Industrial Crops and Products 74 (2015) 485–493

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Industrial Crops and Products

journal homepage: www.elsevier.com/locate/indcrop

Enzyme-assisted extraction of bioactive compounds from bay leaves

(Laurus nobilis L.)
Abdennacer Boulila a , Imed Hassen b , Lamia Haouari a , Feiza Mejri a , Ines Ben Amor b ,
Hervé Casabianca c , Karim Hosni a,∗
a
Laboratoire des Substances Naturelles, Institut National de Recherche et d’Analyse Physico-chimique (INRAP), Biotechpole de Sidi Thabet, 2020 Ariana,
Tunisia
b
Laboratoire des Méthodes et Techniques Analytiques, Institut National de Recherche et d’Analyse Physico-chimique (INRAP), Biotechpole de Sidi Thabet,
2020 Ariana, Tunisia
c
Institut des Sciences Analytiques, Département Service Central d’Analyse, 5 Rue de la Doua, Villeurbanne, 69100 Lyon, France

a r t i c l e i n f o a b s t r a c t

Article history: Bay leaves (Laurus nobilis L.) are widely used as a condiment and their therapeutic benefits are well
Received 31 January 2015 known. These biological properties were attributed to a plethora of highly bioactive secondary metabo-
Received in revised form 7 April 2015 lites namely essential oils and phenolics. However, their recovery from plant matrix is generally limited
Accepted 21 May 2015
by the presence of physical barrier (cell wall). Thus, the use of novel extraction procedures to enhance
Available online 10 June 2015
their release is particularly important. Therefore, the aim of this work is to assess the potential use
of enzyme treatment (cellulase, hemicellulase, xylanase end the ternary mixture of them) as a tool to
Keywords:
improve the extraction efficiency of bioactive compounds from bay leaves. Results showed that enzyme
Enzymes
Laurus nobilis L.
pre-treatment resulted in 243, 227, 240.54 and 0.48% increase in the essential oil yields in samples treated
Essential oils with cellulase, hemicellulase, xylanase and the ternary mixture, respectively. Compositional analysis by
Phenolics GC and GC–MS revealed remarkable enrichment of the essential oils derived from enzyme-treated sam-
Antioxidant activity ples with oxygenated monoterpenes, leading hence to better antioxidant activity as revealed by the
Food processing technology 2,2-diphenyl-1-picrylhydrazyl (DPPH) and azino-bis-(3-ethylbenzothiolzoline 6-sulphonic acid) (ABTS)
assays. The 1,8-cineole, ␣-terpinyl acetate, methyl eugenol, linalool, ␣-pinene, sabinene and ␤-pinene
were found as the most prominent components in all essential oils. Most importantly, enzyme treatment
did not induce transformation of the volatile components, but it contributes to the liberation of some
glycosidically bound volatiles. Moreover, it significantly enhances the release of phenolic compounds
from the hydrodistilled residual leaves and consequently their antioxidant activity. These results suggest
that enzyme pre-treatment could be useful for extracting valuables components, and hold good potential
for use in food, cosmetic and pharmaceutical industries.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction
Laurus nobilis L., commonly known as bay (Lauraceae family) is
one of the oldest known spices, widely used as a condiment and
spice. Bay leaves are often used as a folk remedies and credited
with a long list of medicinal uses, including antioxidant, antimi-
crobial, anti-inflammatory, cytotoxic, anti-asthmatic, anti-arthritic
and analgesic, among others (Sayyah et al., 2003; Kaileh et al., 2007;
Lee et al., 2013). Most of these effects can be related to its high
amount of essential oils and phenolic compounds (Santoyo et al.,
2006). Previous phytochemical investigations revealed that 1,8-
∗ Corresponding author. Tel.: +216 71537666; fax: +216 71537866.
E-mail addresses: karim hosni1972@yahoo.fr, karim.hosni@inrap.rnrt.tn
(K. Hosni).
cineole, linalool and ␣-terpinyl acetate were the basic components
of the essential oil of bay leaves (da Silveira et al., 2014). Epicate-
chin, procyanidin dimer, procyanidin trimer, flavonol and flavone
derivatives were the most prominent phenolic compounds (Diaz
et al., 2014). Due to their intriguing biological activities, secondary
metabolites from bay leaves are widely applied as a flavouring
agent and potential preservative in perfumery, pharmaceutical,
cosmetic and food industries (Ertaş and Alma, 2010). As a conse-
quence, market demand of laurel essential oils and extracts has
increased remarkably. This has prompted the search for new meth-
ods of extraction to improve their recovery without alteration of
the qualitative features of the end product. Traditionally, these
metabolites are recovered from the plant matrix by solid–liquid
extraction employing water (i.e. hydrodistillation or steam dis-
tillation for essential oils) and water, organic or hydroalcoholic
solvents for phenolic compounds. However, a lot of alternative

