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Sweet bay (Laurus nobilis L.) essential oil and its chemical composition, antioxidant activity and leaf micromorphology under different extraction methods
Sustainable Chemistry and Pharmacy 9 (2018) 12–18
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Sweet bay (Laurus nobilis L.) essential oil and its chemical composition, antioxidant activity and leaf micromorphology under different extraction methods
T
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Azin Tabana, Mohammad Jamal Saharkhiza,b, , Mehrdad Niakousaric a
Department of Horticultural Science, Faculty of Agriculture, Shiraz University, Shiraz, Iran Medicinal Plants Processing Research Center, Shiraz University of Medical Sciences, Shiraz, Iran c Department of Food Science and Technology, Faculty of Agriculture, Shiraz University, Shiraz, Iran b
A R T I C LE I N FO
A B S T R A C T
Keywords: Sweet bay Extraction method Essential oil Antioxidant activity, SEM images
This research explored the essential oil (EO) of Laurus nobilis. Various parameters were recorded, including the amount of yield, chemical composition and antioxidant activity. The EO was obtained by hydro-distillation (HD), hydro-steam distillation (HSD), microwave-assisted hydrodistillation (MAHD), and ohmic-assisted hydrodistillation (OAHD). The micromorphology of the plant leaves were studied under the different extraction methods. The yields of EOs obtained by the mentioned extraction methods were 1.40, 0.74, 1.00 and 0.83 (% w/ w), respectively. The main chemical components in EOs obtained by these methods were eucalyptol (34.4–50.0%), α-terpinenyl acetate (14.9–18.8%), terpinene-4-ol (4.7–6.0%) and sabinene (4.9–5.9%). Two of the extraction methods, i.e. OAHD and MAHD, yielded EOs that contained higher proportions of eucalyptol. SEM images of the leaves were taken after extraction. It was observed that the MAHD method had the most destructive effect on secretory cells, while the HSD method failed to damage numerous cells in the leaves. Generally, the results suggest that MAHD and OAHD can be recognized as clean and faster methods because of their shorter processing time and less energy requirements. The HD and HSD methods extracted EOs with more sesquiterpenes, while the MAHD and OAHD methods yielded higher amounts of oxygenated monoterpenes in the EO.
1. Introduction Laurus nobilis L., commonly known as sweet bay, bay leaves or bay laurel, is an evergreen shrub or small tree that measures up to 2.1 m in height. It belongs to the Lauraceae family and is native to the southern Mediterranean region. The species was brought to Iran during the Qajar dynasty period (1794–1921) and is now widely cultivated in south, central and north of Iran as an ornamental plant (Zargari, 1990). The commercial value of L. nobilis results from the essential oil (EO) obtained from its leaves. The chemical composition of sweet bay EO has been studied by different researchers. In most cases, 1,8-cineole and αterpinyl acetate are the major components of L. nobilis EO (daSilveira et al., 2014; Pilar Santamarina et al., 2016; Sellami et al., 2011). The global demand for sweet bay EO is more than 3000 t per year (UNCTAD, 2006). This oil is used in cosmetics (i.e. creams, perfumes and soaps), in food and agriculture sectors (as a preservative, spice and flavouring agent and as a natural pesticide in postharvest crop protection). It is also used for medicinal purposes (i.e. treating rheumatic
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pain, antimicrobial, pulled muscles and digestive disorders) (Sellami et al., 2011; Pacifico et al., 2013; daSilveira et al., 2014; Pilar Santamarina et al., 2016; De Corato et al., 2010; Van Wyk and Wink, 2004). Accordingly, the market demand for sweet bay EO has increased remarkably. This has prompted the search for the best extraction method to improve both yield and quality in sweet bay EO by reducing the extraction time, preventing degradation of thermally susceptible components and using lower energy consumption (Seidi Damyeh et al., 2016). The development of new extraction techniques such as supercritical fluid extraction, microwave-assisted hydrodistillation, and ohmic-assisted hydrodistillation (Seidi Damyeh et al., 2015) has received much attention recently due to their lower amounts of waste water, shorter extraction time and optimal energy costs (Memarzadeh et al., 2015; Seidi Damyeh et al., 2015). Several studies have shown that EO extraction methods can affect the quality and quantity of the EO. In this context, Sefidkon et al. (2007) studied the effects of distillation methods on the EO content and composition of Satureja rechingeri, whereby the hydro-distillation and steam-distillation produced the
Correspondence to: Department of Horticultural Sciences, College of Agriculture, Shiraz University, Shiraz, Iran. E-mail address: [email protected] (M.J. Saharkhiz).
https://doi.org/10.1016/j.scp.2018.05.001 Received 16 February 2018; Received in revised form 28 April 2018; Accepted 1 May 2018
Available online 07 May 2018 2352-5541/ © 2018 Elsevier B.V. All rights reserved.
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2.3. GC/MS identification
highest and the lowest EO yield, respectively. In a study reported by Sefidkon and Rahimi-Bidgoly (2003) on the extraction of EO from Thymus kotschyanus by hydro- and steam- distillation methods showed that the highest amounts of thymol and carvacrol in EO were obtained by the steam-distillation method. Memarzadeh et al. (2015) indicated that the highest EO yields of Satureja bachtiarica were obtained by HD via the Clevenger apparatus and by the microwave-assisted steam hydro-diffusion. Moreover, significant differences were observed among the major constituents of EOs, including thymol and carvacrol, by the different extraction methods. The antioxidant activity of L. nobilis EO has been reported by several authors (Conforti et al., 2006; Ramos et al., 2012; Cherrat et al., 2013). However, no attempts were made to compare the antioxidant activity of EOs extracted by the different extraction methods. The rising market demand for the EO of sweet bay leaves and its various usages prompted us to conduct this experimental research. The objectives of the current study were to compare: (1) the performance of four extraction methods, i.e. hydrodistillation (HD), hydro-steam distillation (HSD), microwaveassisted hydrodistillation (MAHD) and ohmic-assisted hydrodistillation (OAHD) for the extraction of EO from L. nobilis, (2) the EOs chemical composition and antioxidant activity as affected by the different extraction methods, and (3) the structural changes in the oil cells in the plant leaves after extraction.
The extracted EOs were analysed by GC–MS. The GC analysis was performed using an Agilent gas chromatograph series 7890-A equipped with a flame ionization detector (FID). The analysis was carried out on fused silica capillary HP-5 column (30 m × 0.32 mm i.d., with a film thickness of 0.25 µm). The injector and detector temperatures were maintained at 250 °C and 280 °C, respectively. Nitrogen was used as carrier gas at a flow rate of 1 mL/min. The oven temperature program increased from 60° to 210°C, at a rate of 4 °C/min, which was then programmed to 240 °C, at a rate of 20 °C/min. In the end, the condition was maintained isothermally for 8.5 min. The split ratio was 1:50. The GC–MS analysis was carried out via the Agilent gas chromatograph equipped with fused silica capillary HP-5MS column (30 m × 0.25 mm i.d.; film thickness 0.25 µm) coupled with 5975-C mass spectrometer. Helium was used as carrier gas at a flow rate of 1.1 mL/min, with the ionization voltage of 70 eV and 0.1 μL. The samples were injected manually in the split mode. Ion source and interface temperatures were 230 °C and 280 °C, respectively. Mass range was valued between 45 and 550 amu. The oven temperature program was the same as the temperature for the GC. The retention indices for all components were determined by using n-alkanes as standard. The compounds were identified by comparing their retention indices (RI, HP-5) with those reported in the literature and also by comparing their mass spectra with the Wiley GC–MS Library, Adams Library, Mass Finder 2.1 Library data, and published mass spectra data (Adams, 2007; Karami and Rowshan, 2015).
2. Material and methods 2.1. Plant materials
2.4. Antioxidant activity assay L. nobilis leaves were collected in April 2015 from the Eram Botanical Garden, Shiraz University, Shiraz, Iran, (29°36 ´ N and 52°32 ´ W). Laurus nobilis was identified and authenticated by a senior plant taxonomist at Shiraz University Herbarium, Shiraz, Iran. The voucher specimen (802 LL) was deposited in the Herbarium. The leaves were dried in the shade (20–25 °C) for a week in the laboratory. They were packaged in high density polyethylene bags and were then carefully ground and sifted through a mesh screen (with 0.5 mm pores) to obtain uniform leaf material prior to each experiment.
Free radical scavenging activity was measured by 2,2-diphenyl-1picrylhydrazyl (DPPH; Sigma–Aldrich). The effect of EOs on DPPH degradation was estimated according to the method used by Cherrat et al. (2013). Different concentrations (1, 2, 4, 6, 8, 10 μL mL−1) of the EOs were prepared in pure methanol, and then 150 μL of each concentration were added to 50 μL of 1 mmol L−1DPPH. The mixture was then kept at room temperature in the dark for 30 min. The absorbance of the resulting solutions was measured at 517 nm after 30 min. The DPPH radical scavenging ability of each EO extract was calculated according to the following equation:
2.2. EO extraction procedures
%scavenging of DPPH radical = [(Ablank –Asample) / Ablank ] × 100 Where Ablank is the absorbance of the control reaction (containing all reagents except the EOs), and Asample is the absorbance by samples. In order to calculate the sample concentration that can provide 50% inhibition (IC50), the inhibition percentages were plotted against sample concentrations. All tests were carried out in triplicate (Seidi Damyeh et al., 2016).
