Short Communication

Ultrasound-assisted extraction of phenolic compounds from Laurus nobilis L.

and their antioxidant activity
Diana B. Muiz-Mrquez
a
, Guillermo C. Martnez-vila
b
, Jorge E. Wong-Paz
a
, Ruth Belmares-Cerda
a
,
Ral Rodrguez-Herrera
a
, Cristbal N. Aguilar
a,
a
Department of Food Science and Technology, School of Chemistry, Universidad Autonoma de of Coahuila, 25280 Saltillo, Coahuila, Mexico
b
Laboratory of Biotechnology, School of Agronomy, Universidad Autonoma de Nuevo Leon, 66050 Escobedo, NL, Mexico
a r t i c l e i n f o
Article history:
Received 29 September 2012
Received in revised form 25 February 2013
Accepted 25 February 2013
Available online xxxx
Keywords:
Ultrasound
Sonication time
Solid/liquid ratio
Concentration of solvent
a b s t r a c t
Bay leaves (BL) (Laurus nobilis L., Family: Laureceae) are traditionally used to treat some symptoms of gas-
trointestinal problems, such as epigastric bloating, impaired digestion, eructing and atulence. These bio-
logical properties are mainly attributed to its phenolic compounds. In this paper, ultrasound-assisted
extraction of phenolic compounds from Laurus nobilis L. (Laureceae) was studied.
Effects of several experimental factors, such as sonication time, solid/liquid ratio and concentration of
solvent on extraction of phenolic compounds were evaluated through a randomized complete block
design with factorial treatment arrangement (3
3
). The best extraction conditions were: 1 g plant sample
with 12 mL of 35% ethanol, for 40 min, obtaining a yield of phenolic compounds of 17.32 1.52 mg g
1
of
plant. In addition, free radical-scavenging potential of DPPH and lipid oxidation inhibition, by linoleic
acid peroxidation of the selected extract was measured in order to evidence their antioxidant properties.
Results indicated that high amounts of phenolic compounds can be extracted from L. nobilis by ultra-
sound-assisted extraction technology.
2013 Elsevier B.V. All rights reserved.
1. Introduction
Plants of the generous Laurus are shrubs or small trees with
slender twigs, is represented by two species: Laurus azorica and
Laurus nobilis [1]. Laurus nobilis is an evergreen tree, native of the
Mediterranean region; their dried leaves and essential oil are used
as valuable spice and avoring agent in culinary and food industry
[2]. Also, the aqueous extract is used in Turkish folk medicine as
anti-rheumatic, diuretic, as an antidote in snakebites and for treat-
ment of stomachache [3]. These biological activities have been
attributed to a wide range of phytochemical such as: non-polar
avonoids, sesquiterpenoid lactones, isoquinoline alkaloids and
vitamin E, which that could be used as antioxidant and antimicro-
bial compounds [3].
Extraction of antioxidant and antimicrobial compounds from
medicinal plants and spices can be carried out in a variety of ways,
such as traditional extractions (Soxhlet, infusion and maceration),
and supercritical uid extraction. However, although these
methodologies have been employed for decades, it is important
to mention that some of them could be cause degradation of the
targeted bioactive compounds, because of the high temperature
and long extraction times used. Some health risk for analysts be-
cause require relatively large number of toxic solvents [48]. Ultra-
sonic-assisted extraction (UAE) has been proven to be one of the
most important techniques for extracting bioactive compounds
from plants, and it is quite adaptable on a small or large scale,
beside is a potential technology due to the cheaper and fewer
instrumental requirements [9]. Application of UAE offers many
advantages including: less amount of solvent, lower temperatures
and short extraction time, which is very useful for extraction of
thermo-labile and unstable compounds and also increased extrac-
tion yield [9,10]. The mechanism of ultrasonic enhancement is
mainly attributed to behavior of cavitation bubbles upon propaga-
tion of acoustic waves. The collapse of bubbles can produce
chemical, physical, and mechanical effects [11], which result in dis-
ruption of biological cell walls, facilitating release of extractable
compounds and enhancing mass transport of solvent from the
continuous phase into plant cells [9].