http://dx.doi.org/10.1016/j.indcrop.2015.05.050
0926-6690/© 2015 Elsevier B.V. All rights reserved.
486 A. Boulila et al. / Industrial Crops and Products 74 (2015) 485–493
approaches have been recently used to improve the release of
these components from the plant matrix. They include supercritical
fluid extraction, pressurized liquid extraction, microwave-assisted
extraction and ultrasound-assisted extraction (Joo et al., 2011;
Martins et al., 2011). Another procedure widely used to improve
the extraction efficiency of bioactive components from plant matrix
is the enzyme-assisted extraction (EAE). Such coarse approach
involves the use of hydrolytic enzymes to disrupt the cell walls,
predominantly composed by highly complex large polymers such
as cellulose, hemicellulose, lignin and pectin. The EAE has been
successfully used to enhance the recovery of polyphenols from
blackcurrant (Landbo and Mayer, 2001) citrus peel (Li et al., 2006)
and ginger (Chari et al., 2013); lycopene from tomato (Zuorro
et al., 2011); oil from Forsythia suspens (Gai et al., 2013); pro-
tein from olive leaves (Vergara-Barberán et al., 2015); starch from
potato (Ramasamy et al., 2014) and essential oils from garlic
(Sowbhagya et al., 2009), celery seeds (Sowbhagya et al., 2010),
cumin (Sowbhagya et al., 2011), thyme and rosemary (Hosni et al.,
2013) and lavender (Calinescu et al., 2014). Although the enzyme-
assisted approach has largely improved the extraction yield of
bioactive compounds of the aforementioned species, there are no
reports on the application of this procedure on bay leaves. Bearing
this in mind, and taking into account the increased market demand
of bay leaves essential oils and extracts, the present study was
intended to evaluate the potential use of enzymatic pre-treatment
as a tool to improve the extraction efficiency of bioactive com-
pounds from bay leaves.
2. Materials and methods
2.1. Plant material
Air dried bay leaves were purchased from the local market in
Morneg, Tunis, Tunisia. The plant material was ground by using
a Retsch blender Mill (Normandie-Labo, Normandy, France) and
sifted through 0.5 mm mesh screen to obtain a uniform particle
size before use.
2.2. Chemicals and enzymes
Acetonitrile, methanol and ethyl acetate of HPLC grade were
purchased from LabScan (Dublin, Ireland). Analytical grade hexane
was obtained from Acros Organics (New Jersey, USA). Reference
volatile standards including 1,8-cineole, ␣-terpinene, ␦-3-carene
and ␤-caryophyllene were purchased from Sigma–Aldrich (Stein-
heim, Germany). Linalool, ␤-myrcene and terpinen-4-ol were
from Fluka Chemicals (Buchs, Switzerland). Phenolic compound
standards were from Sigma–Aldrich (St. Louis. MO. USA). Anhy-
drous sodium sulphate (Na2 SO4 ) and n-alkanes (C7 –C20 ) were
obtained from Fluka Chemicals (Buchs, Switzerland). Folin-
Ciocalteu, gallic acid, quercetin, 2,2-diphenyl-1-picrylhydrazyl
(DPPH), AlCl3 , 2,2-azino-bis-(3-ethylbenzothiolzoline 6-sulphonic
acid)- di-ammonium salt (ABTS), butylated hydroxytoluene (BHT)
and trolox were purchased from Sigma–Aldrich Inc (Steinheim,
Germany). Cellulase (E.C. 3.2.1.4, 8.9 U/mg), Xylanase (E.C. 3.2.1.8,
102 U/mg) both from Trichoderma viride and hemicellulase (H-
7649, 13.8 U/mg) from Aspergillus niger, were purchased from
Sigma–Aldrich (St. Louis. MO, USA). Water was treated in a Milli-Q
water purification system (ELGA, Purelab UHQ, High Wicomb, UK).
2.3. Enzyme pre-treatment
Dried and ground bay leaves (100 g) were mixed with 500 mL
distilled water containing 10 mg of single enzyme (cellulase, hemi-
cellulase and xylanase) or 30 mg of the ternary enzyme mixture
(cellulase: hemicellulase: xylanase; 1:1:1). The material was stirred
for 1 h at 40 ◦ C, thereafter; the water was removed and the treated
plant material was subjected to hydrodistillation (Sowbhagya et al.,
2011). Control samples were subjected to hydrodistillation without
any treatment.
2.4. Isolation of essential oils
Essential oils from control and treated samples were isolated by
hydrodistillation for 2 h using a Clevenger-type apparatus. The oils
were recovered, weighed, dried over Na2 SO4 and stored in amber
and airtight sealed vials at −20 ◦ C until analyzed. The residual plant
materials (by-product of distillation) were recovered, oven dried at
40 ◦ C and stored for further uses.
2.5. Analysis of essential oils
Analytical gas chromatography was carried out on a HP 6890
(II) gas chromatograph (Agilent Technologies, Palo Alto, CA, USA)
equipped with flame ionisation detector (FID), an apolar HP-5 and
a polar Innowax capillary columns (60 m × 0.25 mm (i.d), 0.25 ␮m
film thickness). Diluted oil samples in hexane were injected with a
split ratio of 1:60 and a continuous flow rate of 1.2 mL/min of chro-
matographic grade nitrogen was used. The oven temperature was
initially held for 1 min at 50 ◦ C, ramped at 2 ◦ C/min up to 300 ◦ C and
held isothermally for 4 min. The injector and FID detector temper-
atures were held at 250 and 300 ◦ C, respectively.
The gas chromatography–mass spectrometry (GC–MS) analyses
were performed on a gas chromatograph HP 6890N interfaced with
an HP 5975 mass spectrometer (Agilent Technologies, Palo Alto, Ca,
USA) with electron impact ionization (70 eV). An HP-5MS capillary
column (60 m × 0.25 mm, 0.25 mm film thickness) was used for the
separation of volatile compounds. The column temperature was
programmed to rise from 40 to 280 ◦ C at a rate of 5 ◦ C/min. The
carrier gas was helium with a flow rate of 1.2 mL/min. Scan time
and mass range were 1 s and 50–550 m/z, respectively.
The volatile compounds were identified by comparison of their
retention indices relative to (C7 –C20 ) n-alkanes, and by match-
ing their mass spectral fragmentation patterns with corresponding
data (Wiley 275.L library) and other published mass spectra
(Adams, 2001), as well as by comparison of their retention indices
with data from the Mass Spectral Library “Terpenoids and Related
Constituents of Essential oils” (Dr. Detlev Hochmuth, Scientific con-
sulting, Hamburg, Germany) using the Mass Finder 3 software
(www.massfinder.com). Relative percentage amounts of the iden-
tified compounds were obtained from the electronic integration of
the FID peak areas without use of the correction factor.
2.6. Extracts preparation from the hydrodistilled leaf residues
Five solvents with increasing polarity (ethyl acetate,
dichloromethane, methanol, 80% methanol and water) were
used for the preparation of different extracts. Oven dried leaf
residues (1 g) were mixed with 20 mL of solvent in an orbital
shaker (150 rpm for 48 h). Each extraction was repeated twice
and the resulting solvent extracts (except for water extract) were
filtered through Wattman #1 filter paper (Bärenstain, Germany)
and evaporated under reduced pressure in a Heidolph rotary
evaporator (Schwabach, Germany). The water extract was frozen
and lyophylized in a Christ-Alpha 2–4 freeze drier (Osterode,
Germany).
2.6.1. Determination of total phenolic (TP) content
Total phenolic (TP) were determined with the Folin–Cieucalteu
(FC) assay according to Lister and Wilson (2001). Briefly, 100 ␮L of
extract was mixed with 500 ␮L of freshly diluted 10-fold FC reagent
in water and 1 mL of 20% sodium carbonate solution. After incuba-
tion for 1 h in the dark, the absorbance was measured at 760 nm
 A. Boulila et al. / Industrial Crops and Products 74 (2015) 485–493 487