The extraction of the EOs from L. nobilis was performed by four different methods; hydro-distillation (HD), hydro-steam distillation (HSD), microwave-assisted hydro-distillation (MAHD), and ohmic-assisted hydrodistillation (OAHD) (Seidi Damyeh et al., 2015) (Fig. 1): (1) HD was carried out in a British Pharmacopeia Clevenger-type apparatus using an electric mantle (400 W) heater for 150 min (Memarzadeh et al., 2015). (2) HSD performed in a Kaiser and Lang apparatus using an electric mantle (400 W) heater for 150 min (Sefidkon et al., 2007). (3) MAHD operated via a microwave that was set to 400 W for 45 min (Memarzadeh et al., 2015) (4) OAHD operated by an ohmic heating system that was set to 120 V for 45 min. A small quantity of sodium chloride (0.3% w/v of Na Cl) provided sufficient electrical conductivity between two electrodes in the heat-up process. The exact amount of NaCl was added to water in other extraction methods (Seidi Damyeh et al., 2015).
2.5. Scanning electron microscopy (SEM) SEM images of dried leaves were taken from untreated samples (L. nobilis leaves) and also from samples treated with HD, HSD, MAHD and OAHD. The dried leaves were fixed on an aluminium sample holder and were then sputtered with gold in a sputter coater. All samples were examined with a scanning electron microscope (TescanVEGA3, Czech Republic) under high-vacuum conditions. An accelerating voltage of 20.0 kV was provided, and the working distance ranged between 23.71 and 46.31 mm (i.e. the distance between the surfaces of the sample and the microscope lens).
For each method, 50 g of L. nobilis was extracted with 0.5 L of distilled water. Results were reported in grams of EOs per 100 g of dried leaves. Each experiment was performed at least in triplicate. The EOs were dried by anhydrous sulphate and stored at 4 °C until further analysis.
2.6. Statistical analysis Data were submitted to an analysis of variance (ANOVA) in SPSS (version15.0.0; IBM Institute Inc., USA) and mean values were compared via the LSD test at 5% level. 13
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Fig. 1. Method scheme; HD: Hydro-distillation; HSD: Hydro-steam distillation; MAHD: Microwave-assisted hydrodistillation; OAHD: Ohmic-assisted hydrodistillation.
3. Results
the extraction.
3.1. Effect of extraction methods on the yield, colour and extraction time of EOs
3.2. Effect of extraction methods on the EOs composition Chemical composition of EOs obtained by different methods was identified via a comprehensive GC/MS analysis (Table 1). Eighteen, nineteen, twenty-five and thirty components represented 95%, 97%, 95% and 94% of the total EOs extracted by MAHD, OAHD, HD and HSD, respectively. The main components of the EOs obtained from the different extraction methods were eucalyptol (34.37–5.07%), α-terpinenyl acetate (14.93–18.78%), terpinene-4-ol (4.72–6.02%) and sabinene (4.95–5.93%) (Table 1). All EOs were found to be rich in oxygenated monoterpenes (such as eucalyptol and α-terpinenyl acetate). Among the extraction methods, OAHD yielded the highest percentages of eucalyptol (50.07%), terpinene-4-ol (6.02%) and linalool (2.72%), although it managed to yield the lowest percentages of other components such as α-terpinenyl acetate (14.93%) and caryophyllene oxide (1.32%). Accordingly, the HD and HSD methods enhance “extraction selectivity” for sesquiterpenes by more than twofold in L. nobilis (Table 1). Despite the differences in the quantity of the main components, small differences can also be observed in the quantity of other components such as α-pinene (2.94–4.11%), β-pinene (2.51–3.22%), cis-sabinene hydrate (0.49–1.03%), eugenol (0.55–1.03%), methyleugenol (2.34–3.99%) and trans-caryophyllene (0–1.33%).
EOs extracted from the leaves of L. nobilis by HD and HSD methods produced a colourless liquid, while those obtained from MAHD and OAHD methods were a pale yellow liquid. Results indicated that the extraction method had a significant effect on the EO yield of sweet bay leaves (P ≤ 0.05). The highest and lowest EO yields were obtained by the HD and HSD methods, respectively (Fig. 2). However, no significant difference was observed between the EO yield of HSD, MAHD and OAHD (Fig. 2). The HD and HSD methods required a longer extraction time (150 min), while OAHD and MAHD required 45 min to complete
3.3. Antioxidant activity Fig. 2. L. nobilis essential oil yield obtained through four extraction methods, HD: Hydro-distillation; HSD: Hydro-steam distillation; MAHD: Microwave-assisted hydrodistillation; OAHD: Ohmic-assisted hydrodistillation. Means with same letter are not significantly different using LSD test (P ≤ 0.05).
The EOs obtained from different methods were tested individually against DPPH free radical to investigate their antioxidant capacity (Table 2). ANOVA analysis of IC50 identified significant differences between the four methods (P < 0.05). LSD test subdivided them into 14
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Table 1 Chemical composition of the L. nobilis essential oils obtained through four extraction methods; HD: Hydro-distillation; HSD: Hydro-steam distillation; MAHD: Microwave-assisted hydrodistillation; OAHD: Ohmic-assisted hydrodistillation. no
Component
MAHD
OAHD
HD
HSD
a
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
α-Thujene α-Pinene Camphene Sabinene β-Pinene β-Myrcene α-Terpinene Eucalyptol γ-Terpinene cis-Sabinene hydrate Linalool Cis ρ Menth− 2en− 1-ol trans-Pinocarveol δ-terpineol Terpinene− 4-ol α - terpineol Myrtenol Bornyl acetate Carvacrol α-terpinenyl acetate Eugenol Neryl acetate β-elemene Methyleugenol trans-Caryophyllene Methyl isoeugenol (E) Spathulenol Caryophyllene oxide caryophylla− 4(12),8(13)dien− 5β-ol β-Eudesmol Total identification Monoterpenes Sesquieterpenes Other compounds Unknown compounds
– 2.94 – 5.25 2.51 – – 49.73 – 1.03 2.51 – 0.64 0.94 5.96 2.88 – 0.85 – 16.92 0.55 0.57 0.5 2.34 0.67 – – 1.83 –
0.59 3.9 0.57 5.69 2.96 – – 50.07 – 0.76 2.72 – 0.73 0.86 6.02 2.40 0.54 0.82 – 14.93 0.63 0.5 – 2.66 – – – 1.32 –
0.6 4.11 0.56 5.93 3.22 0.72 – 37.53 0.65 0.52 1.6 – 0.47 0.78 4.72 2.41 0.38 0.83 – 18.65 0.83 0.63 1.05 2.77 1.33 – – 3.26 0.88
0.48 3.28 0.45 4.95 2.85 0.61 0.36 34.37 0.74 0.49 2.08 0.4 0.64 0.82 5.59 2.1 0.53 0.9 0.4 18.78 1.03 0.57 0.71 3.99 0.8 0.52 1.1 2.30 0.94
928 936 951 978 982 992 1020 1047 1061 1071 1103 1124 1142 1172 1185 1197 1201 1289 1309 1355 1363 1367 1396 1408 1426 1499 1580 1592 1644
930 939 954 975 980 990 1017 1039 1059 1070 1096 1121 1139 1166 1177 1188 1194 1288 1298 1349 1359 1361 1390 1403 1419 1492 1587 1583 1640
– 95.73 95.62 3 2.89 1.39
– 98.67 94.06 1.32 3.29 1.32
0.73 95.16 94.31 7.25 3.6 4.84
1 94.74 82.35 6.85 5.54 5.25
1659 – – – – –
1650 – – – – –
30
KIc
b
Table 2 Antioxidant activity of the L. nobilis essential oils obtained through four extraction methods; HD: Hydro-distillation; HSD: Hydro-steam distillation; MAHD: Microwave-assisted hydrodistillation; OAHD: Ohmic-assisted hydrodistillation.
