Enzyme-assisted extraction of bioactive compounds from bay leaves (Laurus nobilis L.)

Industrial Crops and Products 74 (2015) 485–493

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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

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Article history: Received 31 January 2015 Received in revised form 7 April 2015 Accepted 21 May 2015 Keywords: Enzymes Laurus nobilis L. Essential oils Phenolics Antioxidant activity Food processing technology

a b s t r a c t Bay leaves (Laurus nobilis L.) are widely used as a condiment and their therapeutic benefits are well known. These biological properties were attributed to a plethora of highly bioactive secondary metabolites namely essential oils and phenolics. However, their recovery from plant matrix is generally limited by the presence of physical barrier (cell wall). Thus, the use of novel extraction procedures to enhance 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 improve the extraction efficiency of bioactive compounds from bay leaves. Results showed that enzyme pre-treatment resulted in 243, 227, 240.54 and 0.48% increase in the essential oil yields in samples treated with cellulase, hemicellulase, xylanase and the ternary mixture, respectively. Compositional analysis by GC and GC–MS revealed remarkable enrichment of the essential oils derived from enzyme-treated samples with oxygenated monoterpenes, leading hence to better antioxidant activity as revealed by the 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, antimicrobial, 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 [email protected], [email protected] (K. Hosni). http://dx.doi.org/10.1016/j.indcrop.2015.05.050 0926-6690/© 2015 Elsevier B.V. All rights reserved.

cineole, linalool and ␣-terpinyl acetate were the basic components of the essential oil of bay leaves (da Silveira et al., 2014). Epicatechin, 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 (Ertas¸ and Alma, 2010). As a consequence, market demand of laurel essential oils and extracts has increased remarkably. This has prompted the search for new methods 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 distillation for essential oils) and water, organic or hydroalcoholic solvents for phenolic compounds. However, a lot of alternative

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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); protein 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 enzymeassisted 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 compounds 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 (Steinheim, Germany). Linalool, ␤-myrcene and terpinen-4-ol were from Fluka Chemicals (Buchs, Switzerland). Phenolic compound standards were from Sigma–Aldrich (St. Louis. MO. USA). Anhydrous sodium sulphate (Na2 SO4 ) and n-alkanes (C7 –C20 ) were obtained from Fluka Chemicals (Buchs, Switzerland). FolinCiocalteu, 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 (H7649, 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).

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 chromatographic 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 temperatures 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 matching 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 consulting, Hamburg, Germany) using the Mass Finder 3 software (www.massfinder.com). Relative percentage amounts of the identified 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).

2.3. Enzyme pre-treatment Dried and ground bay leaves (100 g) were mixed with 500 mL distilled water containing 10 mg of single enzyme (cellulase, hemicellulase 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

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 incubation for 1 h in the dark, the absorbance was measured at 760 nm

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

RIa

RIb

Control

Cellulase

␣-Thujene ␣-Pinene Camphene Sabinene ␤-Pinene ␤-Myrcene 3-Carene 1,8-Cineole ␥-Terpinene Linalool Terpinen-4-ol ␣-Terpineol ␣-Terpinyl acetate Eugenol methyl eugenol ␤-Caryophyllene Elemicine ␦-Cadinene Spathulenol Caryophyllene oxide t-Muurolol

932 939 950 976 981 988 1011 1031 1059 1088 1178 1189 1351 1356 1402 1418 1523 1525 1576 1569 1641

1035 1032 1076 1132 1118 1176 1159 1213 1255 1553 1611 1706 1709 2192 2028 1612 2229 1773 2153 2008 2145

0,44 10,17 0,51 7,26 7,12 0,39 1,45 39,76 0,61 10,03 0,87 1,35 13,35 0,07 2,98 1,41 – 0,46 0,82 0,48 0,11

–

Group components Monoterpene hydrocarbons Oxygenated monoterpenes Susquiterpene hydrocarbons Oxygentaed sesquiterpenes Oxygenated/Hydrocarbons Total identified Essential oil yield (% w/w)

27,95 68,41 1,87 1,41 2,34 99,64 0.37

7,34 – 7,38 5,66 – – 48,33 0,56 9,2 1,78 2,13 10,39 0,35 5,22 1,46 – – – 0,13 20,94 77,4 1,46 0,13 3,46 99,93 1.25