Table 1
Chemical composition (% total peak area) of control (untreated) and enzyme-pretreated samples of L. nobilis leaves.

Component RIa RIb Control Cellulase Hemicellulase Xylanase Ternary Mixturea

␣-Thujene 932 1035 0,44 – 0,37 0,42 –

␣-Pinene 939 1032 10,17 7,34 7,65 7,84 5,33
Camphene 950 1076 0,51 – 0,36 0,32 –
Sabinene 976 1132 7,26 7,38 7,27 7,15 6,64
␤-Pinene 981 1118 7,12 5,66 5,49 5,54 4,96
␤-Myrcene 988 1176 0,39 – 0,36 0,37 –
3-Carene 1011 1159 1,45 – – – –
1,8-Cineole 1031 1213 39,76 48,33 42,16 38,38 33,86
␥-Terpinene 1059 1255 0,61 0,56 0,72 0,72 –
Linalool 1088 1553 10,03 9,2 8,36 8,17 8,35
Terpinen-4-ol 1178 1611 0,87 1,78 2,17 2,19 1,27
␣-Terpineol 1189 1706 1,35 2,13 2,51 2,6 1,57
␣-Terpinyl acetate 1351 1709 13,35 10,39 12,35 13,54 18,05
Eugenol 1356 2192 0,07 0,35 0,16 0,12 0,09
methyl eugenol 1402 2028 2,98 5,22 7,37 8,53 12,48
␤-Caryophyllene 1418 1612 1,41 1,46 1,87 2,07 2,95
Elemicine 1523 2229 – – – 0,62 1,41
␦-Cadinene 1525 1773 0,46 – – – –
Spathulenol 1576 2153 0,82 – – 0,55 0,79
Caryophyllene oxide 1569 2008 0,48 0,71 0,78 2,12
t-Muurolol 1641 2145 0,11 0,13 0,09 0,07 0,11