KIr
Extraction method
Concentration (μL/mL)
Inhibition (%)
IC50 (μL/mL)
HD
10 8 6 4 2 1 10 8 6 4 2 1 10 8 6 4 2 1 10 8 6 4 2 1
92.4 91.1 85.9 75.3 56.6 42.1 93.7 91.2 86.2 73.5 52.4 38.6 83.7 72.4 65.8 50.9 40.9 34.6 89.7 84.4 76.9 62.6 44.7 35.4
0.84 ± 0.308 c
HSD
MAHD
OAHD
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2.5 0.7 1.0 0.6 2.8 1.5 0.4 0.3 1.4 1.9 2.5 0.5 7.0 8.6 8.6 6.31 1.9 2.4 1.6 3.8 3.3 4.3 4.9 4.1
1.4 ± bc
3.7 ± 0.9 a
2.5 ± 1.0 ab
IC50 (inhibitory concentration at 50%); Means with same letter are not significantly different as indicated byDuncan multiple range test (P ≤ 0.05).
durations of the different methods. The chemical composition of the different EOs were not similar, which also explains the variety of EO colours. Moreover, by increasing the extraction time in HD and HSD, more chemical components were recovered. As stated before, the HD and HSD methods yielded twice as many components than MAHD and OAHD. Nonetheless, it has been previously reported that other physical properties of the EO could be affected by the number of components in the EO (Pearson, 1976). Three extraction methods, i.e. HSD, MAHD and OAHD provided similar EO yields. The maximum EO yield (1.43%) was obtained by HD which was significantly higher than other methods studied herein, while the lowest yield was observed in HSD. Similar results were observed by Sefidkon et al. (2007) who reported that the highest EO content of Satureja rechingeri was obtained by the HD method. However, the results of a study by Sefidkon et al. (1999) indicated that the HSD and HD methods yielded similar EO contents of Thymus kotschyanus. It is believed that the HD is a reference method used for the quantification of EOs. In fact, the HD has been capable of yielding the highest amount of EO in previous studies (Sefidkon et al., 2007, 1999; Memarzadeh et al., 2015). However, the HSD yielded the lowest amount of EO in this study, compared to other methods, which is probably due to the condition of plant material, mode of communication and mode of charging. Although all extracted EOs contain the same main components (eucalyptol, α-terpinenyl acetate, terpinene-4-ol and sabinene), their respective quantities vary more or less with regard to the extraction technique (Table 1). Previous reports confirm that the main components reported in the present study are the most abundant component in L. nobilis EO (Di Leo Lira et al., 2009; Sellami et al., 2011; Pilar Santamarina et al., 2016). Applying MAHD and OAHD resulted in a drastic increase (about 30%) in the content of eucalyptol compared to HD and HSD methods (49.73% and 50.07% versus 37.53% and 34.37%). Di Leo Lira et al. (2009) indicated that if the distillation time of the sweet bay leaves is programmed to be less than an hour, the concentration of eucalyptol (boiling point: 176 °C; mass: 154) would be higher compared to a prolonged distillation which would yield higher
a Experimentally determined Kovats retention index (KI) relative to C19-C18 n-alkanes on the DB-5MS column. b Literature Kovats retention index (Adams, 2007).
three groups. The HD yielded an EO which exhibited the highest activity against DPPH, while the HSD yielded the weakest activity. However, there was no significant difference between the antioxidant activity of EOs which were obtained by the other methods (i.e. MAHD and OAHD) (Table 2). 3.4. Structural changes during EOs extraction The images of secretory cells in the L. nobilis leaves were taken by scanning electron microscopy (SEM) before and after the extraction process (Fig. 3). Fig. 3(A) is a micrograph of the untreated cells before the extraction. Fig. 3(B) and (C) show the SEM images of sweet bay cells that had undergone HD (150 min) and HSD (150 min), respectively. The SEM images display sweet bay cells after 45 min of OAHD and 45 min of MAHD (Fig. 3D and E, respectively). MAHD demonstrated a destructive effect on secretory cells, while the HSD failed to damage numerous cells in the leaves. The morphological shapes of cells after HD and MAHD are relatively similar (Fig. 3). 4. Discussion In this study, different extraction methods yielded EOs of different colours. So far, no published work has reported the difference in the colour of EOs extracted by different methods. Here, the difference in EO colour could be due to the different extraction temperatures and 15
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Fig. 3. Scanning electron micrographs of the L. nobilis leaves after *EO extraction: (A) untreated-2500X, (B) after HD**− 2500X, (C) after HSD-2500X, (D) after OAHD-2500X, (E) after MAHD-2500X. *EO: Essential oil; **HD: Hydro-distillation; HSD: Hydro-steam distillation; MAHD: Microwave-assisted hydrodistillation; OAHD: Ohmic-assisted hydrodistillation.
heating efficiency of the MAHD and OAHD has been already confirmed by the measurement of the consumed electric energy (Gavahian et al., 2015) where the power consumption was measured for the extraction of 1 mL Mentha piperita EO by MAHD and OAHD versus the electromantle. Previous results indicated a substantial saving in the extraction cost by MAHD and OAHD compared to the conventional HD extraction technique (Gavahian et al., 2015). EO extraction from Satureja macrosiphonia by MAHD and OAHD methods also indicated a remarkable decrease in the extraction time and energy, compared to the conventional HD method (Seidi Damyeh et al., 2015). Furthermore, the MAHD is less tedious as it minimizes the risk of prolonged exposure to heat and therefore minimizes the degradation of compounds. Previous studies have shown that major component of sweet bay EO and their values can affect EO quality and its biological activity such as antibacterial, antiviral, antifungal and antioxidant activity (Sellami et al., 2011; Pilar Santamarina et al., 2016). Sweet bay EO demonstrated the ability to sequester the free radical DPPH (Table 2). In agreement with our results, previous studies have shown that sweet bay EOs exhibit an effective antioxidant activity when tested by the DPPH radical scavenging method and by the β-carotene–linoleic acid method (Cherrat et al., 2013; Ramos et al., 2012). The IC50 (inhibitory concentration at 50%) is a widely used parameter in evaluating the free radical scavenging activity. A lower IC50 value is attributed to a higher antioxidant activity (Shimada et al., 1992). In this study, EOs obtained
proportions of terpinyl acetate (BP: 210 °C; M:196) and methyl eugenol (BP: 255 °C; M: 178). Similar results have been obtained in this study, using HD and HSD methods (150 min) which increased the values of methyl eugenol and α-terpinenyl acetate compared to MAHD and OAHD extractions (45 min). Altogether, it could be stated that the different extraction methods of the EOs influenced the amount of the chemical classes. In particular, sesquiterpenes were extracted in higher amounts using HD and HSD methods, whereas the MAHD and OAHD increased the amount of oxygenated monoterpenes. The latter results are in line with a report by Flamini et al. (2007). The higher amounts of oxygenated components in OAHD and MAHD are probably attributed to the higher dipolar moment and diminution of thermal effects (Seidi Damyeh et al., 2015). It is also interesting to note that the longer extraction time not only can recover more components but also may cause the deterioration of some other components and can reduce their amounts. A very long and direct contact of the plant materials with boiling water can cause the deterioration. Therefore, faster methods such as MAHD and OAHD can minimize the risk of heat-related degradation of the compounds (Flamini et al., 2007; Gavahian et al., 2015; Seidi Damyeh et al., 2015). In terms of the extraction time, OAHD and MAHD warm up the mixture faster than the heating mantle-based methods. The required time for the extraction process is therefore reduced because of the higher extraction rate (0.84% and 1.01% in 45 min). The greater 16
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performed more optimally. In the case of structural changes to the cells after extraction, the MAHD was more successful. It can be concluded that selecting the best technique for EO extraction from L. nobilis is completely dependent on the aim of the extraction. MAHD and OAHD produce good quality EOs at faster rates which could save energy and are more environmental friendly, while the HD can extract EOs with higher yield and stronger antioxidant activity, but HSD does not seem to be a good option for the extraction of L. nobilis in general.
from four extraction methods showed dose-dependent radical scavenging activities, and there was a significant difference between EOs obtained through the different methods. Definitely, the different antiradical activities are due to the respective differences in the chemical composition of EOs. Furthermore, the EOs obtained through HD showed the highest DPPH scavenging, and this correlates with higher amounts of monoterpenes and sesquiterpenes present in the EO (Bartikova et al., 2014). Eucalyptol is known to possess a strong antioxidant activity. (Ramos et al., 2012). Despite the low amount of eucalyptol in the HD essential oil, compared to MAHD and OAHD, the EO obtained by HD exhibited a stronger antioxidant activity. This pattern might be due to a synergistic interaction between minor and major constituents of EOs (Tepe et al., 2005). Results of the present study are in line with those published by Seidi Damyeh et al. (2016) who reported a higher antioxidant activity of the Prangos ferulacea EO which had been extracted by the HD, compared to ultrasound. Bartikova et al. (2014) indicated that many of the biological activities attributed to sesquiterpenes, such as antimicrobial and anti-inflammatory effects, are based on the antioxidant actions of the sesquiterpenes. Thus, the higher DPPH scavenging activity of EOs obtained from HD can be related to the higher amount of sesquiterpenes compared to other extraction methods (Table 2). The leaves of sweet bay have secretory cells (oil cells) which secrete EO (Dickison, 2000). All extraction methods in this study caused physical changes to the cells along the structure of leaves, as internal secretory structures. HD and OAHD extraction methods caused evident ruptures in the cells of sweet bay leaves during the extraction (Fig. 3B and D). However, in HSD, numerous cells in the leaves were left intact (Fig. 3C), and therefore a lower amount of EO was obtained (Fig. 2). In the case of MAHD, a severe disruption of the oil cells happened and cells became wrinkled (Fig. 3E) while those that had undergone other methods were not so damaged and were more rounded in shape. This phenomenon can be associated with the pattern of heat distribution during MAHD extraction. In MAHD, the radiated microwave energy is immediately converted into heat energy which is transferred directly to the plant material (Desai et al., 2010). This heat energy can therefore be localized at particular parts of the plant containing the oil cells, thereby damaging the cells. Meanwhile, the pattern of heat transfer in HD and HSD methods is mainly performed slowly by conduction and convection, and the heat energy first reaches the solvent before heating the targeted plant material (Gavahian et al., 2015). These results of the present study are not in line with those reported by Jeyaratnam et al. (2016) who studied the extraction of EO from Cinnamomum cassia bark, using the HD and MAHD, and the latter method caused less damage to oil glands. This could be due to differences in secretory structures (cells versus glands) and plant organs (leaves versus barks). Regarding the OAHD method, in solid materials such as plant leaves in which the cell structure is intact, electrical conductivity is dependent on electric field power, and is controlled by applying a certain degree of voltage, and cellular breakdown occurs by electro-permeabilization (Seidi Damyeh et al., 2015). However, since more ruptured cell structures are observed in the sample treated with OAHD, the electroporation effect may be observed. In this regard, our data were in good agreement with those published by Seidi Damyeh and Niakousari (2015).
