Industrial Crops and Products 54 (2014) 142148

Contents lists available at ScienceDirect

Industrial Crops and Products
j our nal homepage: www. el sevi er . com/ l ocat e/ i ndcr op
The effect of different extraction techniques on the composition
and antioxidant activity of cherry laurel (Prunus laurocerasus)
leaf and fruit extracts
Ivana T. Karabegovi c
a,
, Sa sa S. Stoji cevi c
a
, Dragan T. Veli ckovi c
b
, Zoran B. Todorovi c
a
,
Nada

C. Nikoli c
a
, Miodrag L. Lazi c
a
a
University of Ni s, Faculty of Technology, 124 Bulevar oslobodjenja St., Leskovac, Serbia
b
College of Agriculture and Food Technology, 1

Cirila i Metodija St., 18400 Prokuplje, Serbia
a r t i c l e i n f o
Article history:
Received 17 September 2013
Received in revised form
27 December 2013
Accepted 30 December 2013
Available online 8 February 2014
Keywords:
Cherry laurel
Extraction techniques
Phenolic compounds
Antioxidant activity
a b s t r a c t
The effects of different extraction techniques, including microwave and ultrasound assisted, classical
and Soxhlet extraction, on the extractive yield, phenolic composition and DPPH-scavenging activity
of cherry laurel leaf and fruit extracts were compared. The total phenolic and avonoid content
was determined according to the FolinCiocalteu and aluminum chloride methods, respectively. The
antioxidant activity of the methanolic extract was evaluated according to the DPPH assay, while the
phenolic composition was determined using the HPLC. The results showed that the extracting techniques
and nature of the plant material signicantly affect the extractive yield and phenolic composition of
the extracts. The different plant material showed signicant differences in the total phenolic content
(119.4 1.1 to 36.2 0.6mg of gallic acid/g of dry extract), the total avonoid content (66.6 0.2 to
12.9 0.2 mg of rutin/g of dry extract) and the antioxidant activity (108.1 7.7 to 271.2 7.6 g/ml)
(p < 0.05). The highest extractive yields for both plant materials (leaves and fruit) were obtained by
the Soxhlet extraction, while the extracts obtained by microwave-assisted extraction contained the
highest amount of phenolic and avonoid compounds and exhibited the best antioxidant activity. High
correlations between phenolic compositions and antioxidant activities of both analyzed extracts were
observed. Independently of the plant material and technique applied, chlorogenic acid was the major
phenolic compound in all the extracts. o-Coumaric acid, quercetin 3-glucoside, luteolin 7-glucoside,
apigenin 7-glucoside, kaempferol 3-glucoside, and naringenin were detected in the leaf extracts for
the rst time, while the presence of vanilic acid, caffeic acid, and rutin was conrmed in the fruit
extracts.
2014 Elsevier B.V. All rights reserved.
1. Introduction
Cherry laurel (Prunus laurocerasus L.), Rosaceae family,
Prunoideae subfamily, is an evergreen shrub or small tree which
owers fromMarch to the rst half of April and bears small cherry
fruits which ripens from July to September. It is native to Asia
Minor, Serbia, Bulgaria, western Europe, the Caucasia, Iran, and
some Mediterranean countries (Kolayli et al., 2003; Sahan et al.,
2012). It is cultivated throughout northern Turkey for its fruits,
and is a popular landscape architecture plant in temperate regions
worldwide (Sulusoglu, 2011).
In Turkish traditional medicine, cherry laurel leaves are used
for its analgesic, antispasmodic, narcotic, and sedative effects

Corresponding author. Tel.: +381 63438585.

E-mail address: ivana.karabegovic@tf.ni.ac.rs (I.T. Karabegovi c).
(Kolayli et al., 2003), as well as for asthma, coughs, and dyspep-
sia treatment (Yes ilada et al., 1999), while its water and ethanolic
extracts showed antifungal (Sahan, 2011), antinociceptive and
anti-inammatory activity without inducing any gastric lesions
(Erdemoglu et al., 2003). However, so far, only three phenolic
compounds, namely: 2-O--d-glucopyranosyl-2-hydroxyphenyl-
acetic acid, (+)-catechin and kaempferol-3-O--d-xylopyranosyl-
(12)-O--d-glucopyranoside have been reported in the extract
of cherry laurel leaves (Akkol et al., 2012). The cherry laurel fruit
and seed are recommended for digestive and respiratory disorders,
bronchitis, eczema, and hemorrhoid treatment (Colak et al., 2005;
Kolayli et al., 2003). The cherry laurel fruit is widely consumed
in the eastern Black Sea region in fresh or dried form, pickled or
processed into jam, juice, marmalade, and alcoholic drinks (Liyana-
Pathirana et al., 2006; Kolayli et al., 2003). The nutritional and
pharmaceutical value of this fruit stems from its phenolic acids,
where chlorogenic, vanillic, caffeic and benzoic acid are the most
0926-6690/$ see front matter 2014 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.indcrop.2013.12.047
I.T. Karabegovic et al. / Industrial Crops and Products 54 (2014) 142148 143
prevalent (Ayaz, 2001; Alasalvar et al., 2005), withfructose, glucose,
sorbitol (Var and Ayaz, 2004), mannitol, ascorbic acid, dietary ber,
antocyanins, some mineral (Kolayli et al., 2003) and tannin con-
tent (Colak et al., 2005). Some of these compounds are well-known
antioxidants.
Interest for bioactive compounds of a natural origin with a
high antioxidant capacity has increased considerably in the past
two decades, mostly due to their potential for the treatment and
prevention of cancer, cardiovascular, chronic and neurodegenera-
tive diseases (Gil-Chvez et al., 2013). Therefore, nding the most
efcient extraction and separation method, as well as the full
characterization of obtained bioactive compounds from natural
matrices are a major challenge for researchers in the food, phar-
maceutical, and cosmetic industry.
The extraction efciency of bioactive components from plant
materials is affected by different factors, such as the extraction
techniques, solvents, time, temperature, solvent-to-plant material
ratio and many others. However, a suitable extracting method and
solvent are crucial to ensuring an efcient extraction of the targeted
nutraceuticals fromplant material (Goli et al., 2005). Conventional
extraction methods, such as Soxhlet and classical extraction (mac-
eration), are despite high solvent, time and energy consumption
still in widespread use. However, over the last decades, some
novel extraction techniques, including ultrasound and microwave-
assisted extraction, have been developed as energy-saving and
environment-friendly processes with cost-effective production of
high quality extracts (Wang and Weller, 2006). Theoretically, the
optimal extraction method should be simple, safe, reproducible,
inexpensive and suitable for industrial application (Vongsak et al.,
2013).
The aims of the current study were to compare the extraction
yields, chemical composition, antioxidant activities, total pheno-
lic, and avonoid content of cherry laurel leaf and fruit extracts,
obtained by classical (CE), Soxhlet extraction (SE), ultrasound
(UAE), and microwave (MWAE) assisted extraction, despite the
widespread use of cherry laurel in traditional medicine, a proper
biochemical characterization of leaf extracts, as well as the effects
of the extractiontechniques onthe cherry laurel leaf or fruit extract
composition has not been reported and compared yet.
2. Materials and methods
2.1. Materials
Cherry laurel (Prunus laurocerasus L.) leaf andfruit samples were
harvested (August 2012) in the Zeleni cje Nature Reserve (moun-
tain Ostrozub, southeastern Serbia, 42