The purposes of this study were: (1) to elucidate the inu-
ence of solid/liquid ratio, extraction time and solvent concentra-
tion on yields of extracted polyphenols, (2) to evaluate free
radical scavenging and antioxidant activity of L. nobilis extracts
and (3) to determine the phenolic prole by HPLC of the selected
extract.
1350-4177/$ - see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ultsonch.2013.02.008

Corresponding author. Address: D.I.A. School of Chemistry, Autonomous

University of Coahuila, Blvd. Venustiano Carranza, Col. Repblica, 25280 Saltillo,
Coahuila, Mxico. Tel.: +52 844 4161238; fax: +52 844 4159534.
E-mail address: cristobal.aguilar@uadec.edu.mx (C.N. Aguilar).
Ultrasonics Sonochemistry xxx (2013) xxxxxx
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Please cite this article in press as: D.B. Muiz-Mrquez et al., Ultrasound-assisted extraction of phenolic compounds from Laurus nobilis L. and their anti-
oxidant activity, Ultrason. Sonochem. (2013), http://dx.doi.org/10.1016/j.ultsonch.2013.02.008
2. Materials and methods
2.1. Plant material
Dried leaves of L. nobilis L. (Lauraceae) were purchased from a
local market in Saltillo, Coahuila, Mexico, during November
2010. The plant material was prepared according to the method-
ology reported by Castillo et al. and Osorio et al. [12,13]. Briey,
L. nobilis leaves were milled and sieved (0.60.8 mm particle
size), ne powder was kept in a sealed plastic bag and stored
at room temperature, under darkness to avoid possible oxidation
of compounds.
2.2. Reagents and standards
As extraction solvents, ultrapure water and ethanol-water
mixtures were used. As reagents in this study were used
FolinCiocalteu reagent (2N), sodium carbonate, 1, 1-diphenyl-2-
picrylhydrazyl (DPPH), linoleic acid, EDTA, NaOH and FeCl
2
. Some
standard of phenolic compounds were used during chromatogra-
phy such as pyrogallol (PG), gallic acid (GA), resorcinol (RS),
chlorogenic acid (CHA), methyl gallate (MG), coumaric acid
(CUA), catechin (CAT), 2-hidroxycinamic acid (HA), ellagic acid
(EA), quercetin (QE) and cinnamic acid (CA), all were purchased
from Sigma Aldrich. The solvents acetonitrile (ACN), acetic acid,
methanol and ethanol were of analytical grade.
2.3. Ultrasound assisted extraction
Ultrasound assisted extraction of phenolic compounds from
L. nobilis was performed in an ultrasonic bath (model 2510, BRAN-
SON) with a useful volume of 10 L (internal dimensions: 34.29
10.16 30.5 cm), by employing some extraction variables includ-
ing: solid/liquid ratio 1:4, 1:8 and 1:12 (g mL
1
), time of sonication
20, 40 and 60 (min) and ethanol concentration 0, 35 and 70 (%)
[14]. Ultrasound equipment operated at a frequency of 40 kHz
and at room temperature.
Samples (3 g) were placed in capped tubes (100 mL) with
extracting agent and were submitted to ultrasonic irradiation at
different extraction conditions. The extracts obtained were ltered
(Whatman No. 41 paper) and centrifuged at 3500 rpm during
10 min (HERMLE Z 232 MKII). Then the solvent was eliminated
by evaporation at 60 C for 48 h (Oven LABNET International,
Inc.) and extracts were stored in micro-tubes at 4 C, until used.
Samples were re-suspended in distilled water at concentrations
of 1 g L
1
for determination of total phenolic compounds, antioxi-
dant analysis and phenolic prole by HPLC. All measurements were
performed in triplicate under same conditions and mean
values SD (standard deviation) were reported.