Acknowledgements The authors wish to extend their thanks and appreciation to Shiraz University Research and Technology Council as well as Shiraz University of Medical Sciences for their financial supports (Grant No: 1396-01–70-15354). We also thank Mohsen Hamedpour-Darabi for editing the research language. References Adams, R.P., 2007. Identification of Essential Oil Components by Gas Chromatography/ Mass Spectrometry, 4th ed. Allured Publishing, Carol Stream,IL. Bartikova, H., Hanusova, V., Skalova, L., Ambroz, M., Bousova, I., 2014. Antioxidant, prooxidant and other biological activities of sesquiterpenes. Curr. Top. Med. Chem. 14 (22), 2478–2494. Cherrat, C., Espina, L., Bakkali, M., Garcia-Gonzalo, D., Pagan, R., Laglaoui, A., 2013. Chemical composition and antioxidant properties of LaurusnobilisL. and Myrtus communis L. essential oils from Morocco and evaluation of their antimicrobial activity acting alone or in combined processes for food preservation. J. Sci. Food Agric. 94, 1197–1204. Conforti, F., Statti, G., Uzunov, D., Menichini, F., 2006. Comparative chemical composition and antioxidant activities of wild and cultivated Laurus nobilis L. leaves and Foeniculum vulgare subsp. piperitum (Ucria) Coutinho seeds. Biol. Pharm. Bull. 29, 2056–2064. daSilveira, S.M., Luciano, F.B., Fronza, N., Cunha Jr., A., Scheuermann, G.N., Werneck Vieira, C.R., 2014. Chemical composition and antibacterial activity of Laurus nobilis essential oil towards food borne pathogens and its application in fresh Tuscan sausage stored at 7 °C. LWT - Food Sci. Technol. 59, 86–93. De Corato, U., Maccioni, O., Trupo, M., Di Sanzo, G., 2010. Use of essential oil of Laurus nobilis obtained by means of a supercritical carbon dioxide technique against post harvest spoilage fungi. Crop Prot. 29, 142–147. Desai, M., Parikh, J., Parikh, P., 2010. Extraction of natural products using microwaves as a heat source. Sep. Purif. Rev. 39 (1–2), 1–32. Di Leo Lira, P., Retta, D., Tkacik, E., Ringuelet, J., Coussio, J.D., Van Baren, C., Bandoni, A.L., 2009. Essential oil and by-products of distillation of bay leaves (Laurus nobilisL.) from Argentina. Ind. Crops Prod. 30, 259–264. Dickison, W.C., 2000. Integrative Plant Anatomy. Novanet: Dalhousie Killam Library SCI QK 641 D53 2000. New York: Academic Press, San Diego. Flamini, G., Tebano, M., Luigi Cioni, P., Ceccarini, L., Simone Ricci, A., Longo, I., 2007. Comparison between the conventional method of extraction of essential oil of Laurus nobilis L. and a novel method which uses microwaves applied in situ, without resorting to an oven. J. Chromatogr. A 1143, 36–40. Gavahian, M., Farahnaky, A., Farhoosh, R., Javidnia, K., Shahidi, F., 2015. Extraction of essential oils from Mentha piperita using advanced techniques: microwave versus ohmic assisted hydrodistillation. Food Bioprod. Process. http://dx.doi.org/10.1016/j. fbp.2015.01.003. Jeyaratnam, N., Hamid Nour, A., Kanthasamy, R., Hamid Nour, A., Yuvaraj, A.R., OlabodeAkindoyo, J., 2016. Essential oil from Cinnamomum cassia bark through hydrodistillation and advanced microwave assisted hydrodistillation. Ind. Crops Prod. 92, 57–66. Karami, A., Rowshan, V., 2015. Temporal analysis of volatile oil compounds from winter season flowering ‘Eureka’ lemon. Anal. Chem. Lett. 5, 31–37. Memarzadeh, S.M., GhasemiPirbalouti, A., AdibNejad, M., 2015. Chemical composition and yield of essential oils from Bakhtiarisavory (Satureja bachtiarica Bunge.) under different extraction methods. Ind. Crops Prod. 76, 809–816. Pacifico, S., Gallicchio, M., Lorenz, P., Potenza, N., Galasso, S., Marciano, S., Fiorentino, A., Stintzing, F.C., Monaco, P., 2013. Apolar Laurus nobilis leaf extracts induce cytotoxicity and apoptosis towards three nervous system cell lines. Food Chem. Toxicol. 62, 628–637. Pearson, D., 1976. The Chemical Analysis of Foods, 7ed. Churchill Livingstone, pp. 6–14. Pilar Santamarina, M., Rosello, J., Gimenez, S., Amparo Blazquez, M., 2016. Commercial Laurus nobilis L. and Syzygium aromaticum L. Merr.& Perry essential oils against postharvest phytopathogenic fungi on rice. LWT - Food Sci. Technol. 65, 325–332. Ramos, C., Teixeira, B., Batista, I., Matos, O., Serrano, C., Neng, N.R., Nogueira, J.M.F., Nunes, M.L., Marques, A., 2012. Antioxidant and antibacterial activity of essential oil and extracts of bay laurel Laurus nobilis Linnaeus (Lauraceae) from Portugal. Nat. Product. Res. 26 (6), 518–529. Sefidkon, F., Abbasi, K., Jamzad, Z., Ahmadi, S., 2007. The effect of distillation methods and stage of plant growth on the essential oil content and composition of Satureja rechinger jamzad.J. Food Chem. 100, 1054–1058. Sefidkon, F., Dabiri, M., Rahimi-Bidgoly, A., 1999. The effect of distillation methods and
5. Conclusion The EOs were extracted from sweet bay samples by using HD, HSD, MAHD and OAHD methods. Different extraction methods yielded EOs with a variety of chemical compositions, antioxidant activities and yield parameters. The main EO components obtained through the different methods were similar. However, drastic differences were observed in the quantity of components, depending on the extraction processes. MAHD and OAHD offered good advantages in terms of their selectivity for eucalyptol, whereas more sesquiterpenes were recovered by the HD technique. Regarding EO yield and antiradical activity, the HD 17
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Satureja macrosiphonia essential oil. J. Food Process. Preserv. 40, 647–656. Sellami, I.H., Wannes, W.A., Bettaieb, I., Berrima, S., Chahed, T., Marzouk, B., Limam, F., 2011. Qualitative and quantitative changes in the essential oil of Laurus nobilis L. leaves as affected by different drying methods. Food Chem. 126, 691–697. Shimada, K., Fujikawa, K., Yahara, K., Nakamura, T., 1992. Antioxidative properties of xanthone on the auto oxidation of soybean in cyclodextrin emulsion. J. Agr. Food Chem. 40, 945–948. Tepe, B., Sokmen, M., Akpulat, H.A., Daferera, D., Polissiou Mand, Sokmen, A., 2005. Antioxidative activity of the essential oils of thymus sipyleussubsp. Sipyleusvar. Sipyleus and thymus sipyleus subsp. Sipyleusvar. rosulans. J. Food Eng. 66, 447–454. UNCTAD, 2006. World Market in the Spices Trade 2000–2004. UNCTAD, Geneva. Van Wyk, B.E., Wink, M., 2004. Medicinal Plants of the World and Their Uses. Timber Press, Portland, Oregon (USA). Zargari A., 1990. Medicinal Plants.Tehran, Iran, 5th ed. Vol.4. Iran, p: 923 (In Persian).