52

N 22

14

E) where the
association Lauroceraso-Fagetum, a rare and specic relict commu-
nity of cherry laurel with beech, is under the special protection of
the Institute for Nature Conservationof Serbia from1950. The plant
material was identied by Prof. N. Randjelovic (Department of Biol-
ogy and Ecology, University of Ni s, Serbia). Fresh leaves (separated
fromtwigs) and edible fruit parts (without the stones) were stored
at 4

C until further uses (no longer than 3 days). The moisture con-
tent determined immediately after harvesting, by drying at 105

C
to a constant weight, was 61.92.9% and 77.43.2% for the leaves
and fruit, respectively.
Methanol (HPLC grade) was obtained from J.T. Baker (Deven-
ter, Holland), FolinCiocalteus reagent, DPPH (2,2-diphenyl-1-
picryhydrazyl), quercetin, quercetin 3-glucoside, luteolin, lute-
olin 7-glucoside, apigenin, apigenin 7-glucuronide, apigenin
7-glucoside, rutin, kaempferol, kaempferol 3-glucoside, myricitrin
and naringenin were purchased fromSigma Chemical Co. (St. Louis,
MO, USA). All the other chemicals were analytical grade chemicals
fromMerck (Darmstadt, Germany).
2.2. Extraction of plant material
2.2.1. Microwave assisted extraction (MWAE)
Milled fresh cherry laurel leaves or fruit (40g) were extracted
using methanol (400ml). The MWAE was performed in an elec-
trically modied microwave oven (SAMSUNG, type M1712N,
Malaysia) at 300W and for 15min. The extraction system has
reached solvent (methanol) boiling temperature for less than 4min
at mentioned operational conditions. At the end of the extraction,
the liquid extract was separated fromthe solid residue by vacuum
ltration and evaporated to dryness on a rotary vacuum evapora-
tor at 40

C. The extracting solvent, plant material-to-solvent ratio,

and extraction time were chosen on the basis of our previous work
where we successfully employed response surface methodology
to optimize the operational conditions of the microwave-assisted
extractionof cherry laurel leaves (Karabegovi c et al., 2013) andfruit
(Karabegovi c et al., 2014).
2.2.2. Ultrasound assisted extraction (UAE)
Samples of milled fresh cherry laurel leaves or fruit (40g) were
extracted with methanol (400ml). Sonication was performed for
15min inanultrasonic cleaning bath(Sonic, Ni s, Serbia; total nomi-
nal power: 350W; and frequency: 40kHz). The temperature was
controlled and maintained at 65

C (1

C) by water circulating
froma thermostated bath by means of a pump. Separation and fur-
ther treatment of the ltrates were the same as described in the
previous section.
2.2.3. Classical extraction (CE)
Milled fresh cherry laurel leaves or fruit (40g) with methanol
(400ml) were taken to an Erlenmayer ask placed in a ther-
mostated water bath for 15min. The temperature was maintained
at 651