2.4. Experimental design and statistical analysis
A randomized complete block design with factorial treatment
arrangement (3
3
) was applied in this study, to determine the best
combinations of extraction variables on yield of phenolic
compounds. The treatments used in this work are shown in
Table 1. Statistical analysis was performed using the Statistica
7.0 software (Statsoft, Tulsa Ok, USA). Data were analyzed using
analysis of variance ANOVA with a condence level of 95% and to
predict second-order polynomial models to dependent Y variable
through regression analysis. The model proposed for the response
variable of Y was:
y b
0
b
1
x
1
b
2
x
2
b
3
x
3
b
11
x
2
1
b
22
x
2
2
b
33
x
2
3
b
12
x
1
x
2
b
13
x
1
x
3
b
23
x
2
x
3
where y is the predicted response; b
0
is the intercept; b
1
, b
2
and b
3
are the linear coefcients of solid/liquid ratio (x
1
), time of sonication
(x
2
) and ethanol concentration (x
3
), respectively; b
11
, b
22
and b
33
are
the squared coefcient of solid/liquid ratio, time of sonication and
ethanol concentration, respectively; b
12
, b
13
and b
23
are the
interaction coefcients of solid/liquid ratio, time of sonication and
ethanol concentration, respectively. All analyses were performed
in triplicate.
2.5. Determination of total phenolic compounds (TPC)
Total phenolic content was analyzed using the FolinCiocalteu
method described by Oliveira et al. and Ventura et al. [15,16], with
some modications. Briey, 800 lL of sample (1 g L
1
) was mixed
with 800 lL of FolinCiocalteu phenol reagent. After 5 min,
800 lL of Na
2
CO
3
0.01 M and 5 mL of distilled water were added
to the mixture. Absorbance was measured at 790 nm in a spectro-
photometer (Varian 50 Bio). The response variable was the amount
of total phenolic compounds in the extracts which was expressed
as mg gallic acid equivalents (GAE)/g of plant material using a
regression equation and a gallic acid calibration curve (R
2
0.995).
The extract with the highest phenolic content was selected for
further analysis and experiments were done three times.
2.6. Antioxidant activity
Antioxidant activity of L. nobilis extract was evaluated by two
complementary methods: DPPH (1, 1-diphenyl-2-picryl-hydrazyl)
free radical scavenging activity measured according to previously
reported methods [17] and lipid oxidation of linoleic acid using
the methodology reported by Martinez et al. [18].
Table 1
Ultrasonic extraction conditions for recovery of phenolic compounds from L. nobilis
extracts using an experimental design (complete factorial 3
3
).
Run Solid/liquid
ratio (g mL
1
)
Time of
sonication
(min)
Ethanol
concentration
(%)
Yield = mg
GAE/g plant
a
1 1:4 (1) 20 (1) 0 (1) 3.52 0.52
2 1:8 (0) 20 (1) 0 (1) 6.56 0.21
3 1:12 (1) 20 (1) 0 (1) 8.98 0.90
4 1:4 (1) 40 (0) 0 (1) 4.40 0.66
5 1:8 (0) 40 (0) 0 (1) 7.25 2.54
6 1:12 (1) 40 (0) 0 (1) 8.62 2.75
7 1:4 (1) 60 (+1) 0 (1) 3.67 0.44
8 1:8 (0) 60 (+1) 0 (1) 7.25 2.51
9 1:12 (1) 60 (+1) 0 (1) 7.50 3.50
10 1:4 (1) 20 (1) 35 (0) 8.02 2.57
11 1:8 (0) 20 (1) 35 (0) 13.10 4.27
12 1:12 (1) 20 (1) 35 (0) 14.39 2.72
13 1:4 (1) 40 (0) 35 (0) 9.61 1.74
14 1:8 (0) 40 (0) 35 (0) 12.90 2.18
15 1:12 (1) 40 (0) 35 (0) 17.32 1.52
16 1:4 (1) 60 (+1) 35 (0) 8.81 1.59
17 1:8 (0) 60 (+1) 35 (0) 15.80 2.46
18 1:12 (1) 60 (+1) 35 (0) 16.69 0.67
19 1:4 (1) 20 (1) 70 (+1) 6.33 1.02
20 1:8 (0) 20 (1) 70 (+1) 13.14 5.31
21 1:12 (1) 20 (1) 70 (+1) 12.53 3.63
22 1:4 (1) 40 (0) 70 (+1) 8.65 0.78
23 1:8 (0) 40 (0) 70 (+1) 15.05 0.94
24 1:12 (1) 40 (0) 70 (+1) 15.58 0.52
25 1:4 (1) 60 (+1) 70 (+1) 9.26 0.75
26 1:8 (0) 60 (+1) 70 (+1) 14.69 2.58
27 1:12 (1) 60 (+1) 70 (+1) 14.15 1.41
Signicant level at p 6 0.05.
a
Mean values (n = 3).