Group components
Monoterpene hydrocarbons 27,95 20,94 22,22 22,36 16,93
Oxygenated monoterpenes 68,41 77,4 75,08 73,53 75,67
Susquiterpene hydrocarbons 1,87 1,46 1,87 2,69 4,36
Oxygentaed sesquiterpenes 1,41 0,13 0,8 1,4 3,02
Oxygenated/Hydrocarbons 2,34 3,46 3,15 2,99 3,70
Total identified 99,64 99,93 99,97 99,98 99,98
Essential oil yield (% w/w) 0.37 1.25 1.19 1.24 0.54

RI: retention index on (a) HP-5 and (b) HP-Innowax; (−) not detected.
a
Ternary mixture (cellulase + hemicellulase + xylanase; 1:1:1).

using a Jasco V-630 UV–vis spectrophotometer (Tokyo, Japan). Gal- bition (IC50 ) expressed as ␮g/mL was determined from the graph
lic acid was used as the standard, and results were expressed as of the free radical scavenging activity (%) against the extract con-
microgram of gallic acid equivalents (␮g GAE/g). centration.

2.6.2. Determination of total flavonoid (TF) content
Total flavonoid (TF) content was determined by the AlCl3 col-
orimetric method (Chang et al., 2002). A 500 ␮L sample aliquot
was mixed with 1.5 mL methanol, 0.1 mL of a 10%AlCl3 solution,
0.1 mL of potassium acetate (1 M), and 2.8 mL of distilled water.
After 30 min incubation at room temperature, the absorbance was
measured at 415 nm. Quercetin was used as a reference standard
and the TF content was expressed as microgram of quercetin equiv-
alents (␮g QE/g).
2.7. Antioxidant activity of different extracts and essential oils
2.7.1. DPPH radical scavenging activity
The DPPH assay followed a reported method (Brand-Williams
et al., 1995) with some modifications. Briefly, 1 mL of different
extracts was added to 2 mL of a 0.1 mM methanolic DPPH solu-
tion. The mixture was shaken vigorously and left to stand at room
temperature in the dark for 1 h. Thereafter, the absorbance was
measured at 515 nm. Quercetin and BHT were used as positive
controls.
The same procedure was used for the evaluation of the free radi-
cal scavenging activity of the essential oils at various concentrations
(20, 50, 100, 200, 500, 1000 and 2000 ␮g/mL).
The scavenging activity was measured as the decrease in
absorbance of the samples versus DPPH standard solution. Results
were expressed as radical scavenging activity percentage (%) of the
DPPH using the following formula:
(A0 − As )
%DPPHradicalscavenging = × 100
A0
where A0 and As are the absorbance of the control and the sample,
respectively. The effective concentration having a 50% radical inhi-
2.7.2. ABTS scavenging activity
The ABTS assay was based on the procedure described by Re
et al. (1999). The solution consisting of 7 mM of ABTS and 2.4 mM
potassium persulfate (1:1 v/v) was reacted in the dark for 12 h at
room temperature. Then it was diluted with methanol to obtain an
absorbance of 0.7 at 734 nm. The diluted ABTS solution (2850 ␮L)
was mixed with 150 ␮L of sample extracts or various concentra-
tions of essential oils (20, 50, 100, 200, 500, 1000 and 2000 ␮g/mL)
or trolox standard. The mixture was left to stand at room tempera-
ture in the dark for 15 min, and then the absorbance was measured
at 734 nm. The antoxidant capacity of test samples was expressed
as IC50 , the concentration necessary to 50% inhibition of ABTS+• .
2.8. Statistical analysis
All measurements were carried out in triplicate and the results
were presented as mean values ± SD. Statistical analyses were per-
formed using a one way analysis of variance (ANOVA) test and the
significance between means was determined by Duncan’s multiple
range test. Differences at P < 0.05 were deemed significant. The SPSS
18.0 software package (Chicago, Illinoi, USA) was used to perform
statistical analysis.
3. Results and discussion
3.1. Effect of enzymes on yield and chemical composition of bay
leaf essential oil
The release of essential oil from enzyme-treated bay leaves was
1.4–3.4-fold higher than that from the untreated samples (Table 1).
The magnitude of such an enhancement was greater by using single
488 A. Boulila et al. / Industrial Crops and Products 74 (2015) 485–493