C. Additional agitation or shaking was not employed. Dry

extracts were obtained using the same procedure as described in
the Section 2.2.1.
2.2.4. Soxhlet extraction (SE)
Milled fresh cherry laurel leaves or fruit (40g) and methanol
(400ml) were extracted by the Soxhlet apparatus for a period of
120min (twelve extraction cycles). Previously, we have found that
the yield of extractive substances did not increase after the twelfth
cycle. The liquid extract was evaporated by a rotary vacuumevap-
orator to a constant mass.
2.3. The quantitative estimation of phenolic compounds and the
antioxidant activity of the extracts
Widely accepted spectrophotometric methods involving the
FolinCiocalteu reagent and aluminum chloride were used for
rapid quantication of the total phenol (TPC) and avonoid (TFC)
compounds, respectively. The determination of the free radical
scavenging activity was measured according to the DPPH method.
All tree methods are rapid, simple and reproducible and can be
founddescribedindetail elsewhere (Stanisavljevi c et al., 2008). The
TPCand TFCwere expressed as mg of gallic acid/g of dry extract and
mg of rutin/g of dry extract, respectively, while EC
50
values were
calculated according to the experimental data by using the sigmoid
non-linear regression model.
2.4. HPLC analysis
The HPLC analyses of the extracts were performed on an Agi-
lent 1100 Series HPLC system (Agilent Technologies) consisted
of micro vacuum degasser, binary pump, thermostated column
compartment and variable wavelength detector under previously
144 I.T. Karabegovic et al. / Industrial Crops and Products 54 (2014) 142148
described conditions and procedures (Viet et al., 1995). The Agi-
lent Eclipse column XDB-C18 4.6mm ID150mm (5m) was
used. The mobile phase was composed of solvent (A) 0.15% (w/v)
phosphoric acid in H
2
O:MeOH (77:23, v/v, pH=2) and solvent (B)
methanol as follows: isocratic 03.6min 100% A+0% B; linear gra-
dient for 24min 80.5% A+19.5% B; isocratic with 80.5% A+19.5% B
up to 30min; linear gradient for 60min 51.8% A+48.2% B; linear
gradient for 67.2min 0% A+100% B; followed by isocratic elution
with 100% B for the last 5min at room temperature at ow rate
of 1ml/min, and UV detection at 350nm. The injection amount
was 20l of extracts solution made by diluting dry extracts with
methanol (5mg/ml) and ltered through Econolter, 25/0.45m,
RC (Agilent Technologies). The phenolic compounds present in
the extracts were identied by comparing their retention times
in HPLC chromatogram with the previously published ones (Viet
et al., 1995) and with commercial standard compounds (exter-
nal standard method). The concentrations of analyzed compounds
present in the samples were calculated by introducing the areas
of peaks into the corresponding calibration curves. Those curves
were made with standard solutions of all standards prepared in
methanol (1.0mg/ml) and diluted of at least 5 concentration lev-
els ranging from5 to 200g/ml which were injected into the HPLC.
The corresponding peak areas were plotted against concentrations.
Statistical analysis showed that all standard solutions have good
linearity within the concentration range examined in this work,
as shown by the high correlation coefcients (R
2
>0.9997). The
amounts of phenolic compounds in the extracts were calculated
as mg/g of dry extract, on the basis of peak areas and by using
calibration curves constructed for each standard.
2.5. Statistical analysis
All of the experiments were repeatedthree times andthe results
are expressed as the mean value standard deviation. Statisti-
cal comparisons were made using one-way analysis of variance
(ANOVA) followed by Tukeys HSD post hoc test for multiple com-
parisons (SPSS, version 17). Differences were considered to be
signicant at p<0.05.
3. Results and discussion
3.1. Extractive yield
The extractive yield from fresh cherry laurel leaves and fruit
obtained by MWAE, UAE, CE and SE are shown in Fig. 1.
As can be seen, the extraction techniques strongly affect the
extractive yields which varied from21.3 to 38.6 and 21.6 to 42.8g
ES/100g of dry plant material in the case of leaves and fruit,
respectively. Statistically signicant differences (p<0.05) between
extractive yields obtained by different extraction techniques were
observed for both plant materials. SE showed the best extraction
efciency independently of the plant material, but for an extrac-
tion time which was eight times longer. Considering that the SE
was performed until complete exhaustion of the plant material,
these yields were accepted as the total content of ES in the leaves
(38.61.22g/100g) and fruit (42.80.74g/100g). Independently
of the plant material, an average ES recovery (extraction efciency
with respect to the total content of ES) of 70% was achieved for
MWAE, while 64% and about 53% were observed for UAE and CE,
respectively, showing that MWAE gave the second highest ES yield.
Since the extracting solvent, plant material-to-solvent ratio, time,
temperature, systemvolume, moisture content in each of the plant
materials were the same for MWAE, UAE and CE, it can be con-
cluded that microwave effects should be responsible for extractive
yield enhances, while the mass transfer limitation in CE compared
to MWAE and UAE causes the lowest recovery percent. The lowest
extractive yield obtained by CE was also expected because, MWAE
and UAE, as relatively novel extraction techniques, were created in
order to improve the recovery of nutraceuticals in comparison to
conventional solvent extractionmethods (Wang andWeller, 2006).
The enhancement of extraction with ultrasonic power is due to the
intensication of mass transfer and solvent penetration into plant
material, as well as cell disruption (Vinatoru, 2001), while a major
benet from microwaves is the rapid, efcient and homogeneous
heating of the total extraction system volume which also results
in the expansion and rupture of cell walls and increased solvent
penetration (Kaufmann and Christen, 2002).
Similar results were observed in the extraction of phenolic con-
stituents fromsea buckthorn (Hippophae rhamnoides) leaves, fruit,
pulp and seed (Sharma et al., 2008), anthraquinones fromMorinda
citrifolia root, the total ES and phenolic compounds fromPinus radi-
ata bark (Asp and Fernndez, 2011), where the SE achieved the
highest extraction yield, followed by MWAE, UAE, while CE gave
the lowest ES yield.
In a previous study Erdemoglu et al. (2003), reported that the
two-stage CE of the cherry laurel leaf at room temperature, with
water and 96% ethanol, yielded 38.1 and 29.3g ES/100g of plant
material, respectively, although there are no reliable data on the
extraction time and the moisture content in the plant material,
while Akkol et al. (2012) obtained 39.3g/100g plant material by
CE with 96% ethanol, at room temperature for 24h. Powdered
air-dried fruit and leaves were submitted to successive solvent
extraction sequentially with dichloromethane, ethyl acetate, ace-
tone, methanol, and distilled water at room temperature for 48h.
In the case of leaves, the highest extractive yield was obtained by
water (16.97w/w %), while for the fruit, methanol gave the best
results (13.52w/w %) (Orhan and Akkol, 2011). Celep et al. (2012)
reported a yield of 13.72% for cherry laurel fruit extracted with 80%
Fig. 1. The extractive yield obtained by different extraction techniques fromcherry laurel leaf (a) and fruit (b).
I.T. Karabegovic et al. / Industrial Crops and Products 54 (2014) 142148 145
Table 1
Total phenol (TPC) and avonoid (TFC) contents in cherry laurel leaf and fruit extracts obtained by different extraction techniques.
*
Extraction technique TPC, mg of gallic acid/g of dry extract TFC, mg of rutin/g of dry extract
Leaf extract Fruit extract Leaf extract Fruit extract
MWAE 119.4 1.1
a
46.3 1.4
a
66.6 0.2
a
13.7 0.5
a
UAE 114.7 1.7
b
42.2 1.8
b
64.4 0.3
ab
13.5 0.4
a
CE 116.0 2.2
ab
41.3 1.3
b
63.5 0.9
bc
13.4 0.2
a
SE 85.4 1.4
c
36.2 0.6
c
61.3 1.6
c
12.9 0.2
a
*
Results were expressed as the mean of triplicates standard deviation and the values with different superscript letters within a column were signicantly different
(Tukeys HSD test, p<0.05).
methanol, at 45