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Please cite this article in press as: D.B. Muiz-Mrquez et al., Ultrasound-assisted extraction of phenolic compounds from Laurus nobilis L. and their anti-
oxidant activity, Ultrason. Sonochem. (2013), http://dx.doi.org/10.1016/j.ultsonch.2013.02.008
2.6.1. Free radical scavenging activity measured by DPPH
A 60 lM solution of DPPH in methanol was prepared and
2950 lL of this solution were added to 50 lL of extract re-sus-
pended in distilled water (1 g L
1
). The mixture was shaken vigor-
ously and allowed to stand at room temperature for 30 min. Then
absorbance was measured at 517 nm (Varian 50 Bio spectropho-
tometer). The capability to scavenge DPPH radical was calculated
using the following equation:
Inhibition % bAbs
control
Abs
sample
=Abs
control
c 100
2.6.2. Lipid oxidation inhibition assay
Antioxidant activity of L. nobilis extract was also determined by
measuring oxidation of linoleic acid. The linoleic acid solution was
prepared by placing 0.56 g of linoleic acid and 1.5 g of Tween 20 in
8 mL of 96% ethanol. Then, 50 lL of plant extract were mixed with
100 lL linoleic acid solution and 1.5 mL of 0.02 M acetate buffer
(pH 4.0) were added. Sample was homogenized in a vortex. The
obtained emulsion was incubated at 37 C for 1 min, 750 lL of
50 M FeCl
2
solution (0.0994 g FeCl
2
and 0.168 g EDTA diluted to
1 L with distilled water) then were added to an induce lipid
oxidation. After 1 h, an aliquot of 250 lL the mixture in the
reaction was added to 1 mL of 0.1 M NaOH in 10% ethanol and to
stop the oxidation process, 2.5 mL of 10% ethanol were added.
Two aliquots (250 lL) were taken, at 1 and 24 h. Each aliquot
was processing as follows: aliquot was added to 1 mL of 0.1 M
NaOH in 10% ethanol to stop oxidation process and diluted with
2.5 mL of 10% ethanol. Absorbance was measured at 232 nm
against 10% ethanol blank and controls contained 50 lL of distilled
water. The percentage of inhibition in linoleic acid oxidation was
calculated using the following equation:
Inhibition % A B=A 100
where A is the difference between absorbance of control sample
after 24 h and 1 h of incubation, and B is the difference between
absorbance of extract sample after 24 h and 1 h of incubation.
2.7. HPLC analysis
Phenolic prole of the selected extract was performed accord-
ing to the antioxidant properties. The samples were previously l-
tered through a 0.22 lmnylon membranes (Millipore) and injected
in the HPLC system (Varian Pro-Star 330) under the following oper-
ation conditions: 5 lm column Optisil ODS (250 mm 4.6 mm)
and an injector 10 lL at 280 nm. The mobile phase was acetic acid
and acetonitrile (3%) with a ow rate of 10 lL min
1
. All standard
solutions of the tested phenolic compounds were injected in the
same manner [19].
3. Results and discussion
Ultrasound-assisted extraction was conducted in 27 runs to
study the effect of different variables on the amount of phenolic
compounds extracted from L. nobilis. The coded and decoded val-
ues of independent and responses variables are presented in Table
1. Phenolic compounds extracted from L. nobilis ranged from
3.52 0.52 to 17.32 1.52 mg g
1
. These levels indicated consider-
able yields of phenolic compounds which may be used as natural
antioxidant [20]. Table 2 shows the estimated regression coef-
cients of each evaluated variable and their interactions. Regression
model was formulated with the signicant regression coefcients
at 95% condence level. Table 3 shows ANOVA of the tted model.
According to ANOVA and coefcient of determination (R
2
) the
model was adequate between the experimental and predicted val-
ues with 95% of condence level. Pareto chart (Fig. 1) shows effect
of the evaluated variables on extraction of phenolic compounds.