Fig. 1. Representative GC/MS chromatograms of the reconstituted hydrolysate from untreated (control) and enzyme-treated samples. (1) ␣-pinene, (2) camphene, (3)
sabinene, (4) 1,8-cineole, (5) linalool, (6) terpinen-4-ol, (7) ␣-terpineol, (8) terpinyl acetate, (9) eugenol, (10) methyl eugenol, (11) ␤-caryophyllene, (12) ␦-cadinene, (13)
elemicine, (14) spathulenol, (15) caryophyllene oxide.

enzyme than the combination of enzymes (cellulase, hemicellu-
lase and xylanase). This increase in recovery can be attributed to
the ability of enzymes to degrade cell wall structure and depoly-
merize plant cell wall polysaccharides, facilitating the release of
essential oil (Gil-Chávez et al., 2013). However, such ability was
reduced when enzymes were combined and used simultaneously,
suggesting a competitive adsorption to the cell wall polysaccha-
rides. This leads to steric hindrance of binding positions of enzyme
to substrate, which negtively influences the breakdown of cell-wall
components (Hyunh et al., 2014).
The possibility of cellulase inhibition by xylan and xylo-
oligomers released from hemicellulose during enzymatic digestion
by hemicellulase and xylanase is suggested too (Qing et al., 2010;
Qing and Wyman, 2011a,b; Zhang et al., 2012). From a mechanis-
tic stand point, it has been found that xylan and xylo-oligomers
have an affinity to cellulose and that their adsorption on cellulose
surface may physically block the access of cellulase to cellulose
(Zhang et al., 2012). At this point, it seems that sequential enzy-
matic pre-treatment of bay leaves could overcome such inhibitory
effect.
 A. Boulila et al. / Industrial Crops and Products 74 (2015) 485–493 489

Fig. 2. Representative GC/MS chromatograms of the hydrolysate from the control and enzyme-treated samples. (1) p-cymene, (2) 1,8-cineole, (3) linalool, (4) terpinen-4-ol,
(5) ␣-terpineol, (6) ␣-terpinyl acetate, (7) copaene, (8) ␤-elemene, (9) methyl eugenol, (10) ␤-caryophyllene, (11) ␥-elemene, (12) ␦-muurolene, (13) allo-aromadendrene,
(14) junipene, (15) BHT, (16) ␦-cadinene, (17) elemicine, (18) spathulenol, (19) caryophyllene oxide, (20) patchoulene, (21) hexadecanoic acid, (22) octadecanoic acid.