C for 4h with continuous stirring, while Kolayli

et al. (2003) reported that water provided an extractive yield of
2.370.4g/100g of fresh plant material. However, we did not nd
any report on the extractive yields fromthe cherry laurel leaves or
fruit using MWAE and UAE to compare with our current data.
Furthermore, extraction yield also signicantly depended on
the moisture content and nature of the plant material. It can be
seen that, independently of the extraction techniques, the extrac-
tive yield from cherry laurel fruits was higher than the extractive
yield fromthe leaves. Differences of extractive yields among differ-
ent plant parts could be ascribed to the different compounds which
may be present in each part of the same plant, as well as to the
extraction ability of methanol for its recovering capacity (Shabir
et al., 2011). Likewise, other researchers also reported different
extractive yields from different parts of the same plants (Barreira
et al., 2008; Ozsoy et al., 2013; Sajid et al., 2012).
3.2. Total phenolic compounds and antioxidant activity
The quality of the extract is estimated in terms of TPC, TF and
antioxidant activity (AA). The TPC and TFC determined in cherry
laurel leaf and fruit extracts are shown in Table 1.
The TPC varied from61.31.6 to 119.41mg of gallic acid/g of
dry extract, while the TFC ranged from 12.90.2 to 66.60.2mg
of rutin/g of dry extract, depending on the applied extraction tech-
nique or the plant material. These results were in good agreement
with the ndings of Orhan and Akkol (2011) that the TPC of leaf and
fruit methanolic extracts, obtained by CE at roomtemperature for
48h, were 113.450.71mg/g of dry extract and 64.633.25mg/g
of dry extract, respectively. However, the ndings of the current
study do not support the previous results that the TFC of methano-
lic extract fromcherrylaurel fruit andleaves couldnot becalculated
due to trace amount (Orhan and Akkol, 2011), that the TPC of water
extract from cherry laurel fruit was 10.42.3mg/100g of water-
soluble extract (Kolayli et al., 2003), as well as the results published
by Celep et al. (2012), that TFC of 80% methanolic extract from
cherrylaurel fruit, obtainedat 45