Hemicellulase

Xylanase

0,37 7,65 0,36 7,27 5,49 0,36

0,42 7,84 0,32 7,15 5,54 0,37

– 42,16 0,72 8,36 2,17 2,51 12,35 0,16 7,37 1,87 – – – 0,71 0,09

– 38,38 0,72 8,17 2,19 2,6 13,54 0,12 8,53 2,07 0,62 – 0,55 0,78 0,07

22,22 75,08 1,87 0,8 3,15 99,97 1.19

22,36 73,53 2,69 1,4 2,99 99,98 1.24

Ternary Mixturea – 5,33 – 6,64 4,96 – – 33,86 – 8,35 1,27 1,57 18,05 0,09 12,48 2,95 1,41 – 0,79 2,12 0,11 16,93 75,67 4,36 3,02 3,70 99,98 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). Gallic acid was used as the standard, and results were expressed as microgram of gallic acid equivalents (␮g GAE/g).

bition (IC50 ) expressed as ␮g/mL was determined from the graph of the free radical scavenging activity (%) against the extract concentration.

2.6.2. Determination of total flavonoid (TF) content Total flavonoid (TF) content was determined by the AlCl3 colorimetric 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 equivalents (␮g QE/g).

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 concentrations of essential oils (20, 50, 100, 200, 500, 1000 and 2000 ␮g/mL) or trolox standard. The mixture was left to stand at room temperature 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+• .

2.7. 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 solution. 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 radical 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: %DPPHradicalscavenging =

(A0 − As ) × 100 A0

where A0 and As are the absorbance of the control and the sample, respectively. The effective concentration having a 50% radical inhi-

2.8. Statistical analysis All measurements were carried out in triplicate and the results were presented as mean values ± SD. Statistical analyses were performed 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

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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, hemicellulase and xylanase). This increase in recovery can be attributed to the ability of enzymes to degrade cell wall structure and depolymerize 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 polysaccharides. 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 xylooligomers 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 mechanistic 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 enzymatic pre-treatment of bay leaves could overcome such inhibitory effect.

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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 identification 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 (1,8cineole > ␣-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 Croatia (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 artefacts 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 compounds 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 reduction 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 application 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 (commercial mixture of enzymes including cellulase, hemicellulase, pectinase, arabinase and xylanase) afforded enriched hydrocarbons 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

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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 pretreatment and/or they were glycosidically bounded. To investigate whether enzyme treatment induces transformations of volatile components or liberates bound volatiles, two additional experiments were undertaken. The first experiment consists on producing a reconstituted hydrolysate by mixing pure essential oil with distilled water and enzymes (cellulase and hemicellulase 1:1) under the same conditions described above (1 h at 40 ◦ C). The control sample 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 subsequently analyzed by GC and GC–MS. In the second experiment and in order to determine if the enzyme treatment was able to liberate 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 procedures 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 literature data clearly underscored that enzyme treatment did not induce transformation of the volatile components, but it contributes presumably 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 consequently 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 hemicellulase 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

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

Extract yield (mg/g) TPC (mg GAE/g) TFC (mg QE/g) *

Water

Methanol

80% methanol

Ethyl acetate

Dichloromethane

26.60 ± 2.18e * 0.50 ± 0.12c 0.15 ± 0.01e

92 ± 5.77a 5.87 ± 0.40a 5.18 ± 0.08a

61.60 ± 3.42b 4.02 ± 0.28b 3.46 ± 0.41b

34.60 ± 4.17d 4.61 ± 0.42b 1.05 ± 0.07d

42 ± 2.12c 4.12 ± 0.60b 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 Control Cellulase Hemicellulase Xylanase Ternary mixture a b c *

5.85 7.12 6.89 6.64 6.33

TFCb ± ± ± ± ±

0.40a,b,* 0.64a 0.62a,b 0.49a 0.54ab

5.18 5.79 5.35 6.33 6.09

DPPHc ± ± ± ± ±

0.08a,b 0.41a 0.22a,b 0.38a 0.78a

734.8 478.65 591.22 690.91 650.75

ABTSc ± ± ± ± ±

42.71a 43.98b 51.64a,b 45.63a 21.57ab

1391.36 1142.91 1371.68 1073.73 1212.74

± ± ± ± ±

64.07a 46.87a,b 38.79a 33.60a,b 34.68ab

TPC was expressed as mg gallic acid equivalents per gram of extract. TFC was expressed as mg quercetin equivalents per gram of extract. 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 underscored 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 including 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 essential 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 residual 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+• radical 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 activity (Table 3). In contrast, the ABTS+• scavenging activity appears to be consistent with the high TF found in xylanase-treated samples 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

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