Research Article Received: 25 March 2014
Revised: 25 November 2014
Accepted article published: 1 December 2014
Published online in Wiley Online Library: 30 December 2014
(wileyonlinelibrary.com) DOI 10.1002/jsfa.7031
Extraction of thymol from different varieties of thyme plants using green solvents David Villanueva Bermejo,a Ivan Angelov,b Gonzalo Vicente,a Roumiana P Stateva,b* Mónica Rodriguez García-Risco,a Guillermo Reglero,a Elena Ibañeza and Tiziana Fornaria Abstract BACKGROUND: Thymol (2-isopropyl-5-methylphenol) is the main monoterpene phenol found in thyme essential oil. This compound has revealed several biological properties, including antibacterial, anti-inflammatory and antioxidant activities. In this work, a comparison was made between the performance of different green solvents (ethanol, limonene and ethyl lactate), by pressurized liquid extraction (PLE) and supercritical fluid extraction (SFE) at different conditions, to extract thymol from three different varieties of thyme (Thymus vulgaris, Thymus zygis and Thymus citriodorus). Additionally, new solubility data of thymol in limonene and ethanol at ambient pressure and temperatures in the range 30–43 ∘ C are reported. RESULTS: The highest thymol recoveries were attained with T. vulgaris (7–11 mg g−1 ). No thymol could be quantified in the PLE samples of T. citriodorus. The highest concentrations of thymol in the extracts were obtained with limonene. Thymol is very soluble in both solvents, particularly in ethanol (∼900 mg g−1 at ∼40 ∘ C), and is the main compound (in terms of peak area) present in the essential oil extracts obtained. CONCLUSION: The three solvents show good capacity to extract thymol from T. vulgaris and T. zygis by PLE. Although PLE proved to be a suitable technology to extract thymol from thyme plants, the highest concentrations of thymol were obtained by SFE with supercritical CO2 . © 2014 Society of Chemical Industry Keywords: pressurized liquid extraction (PLE); supercritical fluid extraction (SFE); thyme; ethyl lactate; limonene
INTRODUCTION
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∗
Correspondence to: Roumiana P. Stateva, Institute of Chemical Engineering, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria. E-mail: [email protected]
a Instituto de Investigación en Ciencias de la Alimentación CIAL (CSIC-UAM), CEI UAM + CSIC, Universidad Autónoma de Madrid, C/Nicolás Cabrera 9, E-28049 Madrid, Spain b Institute of Chemical Engineering, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
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The genus Thymus (family Lamiaceae) comprises over 350 aromatic plant species with quite different botanical characteristics and broad chemical heterogeneity.1 One of the most valued constituents of thyme (especially of the leaves) is the volatile oil (VO), which is formed by terpenes, sesquiterpenes and several oxygenated derivative compounds (alcohols, aldehydes, ketones, acids, phenols, ethers, esters, etc.), all of which are responsible for the characteristic plant odor and flavor.2 Thyme VO is appreciated for food flavoring,3 in cosmetics and perfumery4 and in the pharmaceutical industries.5 The main biological properties that have been recognized as its pharmacological actions are antimicrobial, antitussive, antispasmodic and antioxidant activities.6,7 Thymol and its isomer carvacrol are among the most biologically active constituents of thyme VO. Thymol (2-isopropyl-5methylphenol) is the main monoterpene phenol found in the VO of several varieties of thyme. Thymol and carvacrol have shown anti-inflammatory, immunomodulatory, antioxidant, antibacterial and antifungal properties.8 – 10 The variety most studied is Thymus vulgaris,11 – 13 while attention has also been focused on Thymus zygis,14 a thyme variety widespread over Portugal and Spain, as well as on Thymus citriodorus, a plant cultivated throughout the Mediterranean region.15 The oil of the herb thyme has usually been obtained by either steam distillation (in which case, according to the European