Solid/liquid ratio (x
1
) and ethanol concentration (x
3
) were identi-
ed as the most signicant variables at 95% of condence level,
inuencing phenolic compounds recovery. Both variables showed
positive linear and quadratic effects. However, it was observed that
linear effect of time of sonication was also signicant but with low-
er impact. But interactions between factors did not affect extrac-
tion of phenolic compounds.
To investigate the interactive effects of the evaluated variables
in ultrasound-assisted extraction on yield of phenolic compounds,
three dimensional proles were depicted in this study (Figs. 24).
3.1. Solid/liquid ratio effect
Fig. 2 shows extraction of phenolic compounds as a function of
solid/liquid ratio (x
1
) and time of sonication (x
2
). Results indicated
that effect of solid/liquid ratio (x
1
) on yield of phenolic compounds
of extracts is signicant at 95% of condence level. In addition, this
was the variable that affected more during extraction of phenolic
compounds, because it showed positive linear and quadratics ef-
fects (Fig. 1). In the same gure it can be observed that, when de-
crease the solid/liquid ratio from 1:4 to 1:12 g mL
1
it increases
yield of polyphenols. One of the probable explanation for this phe-
nomenon is that usually usage of larger volume of solvent could
obtain larger amount of bioactive compounds [14], as it could
accelerate diffusion of compounds, which could be favorable for in-
crease polyphenols yield [10]. Our results agree with those re-
ported by Sun et al. [7], who conrmed an increase of yield of
ve isoavones when the solid/liquid ratio was decreased from
1:5 to 1:15 g mL
1
. And interaction between solid/liquid ratio
(x
1
) and time of sonication (x
2
) was not signicant. A lower solid/
liquid ratio when the other factor was xed enhances yield of phe-
nolic compounds from extracts.
Table 2
Regression coefcients estimated for phenolic content extraction from L. nobilis
extracts.
Factor Coefcient estimate Standard error 95% Cl low 95% Cl high
b0 14.73661
a
0.642202 13.45610 16.01713
b1 2.97244
a
0.297282 2.37968 3.56520
b11 1.85794
a
0.514907 2.88463 0.83124
b2 0.62452
a
0.297282 0.03176 1.21729
b22 0.79870 0.514907 1.82539 0.22800
b3 2.86804
a
0.297282 2.27528 3.46080
b33 3.67431
a
0.514907 4.70100 2.64761
b12 0.11787 0.364094 0.84385 0.60811
b13 0.37621 0.364094 0.34977 1.10219
b23 0.56230 0.364094 0.16369 1.28828
a
Signicant factors at p60.05. (R
2
) = 79.34%.
Table 3
Analysis of variance of the regression model parameters.
Source Sum of squares Degrees of freedom Mean square F-value
x
1
477.111
a
1 477.1113 99.97455
x
2
1
62.135
a
1 62.1348 13.01981
x
2
21.062
a
1 21.0616 4.41327
x
2
2
11.482 1 11.4825 2.40605
x
3
444.186
a
1 444.1856 93.07526
x
2
3
243.009
a
1 243.0093 50.92051
x
1
x
2
0.500 1 0.5002 0.10481
x
1
x
3
5.095 1 5.0952 1.06766
x
2
x
3
11.382 1 11.3824 2.38508
Error 338.835 1 4.7723
Total SS 1614.798 80
a
Signicant factors at p60.05.
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Please cite this article in press as: D.B. Muiz-Mrquez et al., Ultrasound-assisted extraction of phenolic compounds from Laurus nobilis L. and their anti-
oxidant activity, Ultrason. Sonochem. (2013), http://dx.doi.org/10.1016/j.ultsonch.2013.02.008
3.2. Time of sonication effect
Fig. 3 shows total phenolic content as a function of time of son-
ication (x
2
) and ethanol concentration (x
3
). Only the linear effect of
time of sonication was signicant but with lower impact than the
other two evaluated variables (solid/liquid ratio and ethanol con-
centration) which were identied as the most signicant variables
inuencing extraction of polyphenols.
