall acceptability, color, texture, flavor, and aroma. The results of the study revealed that OE and FDM have promising antioxidant and antidia-betic activities due to their rich bioactive and fortified compounds. Therefore, FDM can be used to provide health benefits to diabetic patients and future consumers.
... In terms of an environmentally friendly approach and green chemistry principle, nowadays, new, green extraction techniques have become the methods of choice for the isolation of bioactive molecules from plant material [15,16]. Among the green extraction techniques, microwave-assisted (MAE) and ultrasound-assisted extraction (UAE) are widely applied [17][18][19][20][21]. The MAE principle is based on the application of microwave energy, which is absorbed by the moisture present in the matrix, resulting in the swelling and rupture of plant cells, thereby enhancing the release of bioactives [22]. ...
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Black chokeberry juice production results in a large amount of pomace, which is usually regarded as waste. Nevertheless, it contains significant amounts of anthocyanins, which can be utilized as health-promoting components, but also as food colorants. To take advantage of their benefits, green extraction methods such as microwave-assisted (MAE) and ultrasound-assisted extraction (UAE) are widely used for their isolation. This study aimed to evaluate the effects of MAE and UAE parameters (solvent, treatment time, temperature, or ultrasound amplitude) on the extraction yield of anthocyanins from black chokeberry pomace and to compare the effectiveness of these two green extraction methods with conventional reflux extraction, both in terms of total anthocyanins yield and effects on individual compounds. In both techniques, acidification of the extraction solvent did not show a significant effect on anthocyanin content. For MAE, a temperature increase from 40 to 60 °C positively affected the extraction yield, while 4 min was a substantial treatment time for the extraction. Conversely, UAE required 10 min of treatment time with no effect on amplitude. UPLC ESI-MS2 analysis confirmed the presence of 6 anthocyanins in the obtained extracts, with significantly higher levels of cyanidin-3-O-xyloside and cyanidin-3-O-arabinoside were in ones isolated by green extraction techniques.
... The cavities created induce the structural modification of plant tissues, which facilitates the penetration of the solvent into the cells, where the primary and secondary metabolites can dissolve [37]. Several studies demonstrated the efficacy of UAE compared to traditional methods [38,39]. Further studies also highlighted the importance of combining ultrasound with another extraction method, such as pressurized liquid extraction, as a clean and environmentally friendly alternative for extracting phenolic compounds from pomegranate peels [40]. ...
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Colorectal cancer (CRC) is one of the most common cancers in the world. When treating patients, therapeutic agents have side effects; hence, the use of natural compounds found in medicinal plants including pomegranate. Ultrasound assisted extraction (UAE) is a new technique evolving to the detriment of traditional methods such as maceration. In this study, we investigated the antioxidant and anticancer effect of pomegranate peel extracts obtained by maceration and UAE at three different ultrasonic power levels (P1 = 10 W; P2 = 50 W; P3 = 100 W) on HCT-116 colorectal cancer cells. Phytochemical screening highlighted the presence of primary and secondary metabolites in pomegranate peels. In addition, the ethanolic extract obtained by UAE at 50 W was shown to be the most concentrated in phenolic and flavonoid compounds and have the most powerful antioxidant activity, which reached a maximum activity of 92% as determined by DPPH test. Similarly, the MTT cell viability test showed that the extract obtained by UAE at 50 W had the most potent inhibitory effect compared to the other extracts. In conclusion, the UAE at 50 W was shown to be the most suitable and efficient extraction technique to obtain bioactive compounds from pomegranate peel extracts that can be used in the treatment of CRC.
... One alternative is the increasingly common use of sonication as a pretreatment in extraction processes of bioactive compounds in plant matrices [26,27]. The propagation of mechanical ultrasound waves provokes the phenomenon of acoustic cavitation in the sample, which induces a series of compressions and rarefactions in the solvent molecules, leading to bubble formation on the solute surface [28]. These bubbles implode, generating an increased interaction between solute and solvent due to the increased penetrability through the open channels on the sample surface [29]. ...