Pharmacopoeia, it is referred to as the essential oil of the herb) or traditional solid–liquid extraction methods such as Soxhlet, maceration or extraction under reflux. Although these methods are relatively simple, they suffer from several shortcomings, such as a long extraction time and a relatively high solvent consumption, to name just two. Pressurized liquid extraction (PLE), a relatively recent solvent extraction technique, can, in principle, eliminate some of the drawbacks of classical solvent extraction methods. PLE is based on the use of solvents at temperatures above their normal boiling points and at pressures high enough to keep the extraction fluid in the liquid state during the whole extraction process. Thus faster extraction processes can be realized, with higher extraction yields and lower volumes of organic solvents,16 which are essential principles of ‘green extraction’.17 Although several studies have been reported on PLE of VOs from a wide range of raw herbal materials,18 – 22 only a few
www.soci.org communications can be found on PLE of VO from thyme. For instance, Dawidowicz et al.23,24 studied the use of hexane, dichloromethane, ethyl acetate and water at pressurized liquid conditions to extract the VO from T. vulgaris. Higher recoveries of thymol were obtained using the non-green solvents hexane (∼10.7 mg g−1 ), dichloromethane (∼12.2 mg g−1 ) and ethyl acetate (∼12.8 mg g−1 ) in comparison with those obtained using subcritical water extraction (∼7 mg g−1 ). On the other hand, supercritical fluid extraction (SFE) has also been suggested as a valuable alternative to classical extraction methods to recover essential oils from plants and herbs. Several studies related to SFE of thyme leaves have already been published.11,14,25 – 28 In all of them, pure CO2 was employed as extraction solvent and, depending on the extraction conditions, different concentrations of thymol were achieved. In the ongoing search for potential new green extraction solvents that might replace toxic organic solvents, two solvents, namely ethyl lactate and D-limonene, have recently been suggested.17 Ethyl lactate (ethyl 2-hydroxypropanoate) is an agrochemical which is an economically viable alternative to traditional liquid solvents; it is fully biodegradable, non-corrosive, non-carcinogenic and non-ozone-depleting. It was self-affirmed as GRAS (‘generally recognized as safe’) and, owing to its low toxicity, approved by the US Food and Drug Administration (FDA) as a pharmaceutical and food additive. These characteristics have increased the interest of researchers in its potential as a green solvent for food applications.29 – 34 D-Limonene (1-methyl-4-(1-methylethenyl)cyclohexene) is also a non-toxic, non-corrosive, non-carcinogenic and fully biodegradable solvent approved by the FDA as GRAS. This monoterpenic molecule is the major component of essential oils extracted from citrus peels and is a major by-product of the citrus fruit industry.36,37 It has been demonstrated that D-limonene is a valuable alternative to petroleum solvents and it has been used to remove oil from rice bran38 and to extract lycopene from tomato.39 Although high solubility of thymol in ethyl lactate at different temperatures has been reported in a recent contribution,35 to the best of our knowledge, there are still no data published in the open literature related to the extraction of thymol from thyme using either pressurized ethyl lactate or D-limonene. Therefore the aim of the present work is to provide new insights into the potential of several green compressed solvents to extract thymol from different thyme species.
MATERIALS AND METHODS Chemicals Ethanol was HPLC grade from Panreac (Barcelona, Spain). Standards employed were thymol (>99%), carvacrol (98%), camphor (>95%), 𝛼-pinene (>99%), 𝛽-linalool (>97%), verbenone (>97%), D-limonene (90%) and ethyl lactate (>98%) from Sigma-Aldrich (St. Louis, MO, USA). Borneol (>99%) and 1,8-cineole (>98%) were purchased from Fluka (Buch, Switzerland). Eugenol (>99%) and 𝛼-terpineol (>96%) were supplied by Extrasynthèse (Lyon, France). CO2 for SFE (99.999%) was provided by Carburos Metálicos (Madrid, Spain).