stage of plant growth on the essential oil content and composition of thymus kotschyanus Boiss.&Hohenfrom Iran. J. Essent. Oil Res. 11, 459–460. Sefidkon, F., Rahimi-Bidgoly, A., 2003. Quantitative and qualitative variation of essential oil of thymus kotschyanus by different methods of distillation and stage of plant growth. J. Iran. Med. Aromat. Plants Res. 15, 1–22. Seidi Damyeh, M., Niakousari, M., 2015. Impact of ohmic-assisted hydrodistillation on kinetics data, physicochemical and biological properties of Prangos ferulacea Lindle. essential oil: Comparison with conventional hydrodistillation. Innov. Food Sci. Emerg. Technol.(2015). http://dx.doi.org/10.1016/j.ifset.2015.12.009. Seidi Damyeh, M., Niakousari, M., Saharkhiz, M.J., 2016. Ultrasound pretreatment impact on Prangos ferulacea Lindle. and Satureja macrosiphonia Bornm.essential oil extraction and comparing their physicochemical and biological properties. Ind. Crops Prod. 87, 105–115. Seidi Damyeh, M., Niakousari, M., Golmakani, M.T., Saharkhiz, M.J., 2015. Microwave and Ohmic heating Impact on the insitu hydrodistillation and selective extraction of
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Sustainable Chemistry and Pharmacy journal homepage: www.elsevier.com/locate/scp
Sweet bay (Laurus nobilis L.) essential oil and its chemical composition, antioxidant activity and leaf micromorphology under different extraction methods
T
⁎
Azin Tabana, Mohammad Jamal Saharkhiza,b, , Mehrdad Niakousaric a
Department of Horticultural Science, Faculty of Agriculture, Shiraz University, Shiraz, Iran Medicinal Plants Processing Research Center, Shiraz University of Medical Sciences, Shiraz, Iran c Department of Food Science and Technology, Faculty of Agriculture, Shiraz University, Shiraz, Iran b
A R T I C LE I N FO
A B S T R A C T
Keywords: Sweet bay Extraction method Essential oil Antioxidant activity, SEM images
This research explored the essential oil (EO) of Laurus nobilis. Various parameters were recorded, including the amount of yield, chemical composition and antioxidant activity. The EO was obtained by hydro-distillation (HD), hydro-steam distillation (HSD), microwave-assisted hydrodistillation (MAHD), and ohmic-assisted hydrodistillation (OAHD). The micromorphology of the plant leaves were studied under the different extraction methods. The yields of EOs obtained by the mentioned extraction methods were 1.40, 0.74, 1.00 and 0.83 (% w/ w), respectively. The main chemical components in EOs obtained by these methods were eucalyptol (34.4–50.0%), α-terpinenyl acetate (14.9–18.8%), terpinene-4-ol (4.7–6.0%) and sabinene (4.9–5.9%). Two of the extraction methods, i.e. OAHD and MAHD, yielded EOs that contained higher proportions of eucalyptol. SEM images of the leaves were taken after extraction. It was observed that the MAHD method had the most destructive effect on secretory cells, while the HSD method failed to damage numerous cells in the leaves. Generally, the results suggest that MAHD and OAHD can be recognized as clean and faster methods because of their shorter processing time and less energy requirements. The HD and HSD methods extracted EOs with more sesquiterpenes, while the MAHD and OAHD methods yielded higher amounts of oxygenated monoterpenes in the EO.
1. Introduction Laurus nobilis L., commonly known as sweet bay, bay leaves or bay laurel, is an evergreen shrub or small tree that measures up to 2.1 m in height. It belongs to the Lauraceae family and is native to the southern Mediterranean region. The species was brought to Iran during the Qajar dynasty period (1794–1921) and is now widely cultivated in south, central and north of Iran as an ornamental plant (Zargari, 1990). The commercial value of L. nobilis results from the essential oil (EO) obtained from its leaves. The chemical composition of sweet bay EO has been studied by different researchers. In most cases, 1,8-cineole and αterpinyl acetate are the major components of L. nobilis EO (daSilveira et al., 2014; Pilar Santamarina et al., 2016; Sellami et al., 2011). The global demand for sweet bay EO is more than 3000 t per year (UNCTAD, 2006). This oil is used in cosmetics (i.e. creams, perfumes and soaps), in food and agriculture sectors (as a preservative, spice and flavouring agent and as a natural pesticide in postharvest crop protection). It is also used for medicinal purposes (i.e. treating rheumatic
⁎
pain, antimicrobial, pulled muscles and digestive disorders) (Sellami et al., 2011; Pacifico et al., 2013; daSilveira et al., 2014; Pilar Santamarina et al., 2016; De Corato et al., 2010; Van Wyk and Wink, 2004). Accordingly, the market demand for sweet bay EO has increased remarkably. This has prompted the search for the best extraction method to improve both yield and quality in sweet bay EO by reducing the extraction time, preventing degradation of thermally susceptible components and using lower energy consumption (Seidi Damyeh et al., 2016). The development of new extraction techniques such as supercritical fluid extraction, microwave-assisted hydrodistillation, and ohmic-assisted hydrodistillation (Seidi Damyeh et al., 2015) has received much attention recently due to their lower amounts of waste water, shorter extraction time and optimal energy costs (Memarzadeh et al., 2015; Seidi Damyeh et al., 2015). Several studies have shown that EO extraction methods can affect the quality and quantity of the EO. In this context, Sefidkon et al. (2007) studied the effects of distillation methods on the EO content and composition of Satureja rechingeri, whereby the hydro-distillation and steam-distillation produced the
Correspondence to: Department of Horticultural Sciences, College of Agriculture, Shiraz University, Shiraz, Iran. E-mail address: [email protected] (M.J. Saharkhiz).
https://doi.org/10.1016/j.scp.2018.05.001 Received 16 February 2018; Received in revised form 28 April 2018; Accepted 1 May 2018
Available online 07 May 2018 2352-5541/ © 2018 Elsevier B.V. All rights reserved.
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2.3. GC/MS identification
highest and the lowest EO yield, respectively. In a study reported by Sefidkon and Rahimi-Bidgoly (2003) on the extraction of EO from Thymus kotschyanus by hydro- and steam- distillation methods showed that the highest amounts of thymol and carvacrol in EO were obtained by the steam-distillation method. Memarzadeh et al. (2015) indicated that the highest EO yields of Satureja bachtiarica were obtained by HD via the Clevenger apparatus and by the microwave-assisted steam hydro-diffusion. Moreover, significant differences were observed among the major constituents of EOs, including thymol and carvacrol, by the different extraction methods. The antioxidant activity of L. nobilis EO has been reported by several authors (Conforti et al., 2006; Ramos et al., 2012; Cherrat et al., 2013). However, no attempts were made to compare the antioxidant activity of EOs extracted by the different extraction methods. The rising market demand for the EO of sweet bay leaves and its various usages prompted us to conduct this experimental research. The objectives of the current study were to compare: (1) the performance of four extraction methods, i.e. hydrodistillation (HD), hydro-steam distillation (HSD), microwaveassisted hydrodistillation (MAHD) and ohmic-assisted hydrodistillation (OAHD) for the extraction of EO from L. nobilis, (2) the EOs chemical composition and antioxidant activity as affected by the different extraction methods, and (3) the structural changes in the oil cells in the plant leaves after extraction.
The extracted EOs were analysed by GC–MS. The GC analysis was performed using an Agilent gas chromatograph series 7890-A equipped with a flame ionization detector (FID). The analysis was carried out on fused silica capillary HP-5 column (30 m × 0.32 mm i.d., with a film thickness of 0.25 µm). The injector and detector temperatures were maintained at 250 °C and 280 °C, respectively. Nitrogen was used as carrier gas at a flow rate of 1 mL/min. The oven temperature program increased from 60° to 210°C, at a rate of 4 °C/min, which was then programmed to 240 °C, at a rate of 20 °C/min. In the end, the condition was maintained isothermally for 8.5 min. The split ratio was 1:50. The GC–MS analysis was carried out via the Agilent gas chromatograph equipped with fused silica capillary HP-5MS column (30 m × 0.25 mm i.d.; film thickness 0.25 µm) coupled with 5975-C mass spectrometer. Helium was used as carrier gas at a flow rate of 1.1 mL/min, with the ionization voltage of 70 eV and 0.1 μL. The samples were injected manually in the split mode. Ion source and interface temperatures were 230 °C and 280 °C, respectively. Mass range was valued between 45 and 550 amu. The oven temperature program was the same as the temperature for the GC. The retention indices for all components were determined by using n-alkanes as standard. The compounds were identified by comparing their retention indices (RI, HP-5) with those reported in the literature and also by comparing their mass spectra with the Wiley GC–MS Library, Adams Library, Mass Finder 2.1 Library data, and published mass spectra data (Adams, 2007; Karami and Rowshan, 2015).
2. Material and methods 2.1. Plant materials
2.4. Antioxidant activity assay L. nobilis leaves were collected in April 2015 from the Eram Botanical Garden, Shiraz University, Shiraz, Iran, (29°36 ´ N and 52°32 ´ W). Laurus nobilis was identified and authenticated by a senior plant taxonomist at Shiraz University Herbarium, Shiraz, Iran. The voucher specimen (802 LL) was deposited in the Herbarium. The leaves were dried in the shade (20–25 °C) for a week in the laboratory. They were packaged in high density polyethylene bags and were then carefully ground and sifted through a mesh screen (with 0.5 mm pores) to obtain uniform leaf material prior to each experiment.
