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M. DENT et al., Comparison of Conventional and Ultrasound-assisted Extraction…, Chem. Biochem. Eng. Q., 29 (3) 475–484 (2015) 475
Introduction
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: mbrncic@pbf.hr; 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
Netherlands).
Material
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-
in-7-glucoside.
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 p ≤ 0.05.
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
Temp
(°C)
Time
(min) Total phenols
Phenolic acids, mg/100 g
Flavo noids
Flavone glycosides, mg/100 g DPPH
(mmol
TE kg–1)
rosmarinic
acid
Σ hydroxy-
cinnamic acids
Σ hydroxy-
benzoic acids
Σ luteolin
glycosides
Σ apigenin
glycosides
40
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
50
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
60
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
p-value
Extraction
time NS NS NS NS 0.028019 NS NS NS
Extraction
temperature NS 0.003539 NS 0.000227 NS 0.003452 0.036498 NS
Correlation, r 0.7170 0.5070
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 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
optimum.
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 (p ≥ 0.05). 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 (p ≥ 0.05) (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).
Ultra-
sonic
device
(W)
Time
(min)
Total
phenols
Phenolic acids, mg/100 g Flavone glycosides, mg/100 g DPPH
(mmol TE
kg–1)
rosmarinic
acid
Σ hydroxy-
cinnamic acids
Σ hydroxy-
benzoic acids Flavonoids Σ luteolin
glycosides
Σ apigenin
glycosides
100
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
400
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
p-value
Ultrasonic
device 0.015128 NS NS NS NS NS NS 0.030183
Extraction
time NS NS 0.000204 0.000060 NS NS NS NS
Correlation, r0.6742 0.0980
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).
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.
Extraction
method Solvent type
Phenolic acids, mg/100 g Flavone glycosides, mg/100 g
total phenols rosmarinic
acid
hydroxy-
cinnamic acids
hydroxy-
benzoic acids Flavonoids luteolin
glycosides
apigenin
glycosides
Conven-
tional
extraction
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
Ultra-
sound
extraction
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
p-value
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.
Conclusion
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.
ACKNOWLEDGMENT
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
Abbreviations
CE – Conventional extraction
dm – Dry matter
FL – Flavonoids
TP – Total phenols
RA – Rosmarinic acid
UAE – Ultrasound-assisted extraction
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