Another possible explanation of the lower activity of the ternary
enzyme mixture is the presence of lignin on the cell walls (27.61%
in bay leaves) which considerably limits accessibility of cellulase
and hemicellulase to their substrate (Kaya et al., 2000; Van Dyk
and Pletschke, 2012; Miron et al., 2013).
Compared with earlier studies, our results were in line with
those reported for celery seeds (Sowbhagya et al., 2010), cumin
seeds (Sowbhagya et al., 2011), garlic cloves (Sowbhagya et al.,
2009), Fructus forsythiae (Jiao et al., 2012), thyme and rosemary
(Hosni et al., 2013). They also compared the efficiency of individual
enzyme and found that pre-treatment with cellulase gave the best
yields which is consistent with our results.
Analytical gas chromatography and GC–MS allowed identifica-
tion of 21 components covering more than 99% of the total GC
profile. Table 1 lists the chemical components of the essential oils
grouped as classes of compounds. The main components were
1,8-cineole (33.86–48.33%), ␣-terpinyl acetate (10.69–18.05%),
methyl eugenol (2.98–12.48%), linalool (8.37–10.03%), ␣-pinene
(5.33–10.87), sabinene (6.74–7.48%) and ␤-pinene (4.96–7.12%).
When compared with earlier studies, the same volatile profile (1,8-
cineole > ␣-terpinyl acetate > methyl eugenol) was also described
for Italian (Flamini et al., 2007), Jordanian (Al-Kalaldeh et al., 2010)
and Turkish specimens (Chalchat et al., 2011; Tabanca et al., 2013).
In contrast it differed slightly from those originated from Brazil
(da Silveira et al., 2014), Argentina (Lira et al., 2009) and Cro-
atia (Politeo, 2009) where linalool was found as the second main
compounds. The components 3-carene and ␦-cadinene which are
constituents of the essential oil of the control sample were not
present in the enzyme-treated samples showing that they are arte-
facts in the essential oil of bay leaves.
What is striking is the marked enrichment of essential oils
derived from enzyme-pre-treated samples with oxygenated com-
pounds as revealed by the high values of oxygenated/hydrocarbons
(O/H) ratios (from 2.99 to 3.7 in enzyme-treated sample versus 2.34
in the control samples). This observation may confirm that enzyme
pre-treatment resulted in enhanced rate of oxidation following cell
wall disruption (Charoensiddhi and Anprung, 2010). The presence
of oxidases in the enzyme preparation is suggested too. The positive
effect of enzymes on the release of oxygenated components was
also reported for cardamom (Chandran et al., 2012), F. forsythiae
(Jiao et al., 2012), thyme and rosemary (Hosni et al., 2013). In the
former species, it was found that the use of Lumicellulase (a mixture
of cellulase, ␤-glucanase, petinase and xylanase) caused a reduc-
tion of hydrocarbons, whereas, it significantly improve the release
of oxygenated compounds, leading hence to increased O/H ratio
in enzyme-treated cardamom. Similarly, the simultaneous appli-
cation of cellulase, hemicellulase and ␤-glucosidase resulted in
significant increase of linalool, camphor, terpinen-4-ol, ␣-terpineol
and trans-carveol in F. forsythiae (Jiao et al., 2012). In contrast,
pre-treatment of cumin seeds with cellulase and Viscozyme (com-
mercial mixture of enzymes including cellulase, hemicellulase,
pectinase, arabinase and xylanase) afforded enriched hydrocar-
bons essential oils as reflected in the lower O/H ratios (0.53 and
0.44 for cellulase and Viscozyme treated samples, versus 0.58
fro the untreated seeds) (Sowbhagya et al., 2011). Application of
Lumicellulase to black pepper resulted in significant increase of
490 A. Boulila et al. / Industrial Crops and Products 74 (2015) 485–493
Fig. 3. Free radical scavenging activity of the essential oils derived from untreated (control) and enzyme-treated bay leaves in term of their IC50 (␮g/mL) values.
hydrocarbons as revealed by the decrease of O/H ratios (0.19 in
enzyme-treated samples versus 0.21 in the untreated samples)
(Chandran et al., 2012). At this point it seems that the release of
volatile compounds from enzyme-treated samples is dependent on
the plant species/organs, enzymes used, source of enzymes, and
duration of treatment.
Regarding the individual compounds, a remarkable increase
in the amount of 1,8-cineole, methyl eugenol, terpinen-4-ol, ␣-
terpineol and caryophyllene oxide was observed in enzyme-treated
samples. In contrast, the amounts of linalool, ␣- and ␤-pinene
showed reciprocal trends, suggesting presumably their loss during
the pre-treatment step.
Bearing in mind that commercial preparations of cellulase
and hemicellulase were endowed with a ␤-glycosidase activities
(Pogorzelski and Wilkowska, 2007; Sathya and Khan, 2014), it
seems logical to suggest that a part of these components was
generated by enzymatic or chemical transformations during pre-
treatment and/or they were glycosidically bounded. To investigate
whether enzyme treatment induces transformations of volatile
components or liberates bound volatiles, two additional experi-
ments were undertaken. The first experiment consists on producing
a reconstituted hydrolysate by mixing pure essential oil with dis-
tilled water and enzymes (cellulase and hemicellulase 1:1) under
the same conditions described above (1 h at 40 ◦ C). The control sam-
ple was made without enzymes. The insoluble fraction of essential
oil was leftover and the obtained hydrolysate was extracted with
diethyl ether, concentrated under a stream of nitrogen and sub-
sequently analyzed by GC and GC–MS. In the second experiment
and in order to determine if the enzyme treatment was able to lib-
erate bound volatiles, the plant material was mixed with distilled
water and the binary enzyme mixture (cellulase and hemicellulase)
for 1 h at 40 ◦ C. The volatile aglycones were recovered from the
hydrolysate by using diethyl ether and analyzed by GC and GC–MS.
Results of the first experiment showed no apparent effect of
enzymes in the volatile profiles of the reconstituted hydrolysate
(Fig. 1), suggesting that enzymatic pre-treatment did not induces
transformations of the volatile components.
In the second experiment, enzymatic pre-treatment was found
to be associated with remarkable enrichment of the hydrolysate
with volatiles namely 1,8-cineole, ␦-cadinene, methyl eugenol,
linalool, ␣-terpineol, terpinen-4-ol, and caryophyllene oxide
(Fig. 2). Other components including p-cymene, methyl isoeugenol,
butyl hexadecanoate, palmitic acid, stearic acid and ␤-elemene,
among others were found only in the hydrolysate but they were
detected neither in the essential oil nor in the hydrolysate of
the untreated samples, which indicate that may be glycosidically
bounded. However, such assumption should be taken cautiously
until appropriate analytical experiments were achieved.
Nevertheless, most of the components detected in the
hydrolysate of enzymatically-treated samples were previously
reported as glycosidically bound aromas in numerous species such
as Eucalyptus cinerea (Mann et al., 2011), Vitis vinifera (Fenoll et al.,
2009), Rubus glaucus (Meret et al., 2011) and Prunus avium (Wen
et al., 2014), among others. The glycosidically bound compounds
of bay leaves were also analyzed and different compositional
patterns depending on plant origin, extraction and analytical pro-
cedures were reported. For example, Kilic et al. (2005) found that
benzyl alcohol, linalool oxides, 2-hydroxy-1,8-cineole derivatives,
sobrerols and menthadien-8-ols were the main aglycones which
occur as ␤-d-glucopyranosides in bay leaves. Four years later,
Polieto (2009) reported benzyl alcohol, 2-butenoic acid, vanillin,
butanoic acid and benzoic acid as the main glycosidically bound
volatiles of Croatian bay leaves.
Collectively, results of the present study compiled with litera-
ture data clearly underscored that enzyme treatment did not induce
transformation of the volatile components, but it contributes pre-
sumably to the liberation of some glycosidically bound volatiles
which increased their amounts in the resulting essential oil.
From critical point of view, the extraction procedure developed
by Sowbhagya et al. (2011) and adopted herein should be rectified
by including the extracting phase (hydrolysate) in the distillation
process. Such approach will probably leads to better recovery of
volatiles by preventing their loss in the extracting phase and con-
sequently improved the quality of the end product.
3.2. Antioxidant activities of essential oils
With an IC50 value of 254 ␮g/mL, the essential oil from hemicel-
lulase treated-samples showed the highest DPPH radical scavenger
activity (Fig. 3). In the ABTS assay, those derived from xylanase
treated-samples proved to be the most effective ABTS+• radical
scavenger with a mean IC50 value of 595 ␮g/mL (Fig. 3). In both
assays, the control samples were found as the least active ones. It is
worth mentioning that all assessed oils were less effective than the
synthetic antioxidants trolox (IC50 of 11.42 and 40.44 ␮g/mL in the
DPPH and ABTS+• assays, respectively), BHT (IC50 of 25.28 ␮g/mL
 A. Boulila et al. / Industrial Crops and Products 74 (2015) 485–493 491