The experimental condition that maximized extraction of phe-
nolic compounds was 40 and 60 min of sonication and high etha-
nol concentrations until a midpoint (35%) at a xed solid/liquid
ratio. Yet, the interaction between time of sonication and ethanol
concentration on total phenolic content was not signicant at
95% of condence level. It is also important to note that ultrasonic
irradiation time may vary depending on nature and biological
properties of the vegetal material [21]. Xia et al. [14] evaluated
time of sonication on extraction efciencies of phyllyrin from
Forsythia suspense, these authors reported a gradual increase in
yields of phyllyrin from 0 to 60 min, differences in time of sonica-
tion between plant materials might be ascribed to different avail-
ability and class of extractable components, resulting from the
varied chemical composition of plants [22].
3.3. Ethanol concentration effect
Ethanol concentration effect on the extraction of total phenolic
content was also signicant with positive linear and quadratic ef-
fects (Fig. 1). Response surface plot (Fig. 4) shows total phenolic
compounds as a function of solid/liquid ratio (x
2
) and ethanol con-
centration (x
3
). It is shown that ethanol concentration also has a
strong inuence on yields of phenolic compounds, at low ethanol
concentrations (<30%), phenolic content is affected negatively. In
contrast, when high ethanol concentrations are used until a
midpoint (35%) and a low solid/liquid ratio at a xed time of
Fig. 1. Pareto chart showing evaluated variables. Solid/liquid ratio (x
1
), time of sonication (x
2
) and ethanol concentration (x
3
).
Fig. 3. Response surface graph showing interaction between time of sonication (x
2
/
min) and ethanol concentration (x
3
/%).
Fig. 2. Response surface graph showing interaction between solid/liquid ratio (x
1
/
g mL
1
) and time of sonication (x
2
/min).
4 D.B. Muiz-Mrquez et al. / Ultrasonics Sonochemistry xxx (2013) xxxxxx
Please cite this article in press as: D.B. Muiz-Mrquez et al., Ultrasound-assisted extraction of phenolic compounds from Laurus nobilis L. and their anti-
oxidant activity, Ultrason. Sonochem. (2013), http://dx.doi.org/10.1016/j.ultsonch.2013.02.008
sonication, the extraction of phenolic compounds fromL. nobilis ex-
tracts is enhanced. But interaction between solid/liquid ratio and
ethanol concentration was not signicant at 95%of condence level.
Acetone and alcoholic solvents have been commonly used to
extract phenolic compounds from various vegetal sources. Particu-
larly wateralcohols mixtures have revealed to be more efcient in
extracting phenolic compounds [23]. Also its been indicated that
the polarity of solvent might play an important role in extracting
efciency [14]. Galvan et al. [23] mentioned that high concentra-
tion of ethanol can cause protein denaturation, preventing dissolu-
tion of polyphenols and then inuencing extraction. And our
results are in agreement with the ndings of Sultana et al. [22],
who investigated effects of extracting solvents (absolute ethanol,
absolute methanol, aqueous ethanol and aqueous methanol) on
extraction of phenolic compounds from medicinal plants, obtain-
ing the highest yields with alcoholwater mixtures. In addition,
presence of suitable water in the solvent could increase extraction
yields, since water could be helpful to enhance swelling of plant
material, which is favorable to increase contact surface area be-
tween plant matrix and solvent [10].
According to the surface graphs presented in Figs. 24,
extraction of phenolic compounds was maximized when a low
solid/liquid ratio (code level 1). A medium time of sonication (code
level 0) and medium ethanol concentration (code level 0) were em-
ployed, obtaining an experimental yield of 17.32 mg GAE/g plant
and a predicted yield of 19.34 mg GAE/g plant with the predicted
polynomial model. A high determination coefcient (R
2
0.79) ob-
tained with these results, suggests that the theoretical model is
in concordance with the experimental data. This result showed
UAE process efcacy on extraction of the target compound from
vegetal material due to the advantages produced by ultrasound ex-
plained before. Then, the extract corresponding to the run number
15 was selected for determination of antioxidant activity and phe-
nolic prole by HPLC (Table 1).
3.4. Antioxidant activity
3.4.1. DPPH assay
Due to differences among the wide number of systems avail-
able, results of a single method can give only limited information
about antioxidant properties of extracts [24]. In the present study,
we combined two complementary methods based on the scaveng-
ing of DPPH radical and lipid oxidation inhibition.