Cfor 4hwithcontinuous stirring,

was 16.870.38mg quercetin/g extract. The observed differences
in the results can be explained with differences in the extraction
conditions, type of extraction solvent, as well as with environmen-
tal factors, plant varieties, age of the trees, maturity level of the
fruit, post-harvesting conditions or storage (Kolayli et al., 2003).
Further, the results indicated that TPC and TFC varied signi-
cantly among different plant parts (p<0.05), revealing that cherry
laurel leaves were richer in methanol soluble phenolic compounds
than the fruit, which is consistent with the ndings of Orhan and
Akkol (2011). Many studies have also conrmed that leaf extracts
have higher concentrations of phenolic compounds than other
parts of the same plant (Belkhir et al., 2013; Stankovi c, 2002; Vagiri
et al., 2012), but considering that different plants or parts of the
same plant may synthesize and accumulate different secondary
metabolites or different amounts of a specic compound there are
lot of examples where these compounds are concentrated in the
seed, root, peel or fruit (Brahmi et al., 2013; Sultana et al., 2012).
Generally, among different extraction techniques tested in this
study and independently of the plant material, the MWAE showed
slightly higher, while SE showed the lowest values for TPC and TFC
thanthe other techniques. Inextracts obtainedby means of the UAE
and CE, the content of these bioactive compounds, independently
of the plant material, did not vary signicantly (p<0.05). Simi-
larly, many researchers have conrmed the MWAE efciency for
phenolic extraction compared to traditional extraction techniques
(Beejmohun et al., 2007; Casazza et al., 2010; Dhanani et al., 2013;
Pan et al., 2003). Further, taking into consideration the solvent
consumption and time needed for extraction, the MWAE could be
recommended as a rapid and efcient extraction technique despite
the fact that in some cases it could give a lower yield of TPC and
TFC than conventional extraction techniques (Biesaga, 2011; Kalia
et al., 2008). For the extraction of bioactive phenolics frompropo-
lis, the MWAE was found to give higher yield in the shortest time,
compared to the CE and UAE, but due to low extraction selectiv-
ity, the UAE was chosen as the most efcient extraction method
in this case (Trusheva et al., 2007). However, in the case of some
plant material, the UAE was less effective than the CE for phenolic
compounds extraction. This was explained by the oxidation and
degradation of the some bioactive compounds under sonication
of the aqueous solution which results in highly reactive hydroxyl
radicals (Stanisavljevi c et al., 2008; Vinatoru, 2001).
The DPPH method is recommended as a simple and rapid
screening method for obtaining basic information about the AA
of the extracts. It is a commonly and widely used method despite
some disadvantages (Prior et al., 2005; Snchez-Moreno, 2002). An
efcient concentration or EC
50
parameter, dened as the concen-
tration of the sample required to decrease the initial concentration
of DPPH to 50%, has been introduced for the interpretation of the
results, where lower EC
50
values means higher AA.
As can be seen from Table 2 that AA varied signicantly based
on plant material, while the extraction technique has a smaller
inuence. Cherry laurel leaf extracts showed more than twice the
amounts of AA than those in the fruit extracts, while all the tested
extracts showed moderate to strong AA with respect to the refer-
ence compound, Trolox (EC
50
=82.871.67g/ml).
Consistent with our results, the cherry laurel leaf extracts were
foundtohave a better AAthanfruit extracts for all the usedsolvents
(dichloromethane, ethyl acetate, acetone, methanol, and distilled
water) and sample concentrations (0.5, 1 and 2mg/ml) (Orhan and
Table 2
Antioxidant activity (AA) of cherry laurel leaf and fruit extracts obtained by different
extraction techniques.
*
Extraction technique EC
50
, g/ml
Leaf extract Fruit extract
MWAE 108.1 7.7
a
236.9 3.1
a
UAE 117.9 3.5
a
245.7 3.4
b
CE 115.4 2.8
a
237.2 4.6
a
SE 124.5 1.9
b
271.2 7.6
c
*
Results were expressed as the mean of triplicates standard deviation and val-
ues with different superscript letters within a column were signicantly different
(Tukeys HSD test, p<0.05).
146 I.T. Karabegovic et al. / Industrial Crops and Products 54 (2014) 142148
Akkol, 2011). In addition, some previous results also reported high
antioxidant activity for methanolic extracts of cherry laurel fruit
(Karahalil and S ahin, 2011; Halilova and Ercisli, 2010).
The AA from the cherry laurel fruit in the present study were
higher than 79515.8g/ml that were reported for 80% methano-
lic extracts obtained fromthe cherry laurel fruit (Celep et al., 2012).
These results indicate that AA is inuenced by environmental fac-
tors, type of plant parts, maturity level, and harvesting stage, as
has previously been conrmed on the example of the Olea europaea
leaves and fruit (Brahmi et al., 2013).
Then, comparing the AA of cherry laurel leaf and fruit extracts
obtained by the MWAE, UAE and CE, we found that the values
were comparable, while the extracts obtained using SE showed
statistically signicant differences when compared to the extracts
obtained by the other three used techniques. This might be due to
the slight decomposition of the compounds with AA under longer
treatment andintenseheating. But despitetheobserveddifferences
for the AAof the tested extracts, the results are still very similar and
indicate that different extraction techniques did not cause serious
changes in the phenolic content of the extracts.
According to our literature survey, this is the rst report of
TPC, TFC and AA comparison of cherry laurel leaf and fruit extracts
obtained by different extraction techniques. On the basis of these
results, the MWAE was found to be the most convenient tech-
nique for extracting antioxidants from cherry laurel leaves and
fruit, as well as that the effect of the extraction techniques on the