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The demand for bee products has been growing, especially regarding their application in complementary medicine. Apis mellifera bees using Baccharis dracunculifolia D.C. (Asteraceae) as substrate produce green propolis. Among the examples of bioactivity of this matrix are antioxidant, antimicrobial, and antiviral actions. This work aimed to verify the impact of the experimental conditions applied in low- and high-pressure extractions of green propolis, using sonication (60 kHz) as pretreatment to determine the antioxidant profile in the extracts. Total flavonoid content (18.82 ± 1.15–50.47 ± 0.77 mgQE·g−1), total phenolic compounds (194.12 ± 3.40–439.05 ± 0.90 mgGAE·g−1) and antioxidant capacity by DPPH (33.86 ± 1.99–201.29 ± 0.31 µg.mL−1) of the twelve green propolis extracts were determined. By means of HPLC-DAD, it was possible to quantify nine of the fifteen compounds analyzed. The results highlighted formononetin (4.76 ± 0.16–14.80 ± 0.02 mg·g−1) and p-coumaric acid (<LQ—14.33 ± 0.01 mg·g−1) as majority compounds in the extracts. Based on the principal component analysis, it was possible to conclude that higher temperatures favored the release of antioxidant compounds; in contrast, they decreased the flavonoid content. Thus, the obtained results showed that samples pretreated with 50 °C associated with ultrasound displayed a better performance, which may support the elucidation of the use of these conditions.
Curcumin is a valuable bioactive compound and has attracted the attention of many researchers due to its wide range of medicinal effects. In this paper, curcumin solubility in pressurized hot water (PHW) was determined by static method, and for the first time, molecular dynamics simulation of curcumin solubility in PHW was performed. Molecular dynamics simulation is a powerful method to predict the atomic behavior of different structures. Here, the simulations were performed using the COMPASS force field and Velocity Verlet motion algorithm in the Lammps simulation package. The simulation results were compared with the experimental results. The results showed that the solubility of curcumin in PHW increases with increasing temperature and the solubility at 418.15 K was >230 times its solubility at ambient temperature. Also, the diffusion coefficients of curcumin increased with increasing temperature, and it was found that the tendency of water molecules to surround the curcumin molecule, which occurs through O(OH curcumin)-H(water) interaction, increased with increasing temperature. The Absolute Average Relative Deviation (AARD) between the experimental and simulated results for solubility and density data was 9.07% and 7.85%, respectively. As a result, the molecular dynamics simulation method well predicted the solubility behavior of curcumin in PHW.
Based on a green approach, the potential use of waste tea biomass (fiber and second sieving) with rich polyphenol content was investigated as an alternative source of polyphenol to achieve an economic added value. In addition, this study demonstrated a comparative approach to explore the most sustainable green extraction method by the assessment of single ultrasound-assisted extraction (UAE) at various frequencies (20, 35, and 200 kHz) and the hybrid operations of ultrasound (US) and thermal extraction (50 °C and 80 °C). As a result, it has been determined that waste tea biomass, with a polyphenol extraction rate of more than 80%, provides a higher recovery capacity than tea leaf (the highest polyphenol recovery rate of 72.5%) in almost all single operations. Among the single UAE, 20 kHz was expressed as the method succeeding with high recovery rates (84%) within 30 min for fiber waste. In contrast, the hybrid operation consisting of 20 kHz US (20 min) with heating at 80 °C (10 min) yielded the highest extraction efficiency with 92% in the same time interval more economically for second sieving waste tea biomass. Therefore, this study has shown that it is possible to utilize UAE alone or in combination with heat extraction from tea waste for environmentally friendly, rapid, and effective polyphenol extraction.
Citation: Kurek, M.; Benaida-Debbache, N.; Elez Garofulić, I.; Galić, K.; Avallone, S.; Voilley, A.; Waché, Y. Antioxidants and Bioactive Compounds in Food: Critical Review of Issues and Prospects. Antioxidants 2022, 11, 742.
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The effect of extraction solvents (30, 50 and 70 % aqueous solutions of ethanol and acetone, and 100 % distilled water), extraction temperature (60 and 90 C) and extraction time (30, 60 and 90 min) on composition and mass fraction of polyphenolic compounds of Dalmatian wild sage (Salvia officinalis L.) has been investigated. The total polyhenolic content of sage extracts were determined spectrophotometrically using Folin-Ciocalteu method, whereas the individual polyphenols were determined by HPLC UV/PDA method. Results indicated that the main polyphenols in sage extracts were vanillic, caffeic, syringic, salvianolic K and salvianolic I acids, methyl rosmarinate, 6-hydroxyluteolin-7-glucoside, luteolin-7-glucuronide, luteolin-7-glucoside, apigenin-7-glucunoride, apigenin-7-glucoside and rosmarinic acid and luteolin-3-glucuronide as predominant compounds. The mass fractions of total and individual polyphenols significantly depend on the type of extraction solvent, solvent composition and extraction temperature. The results showed that binary solvent systems are more efficient than mono-solvent systems in the extraction of polyphenolic compounds in regard to their relative polarity. The aqueous solutions of ethanol or acetone (30 %), extraction temperature of 60 C and extraction time of 30 min were the most efficient for the extraction of polyphenols from dry sage leaves.