Free radical scavenging activity was measured by 2,2-diphenyl-1picrylhydrazyl (DPPH; Sigma–Aldrich). The effect of EOs on DPPH degradation was estimated according to the method used by Cherrat et al. (2013). Different concentrations (1, 2, 4, 6, 8, 10 μL mL−1) of the EOs were prepared in pure methanol, and then 150 μL of each concentration were added to 50 μL of 1 mmol L−1DPPH. The mixture was then kept at room temperature in the dark for 30 min. The absorbance of the resulting solutions was measured at 517 nm after 30 min. The DPPH radical scavenging ability of each EO extract was calculated according to the following equation:
2.2. EO extraction procedures
%scavenging of DPPH radical = [(Ablank –Asample) / Ablank ] × 100 Where Ablank is the absorbance of the control reaction (containing all reagents except the EOs), and Asample is the absorbance by samples. In order to calculate the sample concentration that can provide 50% inhibition (IC50), the inhibition percentages were plotted against sample concentrations. All tests were carried out in triplicate (Seidi Damyeh et al., 2016).
The extraction of the EOs from L. nobilis was performed by four different methods; hydro-distillation (HD), hydro-steam distillation (HSD), microwave-assisted hydro-distillation (MAHD), and ohmic-assisted hydrodistillation (OAHD) (Seidi Damyeh et al., 2015) (Fig. 1): (1) HD was carried out in a British Pharmacopeia Clevenger-type apparatus using an electric mantle (400 W) heater for 150 min (Memarzadeh et al., 2015). (2) HSD performed in a Kaiser and Lang apparatus using an electric mantle (400 W) heater for 150 min (Sefidkon et al., 2007). (3) MAHD operated via a microwave that was set to 400 W for 45 min (Memarzadeh et al., 2015) (4) OAHD operated by an ohmic heating system that was set to 120 V for 45 min. A small quantity of sodium chloride (0.3% w/v of Na Cl) provided sufficient electrical conductivity between two electrodes in the heat-up process. The exact amount of NaCl was added to water in other extraction methods (Seidi Damyeh et al., 2015).
2.5. Scanning electron microscopy (SEM) SEM images of dried leaves were taken from untreated samples (L. nobilis leaves) and also from samples treated with HD, HSD, MAHD and OAHD. The dried leaves were fixed on an aluminium sample holder and were then sputtered with gold in a sputter coater. All samples were examined with a scanning electron microscope (TescanVEGA3, Czech Republic) under high-vacuum conditions. An accelerating voltage of 20.0 kV was provided, and the working distance ranged between 23.71 and 46.31 mm (i.e. the distance between the surfaces of the sample and the microscope lens).
For each method, 50 g of L. nobilis was extracted with 0.5 L of distilled water. Results were reported in grams of EOs per 100 g of dried leaves. Each experiment was performed at least in triplicate. The EOs were dried by anhydrous sulphate and stored at 4 °C until further analysis.
2.6. Statistical analysis Data were submitted to an analysis of variance (ANOVA) in SPSS (version15.0.0; IBM Institute Inc., USA) and mean values were compared via the LSD test at 5% level. 13
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Fig. 1. Method scheme; HD: Hydro-distillation; HSD: Hydro-steam distillation; MAHD: Microwave-assisted hydrodistillation; OAHD: Ohmic-assisted hydrodistillation.
3. Results
the extraction.
3.1. Effect of extraction methods on the yield, colour and extraction time of EOs
3.2. Effect of extraction methods on the EOs composition Chemical composition of EOs obtained by different methods was identified via a comprehensive GC/MS analysis (Table 1). Eighteen, nineteen, twenty-five and thirty components represented 95%, 97%, 95% and 94% of the total EOs extracted by MAHD, OAHD, HD and HSD, respectively. The main components of the EOs obtained from the different extraction methods were eucalyptol (34.37–5.07%), α-terpinenyl acetate (14.93–18.78%), terpinene-4-ol (4.72–6.02%) and sabinene (4.95–5.93%) (Table 1). All EOs were found to be rich in oxygenated monoterpenes (such as eucalyptol and α-terpinenyl acetate). Among the extraction methods, OAHD yielded the highest percentages of eucalyptol (50.07%), terpinene-4-ol (6.02%) and linalool (2.72%), although it managed to yield the lowest percentages of other components such as α-terpinenyl acetate (14.93%) and caryophyllene oxide (1.32%). Accordingly, the HD and HSD methods enhance “extraction selectivity” for sesquiterpenes by more than twofold in L. nobilis (Table 1). Despite the differences in the quantity of the main components, small differences can also be observed in the quantity of other components such as α-pinene (2.94–4.11%), β-pinene (2.51–3.22%), cis-sabinene hydrate (0.49–1.03%), eugenol (0.55–1.03%), methyleugenol (2.34–3.99%) and trans-caryophyllene (0–1.33%).
EOs extracted from the leaves of L. nobilis by HD and HSD methods produced a colourless liquid, while those obtained from MAHD and OAHD methods were a pale yellow liquid. Results indicated that the extraction method had a significant effect on the EO yield of sweet bay leaves (P ≤ 0.05). The highest and lowest EO yields were obtained by the HD and HSD methods, respectively (Fig. 2). However, no significant difference was observed between the EO yield of HSD, MAHD and OAHD (Fig. 2). The HD and HSD methods required a longer extraction time (150 min), while OAHD and MAHD required 45 min to complete
3.3. Antioxidant activity Fig. 2. L. nobilis essential oil yield obtained through four extraction methods, HD: Hydro-distillation; HSD: Hydro-steam distillation; MAHD: Microwave-assisted hydrodistillation; OAHD: Ohmic-assisted hydrodistillation. Means with same letter are not significantly different using LSD test (P ≤ 0.05).
The EOs obtained from different methods were tested individually against DPPH free radical to investigate their antioxidant capacity (Table 2). ANOVA analysis of IC50 identified significant differences between the four methods (P < 0.05). LSD test subdivided them into 14
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Table 1 Chemical composition of the L. nobilis essential oils obtained through four extraction methods; HD: Hydro-distillation; HSD: Hydro-steam distillation; MAHD: Microwave-assisted hydrodistillation; OAHD: Ohmic-assisted hydrodistillation. no
Component
MAHD
OAHD
HD
HSD
a
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
α-Thujene α-Pinene Camphene Sabinene β-Pinene β-Myrcene α-Terpinene Eucalyptol γ-Terpinene cis-Sabinene hydrate Linalool Cis ρ Menth− 2en− 1-ol trans-Pinocarveol δ-terpineol Terpinene− 4-ol α - terpineol Myrtenol Bornyl acetate Carvacrol α-terpinenyl acetate Eugenol Neryl acetate β-elemene Methyleugenol trans-Caryophyllene Methyl isoeugenol (E) Spathulenol Caryophyllene oxide caryophylla− 4(12),8(13)dien− 5β-ol β-Eudesmol Total identification Monoterpenes Sesquieterpenes Other compounds Unknown compounds
– 2.94 – 5.25 2.51 – – 49.73 – 1.03 2.51 – 0.64 0.94 5.96 2.88 – 0.85 – 16.92 0.55 0.57 0.5 2.34 0.67 – – 1.83 –
0.59 3.9 0.57 5.69 2.96 – – 50.07 – 0.76 2.72 – 0.73 0.86 6.02 2.40 0.54 0.82 – 14.93 0.63 0.5 – 2.66 – – – 1.32 –
0.6 4.11 0.56 5.93 3.22 0.72 – 37.53 0.65 0.52 1.6 – 0.47 0.78 4.72 2.41 0.38 0.83 – 18.65 0.83 0.63 1.05 2.77 1.33 – – 3.26 0.88
0.48 3.28 0.45 4.95 2.85 0.61 0.36 34.37 0.74 0.49 2.08 0.4 0.64 0.82 5.59 2.1 0.53 0.9 0.4 18.78 1.03 0.57 0.71 3.99 0.8 0.52 1.1 2.30 0.94
928 936 951 978 982 992 1020 1047 1061 1071 1103 1124 1142 1172 1185 1197 1201 1289 1309 1355 1363 1367 1396 1408 1426 1499 1580 1592 1644
930 939 954 975 980 990 1017 1039 1059 1070 1096 1121 1139 1166 1177 1188 1194 1288 1298 1349 1359 1361 1390 1403 1419 1492 1587 1583 1640
– 95.73 95.62 3 2.89 1.39
– 98.67 94.06 1.32 3.29 1.32
0.73 95.16 94.31 7.25 3.6 4.84
1 94.74 82.35 6.85 5.54 5.25
1659 – – – – –
1650 – – – – –
30
KIc
b
Table 2 Antioxidant activity of the L. nobilis essential oils obtained through four extraction methods; HD: Hydro-distillation; HSD: Hydro-steam distillation; MAHD: Microwave-assisted hydrodistillation; OAHD: Ohmic-assisted hydrodistillation.