AA and content of TPC or TFC in the extracts depends mostly on
the type, structure and complexity of the plant material, solvent
types, extraction conditions, type of target extract and differences
in the availability of laboratory equipment. However, the choice
of one best extraction technique for all plant material is unjusti-
ed, as even their ranking according to strengths and weaknesses
is difcult (Khoddami et al., 2013).
As shown in Fig. 2, a high correlation is observed between the
AA and TPC and TFC with a correlation coefcient of 0.945 and
0.985, respectively. Many researchers also reported highlinear cor-
relations between these values (Kalia et al., 2008; Tamuly et al.,
2013; Xu and Chang, 2007) conrming that the radical scavenging
activity of plant extracts depends on the amount of polyphenolic
compounds in the extracts.
3.3. The phenolic composition of cherry laurel extracts
The phenolic proles of methanolic extracts from cherry lau-
rel leaves and fruit were analyzed using HPLCDAD (Figs. 3 and 4)
Fig. 2. The correlation between antioxidant activity and total phenolic (black
squares) and avonoid (open squares) compounds in cherry laurel leaf and fruit
extracts obtained by different extraction techniques.
and the results of the identied compounds are summarized in
Tables 3 and 4. A total of 10 compounds were identied by a com-
parison with reference standards, while for the remaining peaks in
the chromatograms, due to a lack of reference compounds, deni-
tive identication was not carried out.
Independently of the plant materials and extraction techniques,
the most common component in the cherry laurel leaf and fruit
extracts was chlorogenic acid, ranging from25.44 to 36.49mg/g of
dry extract, although the o-coumaric acid, quercetin 3-glucoside,
luteolin7-glucoside, apigenin7-glucoside, kaempferol 3-glucoside,
andnaringeninwere present inthe leaf extracts, while vanillic acid,
caffeic acid, and rutin were detected in fruit extracts. By compar-
ing the phenolic compositions of leaf and fruit extracts obtained
by various recovery techniques, the highest content of most of the
identied phenolic compounds were detected in extracts obtained
by MWAE, except for apigenin 7-glucoside and vanillic acid whose
contents were reduced by about 40% when microwaves were used.
A high content of some phenolic compounds in extracts obtained
by MWAE could be attributed to the better increases of tempera-
tureinsidetheplant cells under microwavetreatment whichcauses
the destruction of cell walls and the release of cell content into
the extraction system. The lowest yield of chlorogenic acid in the
extracts obtained by the CE, from both plant materials, could be
due to the degradation at higher temperature for a long time.
Fig. 3. HPLC chromatogramof cherry laurel leaf extract obtained by UAE.
Fig. 4. HPLC chromatogramof cherry laurel fruit extract obtained by MWAE.
I.T. Karabegovic et al. / Industrial Crops and Products 54 (2014) 142148 147
Table 3
The chemical composition of cherry laurel leaf extracts obtained by different extraction techniques.
Compound Pick number Retention time (min) mg/g of dry extract
MWAE UAE CE SE
Chlorogenic acid 1 11.496 30.44 28.67 32.30 25.44
o-Coumaric acid 2 15.907 0.92 0.58 0.53 0.85
Quercetin 3-glucoside 3 24.214 0.58 0.40 0.58 0.63
Luteolin 7-glucoside 4 29.301 22.78 13.57 18.70 14.87
Apigenin 7-glucoside 5 37.815 7.11 9.95 11.62 8.48
Kaempferol 3-glucoside 6 40.564 0.51 0.37 0.35 0.40
Naringenin 7 45.820 0.11 0.09 0.04 0.05
Table 4
The chemical composition of cherry laurel fruit extracts obtained by different extraction techniques.
Compound Pick number Retention time (min) mg/g of dry extract
MWAE UAE CE SE
Chlorogenic acid 1 11.476 35.21 35.17 36.49 30.39
Vanillic acid 2 15.927 1.19 1.58 1.49 1.89
Caffeic acid 3 25.097 1.69 1.12 1.25 0.21
Rutin 4 33.794 0.98 0.81 0.69 0.40
Chlorogenic acid degradation at higher temperatures was previ-
ously shown in the case of phenolic compound extraction from
defatted and ground robusta cherry coffee (Upadhyay et al., 2012).
Our results are in agreement with previous research which has
shown that chlorogenic acid was the main phenolic acid in the
extracts of the cherry laurel fruit (Alasalvar et al., 2005; Karahalil
and S ahin, 2011). Besides chlorogenic acid, other free phenolic
acids (protocatechuic, p-hydroxybenzoic acid, vanillic acid, caffeic
acid, syringic acid, and p-coumaric acid) were identied and quan-
tied in the fruit extract, among which syringic and p-coumaric
acid were present in higher amounts (Alasalvar et al., 2005). Ayaz
et al. (1997) found the same phenolic acids in the fruit of culti-
vars and wild cherry laurel form, but with vanillic acid as the major
compound present in all forms. The highest content of phenolic
acid in the fruit of the cultivar Laurocerasus ofcinalis Oxygemmis
was found for benzoic, followed by caffeic and vanillic acids (Ayaz,
2001). Furthermore, except chlorogenic acid, which was the pre-
dominant phenolic compound in the extracts of the cherry laurel
fruit, hydroxybenzoic acid, vanillic acid, catechin, protocatechuic
acid and p-coumaric acid were also present in high concentrations,
while syringic acid, ferulic acid, rutin and gallic acids were detected
in small amounts. However, caffeic acid, benzoic acid, o-coumaric
acid, abscisic acid, trans-cinnamic acid were not detected in the
same methanolic extracts of the cherry laurel fruit (Karahalil and
S ahin, 2011). The differences between the current and previous
results conrmed the fact that many factors, such as the grow-
ing season, variety, environmental and climatic conditions, plant
disease, soil type, geographic locations, and even maturity could
inuence the concentration and variability of phenolic compounds
within the same fruit type (Sellappan et al., 2002).
Up to now, in the extract of cherry laurel leaves, just
three phenolic compounds (2-O--d-glucopyranosyl-2-hydro-
xyphenyl-acetic acid, kaempferol-3-O--d-xylopyranosyl-(12)-
O--d-glucopyranoside and (+)-catechin) were identied (Akkol
et al., 2012).