According to the obtained results, we can noted that L. nobilis
extract obtained under the best extraction conditions showed
94.73 0.49% DPPH radical scavenging, this correlates with
amount of phenolic compounds present in L. nobilis extract, which
could be responsible for this action, due to high degree of hydrox-
ylation, which is manifested in high capacity to donate protons and
thus stabilize DPPH radical [25]. This is consistent with those re-
sults reported by Elmastas et al. [3], who investigated antioxidant
activity of ethanolic extracts from bay leaves (at 60 lg lL
1
) with
inhibition of 92% of radical DPPH.
3.4.2. Lipid oxidation inhibition assay
In complex systems such as food and food preparations, differ-
ent mechanisms may contribute to oxidative processes, diverse
reactive oxygen species might be generated and various target
structures such as lipids, proteins and carbohydrates can be af-
fected. Therefore, it is important to characterize extracts by a vari-
ety of antioxidant assays [26]. In lipid oxidation inhibition assay,
the general ability of extracts to prevent lipid oxidation was tested;
results showed 73.55 1.91% lipid oxidation inhibition, so bioac-
tive compounds present in L. nobilis extract also have ability to in-
hibit lipid oxidation. Different extracts from this spice have been
previously evaluated for their antioxidant activity and similar re-
sults to our ndings have been reported, Simic et al. [27], deter-
mined antioxidant capacity of methanolic extracts from L. nobilis
based on lipid oxidation inhibition assay and they reported 70.6%
of oxidation inhibition.
Fig. 5. HPLC analysis of an extract obtained by ultrasound-assisted extraction of L. nobilis.
Fig. 4. Response surface graph showing interaction between solid/liquid ratio (x
1
/
g mL
1
) and ethanol concentration (x
3
/%).
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Please cite this article in press as: D.B. Muiz-Mrquez et al., Ultrasound-assisted extraction of phenolic compounds from Laurus nobilis L. and their anti-
oxidant activity, Ultrason. Sonochem. (2013), http://dx.doi.org/10.1016/j.ultsonch.2013.02.008
3.5. HPLC analysis
The HPLC analysis detected and conrmed the presence of two
phenolic acids in L. nobilis extract, peaks on the HPLC chromato-
gram showed a retention times of 14.91 and 18.02 and corre-
sponded to cumaric and 2-hidroxicinamic acid, as shown in
Fig. 5. However, this analysis also revealed two peaks that corre-
sponded to other phenolic compounds not identied which may
contribute to L. nobilis biological activities together with the iden-
tied phenolic acids.
Muchuweti et al. [28] conrmed the presence of caffeic, ferulic
and vanillic acids by HPLC in L. nobilis extracts. And Lu et al. [29]
detected presence of some avonoids, unknown phenolic acids
and rutinin in ethanolic extracts of bay leaves. These differences
in characterization of phenolic compounds from bay leaves ex-
tracts might be due to growing conditions, vegetal specie, pheno-
logical step, and other environmental factors, such as soil and
climatic growth conditions [28]. In addition, different variables
that must be considered in an extraction process, such as solvent,
method and time of extraction are crucial factors in quality and
quantity of the obtained secondary metabolites from plants [5].
4. Conclusions
The results of this study indicated that ultrasound-assisted
extraction is an effective, easy in operation, reliable and feasible
method for extraction of phenolic compounds from vegetal mate-
rials. The best extraction conditions were solid/liquid ratio
1:12 (g mL
1
), and time of sonication 40 (min) with an ethanol
concentration of 35%. In addition, L. nobilis exhibited strong
in vitro antioxidant capacity and can be considered as a good
source of natural antioxidants.
Acknowledgments
Author D.B. Muiz thanks the Mexican Council for Science and
Technology (CONACYT) for the nancial support for the postgrad-
uate program (Master) in Food Science and Technology offered
by the Autonomous University of Coahuila.
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6 D.B. Muiz-Mrquez et al. / Ultrasonics Sonochemistry xxx (2013) xxxxxx
Please cite this article in press as: D.B. Muiz-Mrquez et al., Ultrasound-assisted extraction of phenolic compounds from Laurus nobilis L. and their anti-
oxidant activity, Ultrason. Sonochem. (2013), http://dx.doi.org/10.1016/j.ultsonch.2013.02.008