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The aim of the present study was to reveal an effective extraction procedure for maximization of the yield of steviol glycosides and total phenolic compounds as well as antioxidant activity in stevia extracts. Ultrasound assisted extraction was compared with conventional solvent extraction. The examined solvents were water (100°C/24 h) and 70% ethanol (at 70°C for 30 min). Qualitative and quantitative analyses of steviol glycosides in the extracts obtained were performed using high performance liquid chromatography. Total phenolic compounds, flavonoids, and radical scavenging capacity by 2, 2-azino-di-3-ethylbenzothialozine- sulphonic acid) assay were also determined. The highest content of steviol glycosides, total phenolic compounds, and flavonoids in stevia extracts were obtained when ultrasound assisted extraction was used. The antioxidant activity of the extracts was correlated with the total amount of phenolic compounds. The results indicated that the examined sonication parameters represented as the probe diameter (7 and 22 mm) and treatment time (2, 4, 6, 8, and 10 min) significantly contributed to the yield of steviol glycosides, total phenolic compounds, and flavonoids. The optimum conditions for the maximum yield of steviol glycosides, total phenolic compounds, and flavonoids were as follows: extraction time 10 min, probe diameter 22 mm, and temperature 81.2°C.
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Z. (2010): Texture and pasting properties of ultrasonically treated corn starch. Czech J. Food Sci., 28: 83–93. The effects of high power ultrasound of 24 kHz and ultrasound bath of 24 kHz frequency on the textural and pasting properties of corn starch suspensions was examined. Suspensions were treated with different intensities and treat-ment times (15 min and 30 min) using an ultrasound probe set and bath. The treatments with high power ultrasound probes caused a significant lowering of the starting gelatinisation temperatures of corn starch. The ultrasound treat-ment caused disruption of starch granules by cavitational forces and made the granules more permeable to water. The highest viscosity was observed for the treatment with 300 W probe. Also, a statistically significant increase in solubility in water (20°C) was observed, being caused by the disruption of starch granules and molecules by ultrasound treatment. When applying more powerful ultrasound, starch granules, specifically in the amorphous region, are much more mechanically damaged. The texture profile analyses of the starch gel prepared from the suspensions that had been treated with ultrasound probe presented higher hardness and higher values of adhesiveness and cohesiveness when compared with untreated suspensions or those treated with ultrasound bath. Micrography showed an obvious impact of ultrasound on the structure of starch granules. Ultrasound treatment ruptures and mechanically damages the starch granules causing collapse of cavitation bubbles which induces high pressure gradients and high local veloci-ties of the liquid layers in their vicinity.
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In order to determine the optimal conditions for the extraction of sage polyphenols, the influence of extraction solvent (water, 30 % (by volume) aqueous ethanol, and 30 % (by volume) aqueous acetone), extraction time (3, 5, 7, 9 and 11 min) and microwave power (500, 600, 700 W) on composition and concentration of phenolic compounds of dry wild sage (Salvia officinalis L.) during microwave assisted extraction (MAE) was studied. Optimized MAE method was compared with conventional extraction (CE). Based on the amount of total phenols, microwave power of 500 W and extraction time of 9 minutes were selected as optimal extraction conditions, resulting in higher content of polyphenols when compared with CE. Ethanol and acetone solutions were equally effective extraction solvents, both producing higher extraction capacity than water. Using HPLC coupled with UV/PDA, fourteen polyphenols were identified (caffeic and rosmarinic acids derivates, luteolin- and apigenin- glycosides) with rosmarinic acid and luteolin glycosides in the highest concentrations. The mass fractions of all individual polyphenols were higher in MAE extracts than in CE ones.
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Sagerinic acid, a novel cyclobutane and salvianolic acid K, derived from rosmarinic acid,were isolated together with the parent compound from polar solvent extracts of Salvia officinalis.Their chemical structures were elucidated by NMR and, for sagerinic acid, the stereochemistry ofthe substituents on the cyclobutane moiety was established as 3β,4a-diaryl-1a,2β-dicarboxylicacid diester (m-truxinate form).