4. Conclusion
Several extraction techniques such as MWAE, UAE, CE and SE
were compared for cherry laurel leaf and fruit extraction on the
basis of theextractiveyields, antioxidant activities andextract com-
position. The results showed that the extraction techniques and
nature of the plant material strongly affect the extractive yields
(p<0.05). The SE achieved the highest extraction yield but for an
eight-fold longer extraction time, followed by MWAE ensuring the
extractive yield of 70% of the maximal yield, while the CE gave
the lowest yield. The maximal total phenol (119.41.1mg of gal-
lic acid/g of dry extract), avonoid (66.60.2mg of rutin/g of dry
extract) content and antioxidant activity (EC
50
=108.17.7g/ml)
were studied for the extract of cherry laurel leaves obtained by
means of MWAE. Polyphenolic compositions of the extracts from
thesameplant material obtainedbydifferent extractiontechniques
were found to be similar to each other. Chlorogenic acid was the
major phenolic compoundintheextracts frombothplant parts, and
variedfrom25.44to36.49mg/gof dryextract. Another sixpolyphe-
nolic compounds were detected and quantied in the leaf extracts
of cherry laurel: chlorogenic acid, o-coumaric acid, quercetin 3-
glucoside, luteolin 7-glucoside, apigenin 7-glucoside, kaempferol
3-glucoside, and naringenin, while vanillic acid, caffeic acid, and
rutin were detected in the fruit extracts. High antioxidant activity
and a high content of phenolic compounds makes the cherry laurel
very interesting and valuable as a source of antioxidant and bioac-
tive compounds. Further investigations are needed to complete the
identication of cherry laurel leaf and fruit phenolic proles. In
addition, the MWAE has proven to be more acceptable than other
techniques for economic and environmental reasons.
Acknowledgement
This project was supportedbythe Ministryof Education, Science
and Technological Development of the Republic of Serbia, project
OI 172047.
References
Akkol, E.K., Krmzbekmez, H., Kc kboyac, N., Gren, A.C., Yesilada, E., 2012. Iso-
lation of active constituents from cherry laurel (Laurocerasus ofcinalis Roem.)
leaves through bioassay-guided procedures. J. Ethnopharmacol. 139, 527532.
Alasalvar, C., Al-Farsi, M., Shahidi, F., 2005. Compositional characteristics andantiox-
idant components of cherry laurel varieties and pekmez. J. Food Sci. 70 (1),
S47S52.
Asp, E., Fernndez, K., 2011. The effect of different extraction techniques on extrac-
tion yield, total phenolic, and anti-radical capacity of extracts fromPinus radiata
bark. Ind. Crops Prod. 34 (1), 838844.
Ayaz, F.A., 2001. Changes in phenolic acids of cherry laurel (Laurocerasus ofcinalis
Oxygemmis) fruit during maturation. Acta Biol. Cracov. Bot. 43, 2326.
Ayaz, F.A., Kadioglu, A., Reunanen, M., Var, M., 1997. Phenolic acid and fatty acid
composition in the fruits of Laurocerasus ofcinalis Roem. and its cultivars. J.
Food Compos. Anal. 10 (4), 350357.
Barreira, J.C.M., Ferreira, I.C.F.R., Oliveira, M.B.P.P., Pereira, J.A., 2008. Antioxidant
activities of the extracts fromchestnut ower, leaf, skins and fruit. Food Chem.
107 (3), 11061113.
148 I.T. Karabegovic et al. / Industrial Crops and Products 54 (2014) 142148
Beejmohun, V., Fliniaux, O., Grand, ., Lamblin, F., Bensaddek, L., Christen, P., Koven-
sky, J., Fliniaux, M., Mesnard, F., 2007. Microwave-assistedextractionof the main
phenolic compounds in axseed. Phytochem. Anal. 18 (4), 275282.
Belkhir, M., Rebai, O., Dhaouadi, K., Sioud, B., Amri, M., Fattouch, S., 2013. Antiox-
idant and antimicrobial activities of Tunisian azarole (Crataegus azarolus L.)
leaves and fruit pulp/peel polyphenolic extracts. Int. J. Food Prop. 16 (6),
13801393.
Biesaga, M., 2011. Inuence of extraction methods on stability of avonoids. J. Chro-
matogr. A 1218 (18), 25052512.
Brahmi, F., Mechri, B., Dhibi, M., Hammami, M., 2013. Variations in phenolic com-
pounds and antiradical scavenging activity of Olea europaea leaves and fruits
extracts collected in two different seasons. Ind. Crops Prod. 49, 256264.
Casazza, A.A., Aliakbarian, B., Mantegna, S., Cravotto, G., Perego, P., 2010. Extraction
of phenolics from Vitis vinifera wastes using non-conventional techniques. J.
Food Eng. 100 (1), 5055.
Celep, E., Aydin, A., Yesilada, E., 2012. A comparative study on the in vitro antiox-
idant potentials of three edible fruits: Cornelian cherry, Japanese persimmon
and cherry laurel. Food Chem. Toxicol. 50 (9), 33293335.
Colak, A., zen, A., Dincer, B., Gner, S., Ayaz, F.A., 2005. Diphenolases from two
cultivars of cherry laurel (Laurocerasus ofcinalis Roem.) fruits at an early stage
of maturation. Food Chem. 90 (4), 801807.
Dhanani, T., Shah, S., Gajbhiye, N.A., Kumar, S., 2013. Effect of extraction meth-
ods on yield, phytochemical constituents and antioxidant activity of Withania
somnifera. Arab. J. Chem. (in press).
Gil-Chvez, G.J., Villa, J.A., Ayala-Zavala, F.J., Heredia, J.B., Sepulveda, D., Yahia, E.M.,
Gonzlez-Aguilar, G.A., 2013. Technologies for extraction and production of
bioactive compounds to be used as nutraceuticals and food ingredients: an
overview. Compr. Rev. Food Sci. F. 12, 523.
Goli, A.H., Barzegar, M., Sahari, M.A., 2005. Antioxidant activity and total phenolic
compounds of pistachio (Pistachia vera) hull extracts. Food Chem. 92, 521525.
Erdemoglu, N., Kpeli, E., Yes ilada, E., 2003. Anti-inammatory and antinocicep-
tive activity assessment of plants used as remedy in Turkish folk medicine. J.
Ethnopharmacol. 89 (1), 123129.
Halilova, H., Ercisli, S., 2010. Several physico-chemical characteristics of cherry lau-
rel (Laurocerasus ofcinalis Roem.) fruits. Biotechnol. Biotechnol. Equip. 24 (3),
19701973.
Kalia, K., Sharma, K., Singh, H.P., Singh, B., 2008. Effects of extraction methods on
phenolic contents and antioxidant activity in aerial parts of Potentilla atrosan-
guinea lodd. and quantication of its phenolic constituents by RP-HPLC. J. Agric.
Food Chem. 56 (21), 1012910134.
Karabegovi c, I.T., Stoji cevi c, S.S., Veli ckovi c, D.T., Nikoli c, N.