KIr
Extraction method
Concentration (μL/mL)
Inhibition (%)
IC50 (μL/mL)
HD
10 8 6 4 2 1 10 8 6 4 2 1 10 8 6 4 2 1 10 8 6 4 2 1
92.4 91.1 85.9 75.3 56.6 42.1 93.7 91.2 86.2 73.5 52.4 38.6 83.7 72.4 65.8 50.9 40.9 34.6 89.7 84.4 76.9 62.6 44.7 35.4
0.84 ± 0.308 c
HSD
MAHD
OAHD
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2.5 0.7 1.0 0.6 2.8 1.5 0.4 0.3 1.4 1.9 2.5 0.5 7.0 8.6 8.6 6.31 1.9 2.4 1.6 3.8 3.3 4.3 4.9 4.1
1.4 ± bc
3.7 ± 0.9 a
2.5 ± 1.0 ab
IC50 (inhibitory concentration at 50%); Means with same letter are not significantly different as indicated byDuncan multiple range test (P ≤ 0.05).
durations of the different methods. The chemical composition of the different EOs were not similar, which also explains the variety of EO colours. Moreover, by increasing the extraction time in HD and HSD, more chemical components were recovered. As stated before, the HD and HSD methods yielded twice as many components than MAHD and OAHD. Nonetheless, it has been previously reported that other physical properties of the EO could be affected by the number of components in the EO (Pearson, 1976). Three extraction methods, i.e. HSD, MAHD and OAHD provided similar EO yields. The maximum EO yield (1.43%) was obtained by HD which was significantly higher than other methods studied herein, while the lowest yield was observed in HSD. Similar results were observed by Sefidkon et al. (2007) who reported that the highest EO content of Satureja rechingeri was obtained by the HD method. However, the results of a study by Sefidkon et al. (1999) indicated that the HSD and HD methods yielded similar EO contents of Thymus kotschyanus. It is believed that the HD is a reference method used for the quantification of EOs. In fact, the HD has been capable of yielding the highest amount of EO in previous studies (Sefidkon et al., 2007, 1999; Memarzadeh et al., 2015). However, the HSD yielded the lowest amount of EO in this study, compared to other methods, which is probably due to the condition of plant material, mode of communication and mode of charging. Although all extracted EOs contain the same main components (eucalyptol, α-terpinenyl acetate, terpinene-4-ol and sabinene), their respective quantities vary more or less with regard to the extraction technique (Table 1). Previous reports confirm that the main components reported in the present study are the most abundant component in L. nobilis EO (Di Leo Lira et al., 2009; Sellami et al., 2011; Pilar Santamarina et al., 2016). Applying MAHD and OAHD resulted in a drastic increase (about 30%) in the content of eucalyptol compared to HD and HSD methods (49.73% and 50.07% versus 37.53% and 34.37%). Di Leo Lira et al. (2009) indicated that if the distillation time of the sweet bay leaves is programmed to be less than an hour, the concentration of eucalyptol (boiling point: 176 °C; mass: 154) would be higher compared to a prolonged distillation which would yield higher
a Experimentally determined Kovats retention index (KI) relative to C19-C18 n-alkanes on the DB-5MS column. b Literature Kovats retention index (Adams, 2007).
three groups. The HD yielded an EO which exhibited the highest activity against DPPH, while the HSD yielded the weakest activity. However, there was no significant difference between the antioxidant activity of EOs which were obtained by the other methods (i.e. MAHD and OAHD) (Table 2). 3.4. Structural changes during EOs extraction The images of secretory cells in the L. nobilis leaves were taken by scanning electron microscopy (SEM) before and after the extraction process (Fig. 3). Fig. 3(A) is a micrograph of the untreated cells before the extraction. Fig. 3(B) and (C) show the SEM images of sweet bay cells that had undergone HD (150 min) and HSD (150 min), respectively. The SEM images display sweet bay cells after 45 min of OAHD and 45 min of MAHD (Fig. 3D and E, respectively). MAHD demonstrated a destructive effect on secretory cells, while the HSD failed to damage numerous cells in the leaves. The morphological shapes of cells after HD and MAHD are relatively similar (Fig. 3). 4. Discussion In this study, different extraction methods yielded EOs of different colours. So far, no published work has reported the difference in the colour of EOs extracted by different methods. Here, the difference in EO colour could be due to the different extraction temperatures and 15
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Fig. 3. Scanning electron micrographs of the L. nobilis leaves after *EO extraction: (A) untreated-2500X, (B) after HD**− 2500X, (C) after HSD-2500X, (D) after OAHD-2500X, (E) after MAHD-2500X. *EO: Essential oil; **HD: Hydro-distillation; HSD: Hydro-steam distillation; MAHD: Microwave-assisted hydrodistillation; OAHD: Ohmic-assisted hydrodistillation.
heating efficiency of the MAHD and OAHD has been already confirmed by the measurement of the consumed electric energy (Gavahian et al., 2015) where the power consumption was measured for the extraction of 1 mL Mentha piperita EO by MAHD and OAHD versus the electromantle. Previous results indicated a substantial saving in the extraction cost by MAHD and OAHD compared to the conventional HD extraction technique (Gavahian et al., 2015). EO extraction from Satureja macrosiphonia by MAHD and OAHD methods also indicated a remarkable decrease in the extraction time and energy, compared to the conventional HD method (Seidi Damyeh et al., 2015). Furthermore, the MAHD is less tedious as it minimizes the risk of prolonged exposure to heat and therefore minimizes the degradation of compounds. Previous studies have shown that major component of sweet bay EO and their values can affect EO quality and its biological activity such as antibacterial, antiviral, antifungal and antioxidant activity (Sellami et al., 2011; Pilar Santamarina et al., 2016). Sweet bay EO demonstrated the ability to sequester the free radical DPPH (Table 2). In agreement with our results, previous studies have shown that sweet bay EOs exhibit an effective antioxidant activity when tested by the DPPH radical scavenging method and by the β-carotene–linoleic acid method (Cherrat et al., 2013; Ramos et al., 2012). The IC50 (inhibitory concentration at 50%) is a widely used parameter in evaluating the free radical scavenging activity. A lower IC50 value is attributed to a higher antioxidant activity (Shimada et al., 1992). In this study, EOs obtained
proportions of terpinyl acetate (BP: 210 °C; M:196) and methyl eugenol (BP: 255 °C; M: 178). Similar results have been obtained in this study, using HD and HSD methods (150 min) which increased the values of methyl eugenol and α-terpinenyl acetate compared to MAHD and OAHD extractions (45 min). Altogether, it could be stated that the different extraction methods of the EOs influenced the amount of the chemical classes. In particular, sesquiterpenes were extracted in higher amounts using HD and HSD methods, whereas the MAHD and OAHD increased the amount of oxygenated monoterpenes. The latter results are in line with a report by Flamini et al. (2007). The higher amounts of oxygenated components in OAHD and MAHD are probably attributed to the higher dipolar moment and diminution of thermal effects (Seidi Damyeh et al., 2015). It is also interesting to note that the longer extraction time not only can recover more components but also may cause the deterioration of some other components and can reduce their amounts. A very long and direct contact of the plant materials with boiling water can cause the deterioration. Therefore, faster methods such as MAHD and OAHD can minimize the risk of heat-related degradation of the compounds (Flamini et al., 2007; Gavahian et al., 2015; Seidi Damyeh et al., 2015). In terms of the extraction time, OAHD and MAHD warm up the mixture faster than the heating mantle-based methods. The required time for the extraction process is therefore reduced because of the higher extraction rate (0.84% and 1.01% in 45 min). The greater 16
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performed more optimally. In the case of structural changes to the cells after extraction, the MAHD was more successful. It can be concluded that selecting the best technique for EO extraction from L. nobilis is completely dependent on the aim of the extraction. MAHD and OAHD produce good quality EOs at faster rates which could save energy and are more environmental friendly, while the HD can extract EOs with higher yield and stronger antioxidant activity, but HSD does not seem to be a good option for the extraction of L. nobilis in general.