Table 2
Effects of different solvents on extract yield, TP and TF in residual hydrodistilled bay leaves.

Solvent

Water Methanol 80% methanol Ethyl acetate Dichloromethane

Extract yield (mg/g) 26.60 ± 2.18e * 92 ± 5.77a 61.60 ± 3.42b 34.60 ± 4.17d 42 ± 2.12c
TPC (mg GAE/g) 0.50 ± 0.12c 5.87 ± 0.40a 4.02 ± 0.28b 4.61 ± 0.42b 4.12 ± 0.60b
TFC (mg QE/g) 0.15 ± 0.01e 5.18 ± 0.08a 3.46 ± 0.41b 1.05 ± 0.07d 1.98 ± 0.13c
*
Values given are mean ± standard deviation of triplicate; superscripts with different letter within the same row are significantly (p < 0.05) different.

Table 3
Effects of enzyme-treatment TP, TF and radical scavenging activity of the methanol extract of residual hydrodistilled bay leaves.

TPCa TFCb DPPHc ABTSc

Control 5.85 ± 0.40a,b,* 5.18 ± 0.08a,b 734.8 ± 42.71a 1391.36 ± 64.07a

Cellulase 7.12 ± 0.64a 5.79 ± 0.41a 478.65 ± 43.98b 1142.91 ± 46.87a,b
Hemicellulase 6.89 ± 0.62a,b 5.35 ± 0.22a,b 591.22 ± 51.64a,b 1371.68 ± 38.79a
Xylanase 6.64 ± 0.49a 6.33 ± 0.38a 690.91 ± 45.63a 1073.73 ± 33.60a,b
Ternary mixture 6.33 ± 0.54ab 6.09 ± 0.78a 650.75 ± 21.57ab 1212.74 ± 34.68ab
a
TPC was expressed as mg gallic acid equivalents per gram of extract.
b
TFC was expressed as mg quercetin equivalents per gram of extract.
c
IC50 (␮g/mL) values of DPPH and ABTS.
*
Values given are mean ± standard deviation of triplicate; superscripts with different letter within the same column are significantly (p < 0.05) different.