C., Lazi c, M.L., 2013.

Optimization of microwave-assisted extraction and characterization of pheno-
lic compounds in cherry laurel (Prunus laurocerasus) leaves. Sep. Purif. Technol.
120, 429436.
Karabegovi c, I.T., Stoji cevi c, S.S., Veli ckovi c, D.T., Nikoli c, N.

C., Lazi c, M.L., 2014.

Optimization of microwave-assisted extraction of cherry laurel fruit. Sep. Sci.
Technol. 49 (3), 416423.
Karahalil, F.Y., S ahin, H., 2011. Phenolic composition and antioxidant capacity of
cherry laurel (Laurocerasus ofcinalis Roem.) sampled from Trabzon region,
Turkey. Afr. J. Biotechnol. 10 (72), 1629316299.
Kaufmann, B., Christen, P., 2002. Recent extraction techniques for natural products:
microwave-assisted extraction and pressurized solvent extraction. Phytochem.
Anal. 13, 105113.
Khoddami, A., Wilkes, M.A., Roberts, T.H., 2013. Techniques for analysis of plant
phenolic compounds. Molecules 18 (2), 23282375.
Kolayli, S., Kc k, M., Duran, C., Candan, F., Dinc er, B., 2003. Chemical andantioxidant
properties of Laurocerasus ofcinalis Roem. (cherry laurel) fruit grown in the
Black Sea region. J. Agric. Food Chem. 51 (25), 74897494.
Liyana-Pathirana, C.M., Shahidi, F., Alasalvar, C., 2006. Antioxidant activity of cherry
laurel fruit (Laurocerasus ofcinalis Roem.) and its concentrated juice. Food
Chem. 99 (1), 121128.
Orhan, I.E., Akkol, E.K., 2011. Estimation of neuroprotective effects of Laurocera-
sus ofcinalis Roem. (cherry laurel) by in vitro methods. Food Res. Int. 44 (3),
818822.
Ozsoy, N., Kultur, S., Melikoglu, G., Can, A., 2013. Screening of the antioxidant poten-
tial of the leaves and owers fromRosa horrida Fischer. J. Med. Plants Res. 7 (10),
573578.
Pan, X., Niu, G., Liu, H., 2003. Microwave-assisted extraction of tea polyphenols and
tea caffeine fromgreen tea leaves. Chem. Eng. Process. 42 (2), 129133.
Prior, R.L., Wu, X., Schaich, K., 2005. Standardized methods for the determination of
antioxidant capacity and phenolics in foods and dietary supplements. J. Agric.
Food Chem. 53 (10), 42904302.
Sajid, Z.I., Anwar, F., Shabir, G., Rasul, G., Alkharfy, K.M., Gilani, A.H., 2012. Antioxi-
dant, antimicrobial properties and phenolics of different solvent extracts from
bark, leaves and seeds of Pongamia pinnata (L.) pierre. Molecules 17 (4),
39173932.
Sahan, Y., 2011. Effect of Prunus laurocerasus L. (cherry laurel) leaf extracts ongrowth
of bread spoilage fungi. Bulg. J. Agric. Sci. 17 (1), 8392.
Sahan, Y., Cansev, A., Celik, G., Cinar, A., 2012. Determination of various chemical
properties, total phenolic contents, antioxidant capacity and organic acids in
Laurocerasus ofcinalis fruits. Acta Hortic. 939, 359366.
Snchez-Moreno, C., 2002. Review: methods used to evaluate the free radical scav-
enging activity in foods and biological systems. Food Sci. Technol. Int. 8 (3),
121137.
Sellappan, S., Akoh, C.C., Krewer, G., 2002. Phenolic compounds and antioxidant
capacity of Georgia-grown blueberries and blackberries. J. Agric. Food Chem. 50
(8), 24322438.
Shabir, G., Anwar, F., Sultana, B., Khalid, Z.M., Afzal, M., Khan, Q.M., Ashrafuzzaman,
M., 2011. Antioxidant and antimicrobial attributes and phenolics of different
solvent extracts fromleaves, owers andbarkof GoldMohar [Delonixregia(Bojer
ex Hook.) Raf.]. Molecules 16 (9), 73027319.
Sharma, U.K., Sharma, K., Sharma, N., Sharma, A., Singh, H.P., Sinha, A.K., 2008.
Microwave-assisted efcient extraction of different parts of Hippophae rham-
noides for the comparative evaluation of antioxidant activity and quantication
of its phenolic constituents by reverse-phase high-performance liquid chro-
matography (RP-HPLC). J. Agric. Food Chem. 56 (2), 374379.
Stanisavljevi c, I.T., Stoji cevi c, S.S., Veli ckovi c, D.T., Lazi c, M.L., Veljkovi c, V.B., 2008.
Screening the antioxidant and antimicrobial properties of the extracts from
plantain (Plantago major L.) leaves. Sep. Sci. Technol. 43 (14), 36523662.
Stankovi c, M.Z., 2002. Bioaktivni proizvodi prirodnog porekla, 12. Zbornik radova
Tehnolo skog fakulteta, Leskovac, pp. 3351.
Sultana, B., Hussain, Z., Asif, M., Munir, A., 2012. Investigation on the antioxidant
activity of leaves, peels, stems bark, and kernel of mango (Mangifera indica L.). J.
Food Sci. 77 (8), C849C852.
Sulusoglu, M., 2011. The cherry laurel (Prunus laurocerasus L.) tree selection. Afr. J.
Agric. Res. 6, 35743582.
Tamuly, C., Saikia, B., Hazarika, M., Bora, J., Bordoloi, M.J., Sahu, O.P., 2013. Correlation
between phenolic, avonoid, and mineral contents with antioxidant activity of
underutilized vegetables. Int. J. Veg. Sci. 19 (1), 3444.
Trusheva, B., Trunkova, D., Bankova, V., 2007. Different extraction methods of bio-
logically active components from propolis: a preliminary study. Chem. Cent. J.
1 (1), 13.
Upadhyay, R., Ramalakshmi, K., Jagan Mohan Rao, L., 2012. Microwave-assisted
extraction of chlorogenic acids from green coffee beans. Food Chem. 130 (1),
184188.
Vagiri, M., Ekholm, A., Andersson, S.C., Johansson, E., Rumpunen, K., 2012. An opti-
mized method for analysis of phenolic compounds in buds, leaves, and fruits of
black currant (Ribes nigrumL.). J. Agric. Food Chem. 60 (42), 1050110510.
Var, M., Ayaz, F.A., 2004. Changes in sugar composition in cherry laurel (cv
oxygemmis) fruit during development and ripening. Pakistan J. Bot. 36 (2),
389394.
Viet, M., Beckert, C., Hhne, C., Bauer, K., Geiger, H., 1995. Interspecic and intraspe-
cic variation of phenolics in the genus Equiesetum subgenus Equiesetum.
Phytochemistry 38, 881891.
Vinatoru, M., 2001. An overviewof the ultrasonically assisted extraction of bioactive
principles fromherbs. Ultrason. Sonochem. 8, 303313.
Vongsak, B., Sithisarn, P., Mangmool, S., Thongpraditchote, S., Wongkrajang, Y., Grit-
sanapan, W., 2013. Maximizing total phenolics, total avonoids contents and
antioxidant activity of Moringa oleifera leaf extract by the appropriate extraction
method. Ind. Crops Prod. 44, 566571.
Wang, L., Weller, C.L., 2006. Recent advances in extraction of nutraceuticals from
plants. Trends Food Sci. Technol. 17, 300312.
Xu, B.J., Chang, S.K.C., 2007. A comparative study on phenolic proles and antiox-
idant activities of legumes as affected by extraction solvents. J. Food Sci. 72,
S159S166.
Yes ilada, E., Sezik, E., Honda, G., Takasihi, Y., Takeda, Y., Tanaka, T., 1999. Traditional
medicine in Turkey IX. Folk medicine in Noth-West Anatolia. J. Ethnopharmacol.
64 (3), 195210.