Drying is a process frequently used in food industry, often based on the use of conventional methods using heat exchange by conduction or convection. This kind of method may lead to quality loss in structure, texture and sensory characteristics of final products. Consequently, the need for research of new drying methods arises. One of such methods is power ultrasound aided drying. The aim of this work was to investigate the impact of high power ultrasound pre-treatment on drying rate and textural properties of the infra red dried apple slices. Ultrasound device working at a frequency of 24 kHz with a power capacity of 200 W was used for ultrasound pre-treatment. The amplitudes used for ultrasonic pre-treatment were 50 and 100%. The results showed that the use of different amplitudes of ultrasound reduces the time of drying and allows elimination of more water from the apple slices. Usage of 50 and 100% of ultrasonic amplitude in great extent shortened the duration of drying (up to 40%). The results showed that hardness of samples gradually increases (50% amplitude - 97.260 N; 100% of amplitude - 217.90 N) with increase of ultrasound intensity. As a result, hardness of untreated apple slices (41.037N) was significantly lower (p < 0.05).
The main goal of this research is to analyze the influence of ultrasonic probe diameters (7 and 10 mm) of high-intensity ultrasound with constant frequency (30 kHz) on the degree of homogenization (variance) of cow milk. Influence of different probe diameters on the physical properties of cow milk was also tested. Changes in temperature, pH and density were measured under the following operational conditions of the ultrasonic device: amplitude, A = 20, 60,100%; and applied cycle, c = 0.6, 0.8, 1, with various treatment duration, t = 2, 6, 10, 15 min. Obtained results are processed in the "Statistica 8" software. Microscopic images of fat globules were edited in Image J software, while size of fat globules was represented with Log-normal distribution. Statistical analysis was conducted and influence of probe diameters on physical properties was expressed over p-value (p < 0.05) and β- standardized coefficient analyses of variance (ANOVA). Applications of different probe diameter have significant influence on all physical properties and variance. With increase of the amplitude and time, significant influence on variance (degree of homogenization) is observed.
Nowadays, there is an increasing trend to develop new extraction methods by using non-conventional technologies as an alternative to classic solid-liquid extraction. Ultrasounds treatment is an alternative cheap, effective and reproducible method for the improved recovery of bioactive compound extracts from plant foods, by-products and algae. The goal of the current article is to revise the impact of ultrasound-assisted extraction on the recovery of polyphenols, carotenoids and chlorophylls from plant and algae materials. At this stage of development, there is a need to optimize ultrasound treatment conditions based on ultrasonic power, the temperature and the sonication duration in order to maximize the antioxidant capacity of the obtained extract, mainly attributed to bioactive compounds. Thereby, the modeling attempts of this technology were also discussed.
Conference Paper
There has been concern expressed over potential toxicology relating to the long- term effects of commonly used synthetic antioxidants such as butylated hydroanisole (BHA), butylated hydroxytoluene (BHT) and propyl gallate (PG). This has increased the interest in the isolation of natural antioxidants and provides the motivation for this research. The benefit of using ultrasound in plant extraction has already been demonstrated for a number of compounds of interest to both the pharmacology and food industries [1]. The herb Rosmarinus Officinalis is a rich source of antioxidant and was the target for our work on ultrasonically enhanced extraction. The aim was to compare conventional solvent extraction and ultrasonic methods for the isolation of two major antioxidants, carnosic acid and rosmarinic acid, from both fresh and dried leaves of the herb. The solvents used were ethanol, butanone and ethyl acetate and the extractions were performed under conventional and ultrasonic conditions. In general sonication proved to be a more effective extraction technique than using solvent at the same temperature with most of the material extracted within 15 to 30 minutes. Sonication also had the advantage that it reduced the dependence of the extraction efficiency on the solvent. This was particularly important in the case of ethanol (the least expensive solvent) which was a poor solvent under conventional conditions but showed greatly enhanced yields with ultrasound. The extraction of dried herb proved more efficient than extraction of fresh material. We believe that this may be due, in part, to the water present in the latter since extraction of the dried herb proved to be less effective in solvents containing water. Sonication appears to have great potential as a method for the extraction of anti¬oxidant materials with comparable levels of extracted carnosic acid obtained employing either an ultrasonic bath or the more powerful ultrasonic probe. This indicates that there is potential for scale up of the extraction process since the acoustic energy requirements appear to be those provided by a conventional bath system. An additional benefit appears to be that ultrasound both improves the amount of material extracted and reduces the differences in extraction efficiency between the solvents commonly used. 1. M.Vinatoru, M.Toma and T.J.Mason, Ultrasonically assisted extraction of bioactive principles from plants and their constituents, Advances in Sonochemistry, Volume 5, ed. T.J.Mason, JAI Press, 209-248, 1999.