for the DPPH assay) and quercetin (IC50 of 5.75 ␮g/mL for the DPPH
assay).
On the other hand, the observed activity was not associated
with their major component 1,8-cineole, which is consistent with
the findings of Ojeda-Sana et al. (2013). The author’s study under-
scored that rosemary myrcene-rich oil was more active than those
rich in 1,8-cineole. They also compared the antioxidant activity of
individual compounds and found that 1,8-cineole was not active
in the DPPH assay, while thymol, myrcene and ␣-pinene were
the strongest antioxidants. In another report from India, Mishra
et al. (2013) assessed the radical scavenging activity of individual
essential oil components and different combinations and found that
eugenol, myrcene, ␤-caryophyllene and the combinations includ-
ing at least one of these components, particularly eugenol were the
strongest free radical scavengers. More recently, Horvathova et al.
(2014) compared the antioxidant activity of 1,8-cineole, eugenol,
carvacrol, thymol and borneol by using the DPPH and hydroxyl
radicals assays and found that 1,8-cineole lacks such activity.
At this point, it seems that the antioxidant activity of the essen-
tial oil of bay leaves may be due to complex interaction between its
different components, which may produce additive, synergistic or
antagonistic effects (Hosni et al., 2013; Riachi and De Maria, 2015).
3.3. Effects of enzymes on extraction yield, TP and TF contents of
the hydrodistilled residues of bay leaves.
It has previously been reported that the exhausted bay leaves
after distillation could be considered as a good source of fibrous feed
having high and digestibility values for ruminants (Lira et al., 2009).
However, data regarding their content on antioxidants namely
phenols and flavonoids are lacking. With regard to this topic, the
hydrodistilled residues of bay leaves were evaluated for their TP
and TF contents.
3.3.1. Solvent selection
Extracting solvent had a great impact in the extraction yield,
TP and TF. Alcoholic solvent have been commonly used to extract
phenolic compounds from plant raw materials (d’Alessandro et al.,
2012). In the present study, five solvents of different polarity were
used to determine the most efficient extracting solvent in term
of extract yield, TP and TF. Results depicted in Table 2 show that
methanol gives the highest extract yield, TP and TF. In contrast,
the values of the latter parameters were significantly (p < 0.05)
lower in water and the less polar solvents ethyl acetate and
dichloromethane. These results were in good agreement with those
reported by Meneses et al.(2009) who explained the fact by the
higher solubility of phenolic compounds in solvents less polar than
water.
Given its good efficiency in the extraction of phenolics, methanol
was chosen as extracting solvent for the rest of experiments.
3.3.2. Antioxidant activity of methanol extracts
The antioxidant activity of methanolic extracts from the resid-
ual hydrodistilled bay leaves was assessed by using DPPH and ABTS
assays. Results presented in Table 3 revealed that the extracts from
cellulase treated samples were the most active against the DPPH
radicals with an IC50 value of 478.65 ␮g/mL, while those derived
from xylanase-treated samples exhibited the highest ABTS+• rad-
ical scavenging activity (IC50 = 1073.73 ␮g/mL). The latter extracts
were not significantly (p > 0.05) different than those derived from
samples treated with hemicellulase and the ternary mixture.
As for essential oils, extracts from untreated samples were found
as the least active in both assays. The DPPH scavenging activity of
methanol extract is likely due to the high content of TP observed in
the residual bay leaves derived from cellulase-treated samples and
confirms the usual correlations between TP and antioxidant activ-
ity (Table 3). In contrast, the ABTS+• scavenging activity appears
to be consistent with the high TF found in xylanase-treated sam-
ples despite the non significant (p > 0.05) differences with samples
treated with cellulase and the ternary enzyme mixture. These
results were in accordance with those reported by Pacifico et al.
(2013) and Diaz et al. (2014) who reported that methanol extract
of L. nobilis leaves was effective against DPPH and ABTS+• radicals
at higher concentrations (IC50 > 100 ␮g/mL in both assays).
In general, the methanolic extracts of the residual hydrodistilled
bay leaves could be considered as affective scavengers of DPPH
and ABTS radicals, reflecting their ability to donate electrons or
hydrogen atoms to inactivate radical species. This observation is
important when looking for the integral exploitation of bay leaves.
4. Conclusions
In view of these results, it can be concluded that the recovery of
essential oils from bay leaves can be greatly enhanced by the use of
enzymes with cellulolytique and hemicellulolytic activities. While
there are a little change in the qualitative traits of the essential
492 A. Boulila et al. / Industrial Crops and Products 74 (2015) 485–493
oils, enzyme-pre-treatment induces remarkable variations in the
proportion of individual components and enhanced the amounts
of oxygenated monoterpenes leading hence to a better antioxidant
activity of the essential oils derived from enzyme-treated samples.
It was also found that enzyme-assisted extraction showed an effi-
cient way to extract natural antioxidants from leaf hydrodistilled
residues. Consequently, the exploitation of this material (consid-
ered as waste) could be considered as a promising alternative
to synthetic antioxidants, making them particularly attractive for
food, cosmetic and pharmaceutical industries.
Acknowledgments
The authors are indebted to the Direction Générale de la
Recherche Scientifique (DGRST, Tunisia) and the Centre National
de la Recherche Scientifique (CNRS, France) and the Laboratoire
International Associé (LIA) for financial support (Research Project
PHC-Utique N◦ 13G0904).
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