from four extraction methods showed dose-dependent radical scavenging activities, and there was a significant difference between EOs obtained through the different methods. Definitely, the different antiradical activities are due to the respective differences in the chemical composition of EOs. Furthermore, the EOs obtained through HD showed the highest DPPH scavenging, and this correlates with higher amounts of monoterpenes and sesquiterpenes present in the EO (Bartikova et al., 2014). Eucalyptol is known to possess a strong antioxidant activity. (Ramos et al., 2012). Despite the low amount of eucalyptol in the HD essential oil, compared to MAHD and OAHD, the EO obtained by HD exhibited a stronger antioxidant activity. This pattern might be due to a synergistic interaction between minor and major constituents of EOs (Tepe et al., 2005). Results of the present study are in line with those published by Seidi Damyeh et al. (2016) who reported a higher antioxidant activity of the Prangos ferulacea EO which had been extracted by the HD, compared to ultrasound. Bartikova et al. (2014) indicated that many of the biological activities attributed to sesquiterpenes, such as antimicrobial and anti-inflammatory effects, are based on the antioxidant actions of the sesquiterpenes. Thus, the higher DPPH scavenging activity of EOs obtained from HD can be related to the higher amount of sesquiterpenes compared to other extraction methods (Table 2). The leaves of sweet bay have secretory cells (oil cells) which secrete EO (Dickison, 2000). All extraction methods in this study caused physical changes to the cells along the structure of leaves, as internal secretory structures. HD and OAHD extraction methods caused evident ruptures in the cells of sweet bay leaves during the extraction (Fig. 3B and D). However, in HSD, numerous cells in the leaves were left intact (Fig. 3C), and therefore a lower amount of EO was obtained (Fig. 2). In the case of MAHD, a severe disruption of the oil cells happened and cells became wrinkled (Fig. 3E) while those that had undergone other methods were not so damaged and were more rounded in shape. This phenomenon can be associated with the pattern of heat distribution during MAHD extraction. In MAHD, the radiated microwave energy is immediately converted into heat energy which is transferred directly to the plant material (Desai et al., 2010). This heat energy can therefore be localized at particular parts of the plant containing the oil cells, thereby damaging the cells. Meanwhile, the pattern of heat transfer in HD and HSD methods is mainly performed slowly by conduction and convection, and the heat energy first reaches the solvent before heating the targeted plant material (Gavahian et al., 2015). These results of the present study are not in line with those reported by Jeyaratnam et al. (2016) who studied the extraction of EO from Cinnamomum cassia bark, using the HD and MAHD, and the latter method caused less damage to oil glands. This could be due to differences in secretory structures (cells versus glands) and plant organs (leaves versus barks). Regarding the OAHD method, in solid materials such as plant leaves in which the cell structure is intact, electrical conductivity is dependent on electric field power, and is controlled by applying a certain degree of voltage, and cellular breakdown occurs by electro-permeabilization (Seidi Damyeh et al., 2015). However, since more ruptured cell structures are observed in the sample treated with OAHD, the electroporation effect may be observed. In this regard, our data were in good agreement with those published by Seidi Damyeh and Niakousari (2015).
Acknowledgements The authors wish to extend their thanks and appreciation to Shiraz University Research and Technology Council as well as Shiraz University of Medical Sciences for their financial supports (Grant No: 1396-01–70-15354). We also thank Mohsen Hamedpour-Darabi for editing the research language. References Adams, R.P., 2007. Identification of Essential Oil Components by Gas Chromatography/ Mass Spectrometry, 4th ed. Allured Publishing, Carol Stream,IL. Bartikova, H., Hanusova, V., Skalova, L., Ambroz, M., Bousova, I., 2014. Antioxidant, prooxidant and other biological activities of sesquiterpenes. Curr. Top. Med. Chem. 14 (22), 2478–2494. Cherrat, C., Espina, L., Bakkali, M., Garcia-Gonzalo, D., Pagan, R., Laglaoui, A., 2013. Chemical composition and antioxidant properties of LaurusnobilisL. and Myrtus communis L. essential oils from Morocco and evaluation of their antimicrobial activity acting alone or in combined processes for food preservation. J. Sci. Food Agric. 94, 1197–1204. Conforti, F., Statti, G., Uzunov, D., Menichini, F., 2006. Comparative chemical composition and antioxidant activities of wild and cultivated Laurus nobilis L. leaves and Foeniculum vulgare subsp. piperitum (Ucria) Coutinho seeds. Biol. Pharm. Bull. 29, 2056–2064. daSilveira, S.M., Luciano, F.B., Fronza, N., Cunha Jr., A., Scheuermann, G.N., Werneck Vieira, C.R., 2014. Chemical composition and antibacterial activity of Laurus nobilis essential oil towards food borne pathogens and its application in fresh Tuscan sausage stored at 7 °C. LWT - Food Sci. Technol. 59, 86–93. De Corato, U., Maccioni, O., Trupo, M., Di Sanzo, G., 2010. Use of essential oil of Laurus nobilis obtained by means of a supercritical carbon dioxide technique against post harvest spoilage fungi. Crop Prot. 29, 142–147. Desai, M., Parikh, J., Parikh, P., 2010. Extraction of natural products using microwaves as a heat source. Sep. Purif. Rev. 39 (1–2), 1–32. Di Leo Lira, P., Retta, D., Tkacik, E., Ringuelet, J., Coussio, J.D., Van Baren, C., Bandoni, A.L., 2009. Essential oil and by-products of distillation of bay leaves (Laurus nobilisL.) from Argentina. Ind. Crops Prod. 30, 259–264. Dickison, W.C., 2000. Integrative Plant Anatomy. Novanet: Dalhousie Killam Library SCI QK 641 D53 2000. New York: Academic Press, San Diego. Flamini, G., Tebano, M., Luigi Cioni, P., Ceccarini, L., Simone Ricci, A., Longo, I., 2007. Comparison between the conventional method of extraction of essential oil of Laurus nobilis L. and a novel method which uses microwaves applied in situ, without resorting to an oven. J. Chromatogr. A 1143, 36–40. Gavahian, M., Farahnaky, A., Farhoosh, R., Javidnia, K., Shahidi, F., 2015. Extraction of essential oils from Mentha piperita using advanced techniques: microwave versus ohmic assisted hydrodistillation. Food Bioprod. Process. http://dx.doi.org/10.1016/j. fbp.2015.01.003. Jeyaratnam, N., Hamid Nour, A., Kanthasamy, R., Hamid Nour, A., Yuvaraj, A.R., OlabodeAkindoyo, J., 2016. Essential oil from Cinnamomum cassia bark through hydrodistillation and advanced microwave assisted hydrodistillation. Ind. Crops Prod. 92, 57–66. Karami, A., Rowshan, V., 2015. Temporal analysis of volatile oil compounds from winter season flowering ‘Eureka’ lemon. Anal. Chem. Lett. 5, 31–37. Memarzadeh, S.M., GhasemiPirbalouti, A., AdibNejad, M., 2015. Chemical composition and yield of essential oils from Bakhtiarisavory (Satureja bachtiarica Bunge.) under different extraction methods. Ind. Crops Prod. 76, 809–816. Pacifico, S., Gallicchio, M., Lorenz, P., Potenza, N., Galasso, S., Marciano, S., Fiorentino, A., Stintzing, F.C., Monaco, P., 2013. Apolar Laurus nobilis leaf extracts induce cytotoxicity and apoptosis towards three nervous system cell lines. Food Chem. Toxicol. 62, 628–637. Pearson, D., 1976. The Chemical Analysis of Foods, 7ed. Churchill Livingstone, pp. 6–14. Pilar Santamarina, M., Rosello, J., Gimenez, S., Amparo Blazquez, M., 2016. Commercial Laurus nobilis L. and Syzygium aromaticum L. Merr.& Perry essential oils against postharvest phytopathogenic fungi on rice. LWT - Food Sci. Technol. 65, 325–332. Ramos, C., Teixeira, B., Batista, I., Matos, O., Serrano, C., Neng, N.R., Nogueira, J.M.F., Nunes, M.L., Marques, A., 2012. Antioxidant and antibacterial activity of essential oil and extracts of bay laurel Laurus nobilis Linnaeus (Lauraceae) from Portugal. Nat. Product. Res. 26 (6), 518–529. Sefidkon, F., Abbasi, K., Jamzad, Z., Ahmadi, S., 2007. The effect of distillation methods and stage of plant growth on the essential oil content and composition of Satureja rechinger jamzad.J. Food Chem. 100, 1054–1058. Sefidkon, F., Dabiri, M., Rahimi-Bidgoly, A., 1999. The effect of distillation methods and
5. Conclusion The EOs were extracted from sweet bay samples by using HD, HSD, MAHD and OAHD methods. Different extraction methods yielded EOs with a variety of chemical compositions, antioxidant activities and yield parameters. The main EO components obtained through the different methods were similar. However, drastic differences were observed in the quantity of components, depending on the extraction processes. MAHD and OAHD offered good advantages in terms of their selectivity for eucalyptol, whereas more sesquiterpenes were recovered by the HD technique. Regarding EO yield and antiradical activity, the HD 17
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stage of plant growth on the essential oil content and composition of thymus kotschyanus Boiss.&Hohenfrom Iran. J. Essent. Oil Res. 11, 459–460. Sefidkon, F., Rahimi-Bidgoly, A., 2003. Quantitative and qualitative variation of essential oil of thymus kotschyanus by different methods of distillation and stage of plant growth. J. Iran. Med. Aromat. Plants Res. 15, 1–22. Seidi Damyeh, M., Niakousari, M., 2015. Impact of ohmic-assisted hydrodistillation on kinetics data, physicochemical and biological properties of Prangos ferulacea Lindle. essential oil: Comparison with conventional hydrodistillation. Innov. Food Sci. Emerg. Technol.(2015). http://dx.doi.org/10.1016/j.ifset.2015.12.009. Seidi Damyeh, M., Niakousari, M., Saharkhiz, M.J., 2016. Ultrasound pretreatment impact on Prangos ferulacea Lindle. and Satureja macrosiphonia Bornm.essential oil extraction and comparing their physicochemical and biological properties. Ind. Crops Prod. 87, 105–115. Seidi Damyeh, M., Niakousari, M., Golmakani, M.T., Saharkhiz, M.J., 2015. Microwave and Ohmic heating Impact on the insitu hydrodistillation and selective extraction of
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