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Thyme leaf preparation The thyme (T. vulgaris, T. zygis and T. citriodorus) samples consisted of dried leaves obtained from a herbalist’s producer (Murcia,
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Spain). The leaves were ground in a cooled mill. Particles of size smaller than 250 μm were separated using sieves and employed in the experiments. The whole sample was stored at −20 ∘ C until used. Solubility measurements Solubility measurements of thymol in the liquid solvents were made at atmospheric pressure as a function of temperature over the range 30–43 ∘ C. For all solutions studied, the liquid solvent (either D-limonene or ethanol) and thymol in excess were placed in glass vessels (10 mL) with magnetic stir bars. The vessels were put inside a water bath heated by a magnetic hotplate stirrer (RCT classic IKAMAG® safety control, IKA Works GmbH & Co. KG, Staufen, Germany), which was used to agitate the samples for 24 h at a fixed temperature, controlled by an electronic contact thermometer with probe (VWR VT-5, VWR International, LLC, West Chester, PA, USA) having an accuracy of 0.2 K. After equilibrium had been reached, the stirring was stopped and the vessels were left still for more than 48 h to allow complete phase separation. Samples of clear saturated liquid solution (100 𝜇L) were taken using a micropipette and placed in glass vials. Samples were appropriately diluted for gas chromatography/mass spectrometry (GC/MS) analysis. Pressurized liquid extraction Thymol extractions were carried out in an Accelerated Solvent Extraction system ASE 350 (Dionex Corporation, Sunnyvale, CA, USA) equipped with a solvent controller unit. The cells employed (10 mL capacity) were placed in an oven; each cell was filled with ∼1 g of solid sample. After loading the sample into the extraction cell, the cell was filled with the corresponding solvent up to a pressure of 10 MPa (which ensured the liquid state of the three solvents employed at the three temperatures studied) and heated to the selected temperature. In order to prevent over-pressurization of the cell, a static valve opened and closed automatically when the cell pressure exceeded the set point. The solvent that escaped during this venting was collected in the collection vial. Then a static extraction continued in which all system valves were closed. After extraction, the cell was washed with solvent and subsequently the solvent was purged from the cell using nitrogen gas until complete depressurization was accomplished. Supercritical fluid extraction Extractions were carried out using a pilot-plant supercritical fluid extractor (SF2000, Thar Technology, Pittsburgh, PA, USA) comprising a 2 L cylindrical extraction cell and two separators (S1 and S2), each of 0.5 L capacity, with independent control of temperature (±2 K) and pressure (±0.1 MPa). The extraction device also includes a recirculation system, where CO2 is condensed and pumped up to the extraction pressure desired. For each experiment, the extraction vessel was packed with 0.55 kg of thyme leaves. Following the results of previous kinetic studies on T. vulgaris, the overall extraction time was set at 4 h.40 The SFE assays for the three thyme species considered were performed at 40 ∘ C, pressures in the range 15–40 MPa and CO2 /thyme ratios of 25–35 kg kg−1 (CO2 flow rate of 60–70 g min−1 ). The temperature was set to 30 ∘ C in the separators, where decompression was accomplished to 5 MPa (CO2 recirculation system pressure). The SFE assays of T. vulgaris correspond to previously reported data,11 while those of T. zygis and T. citriodorus were accomplished in this work.
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Extraction of thymol from thyme using green solvents
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Table 1. Experimental solubilities of thymol in ethanol and limonene (this work) and ethyl lactate35 Ethanol T (∘ C)
30.9 33.8 37.2 40.4 43.0
Limonene
Solubility (mg g−1 )
T (∘ C)
744 821 885 946 977
31.1 34.5 37.0 40.3
Ethyl lactate
Solubility (mg g−1 )
T (∘ C)
527 593 674 726
28.0 30.5 34.5 35.4 36.3 38.0 40.3 44.8
Solubility (mg g−1 ) 746 766 804 817 830 843 872 918
GC/MS analysis PLE and SFE samples and samples corresponding to the solid–liquid solubility measurements were analyzed in an Agilent 7890A system (Agilent Technologies, Santa Clara, CA, USA) comprising a split/splitless injector, a flame ionization detector, an electronic pressure control, a G4513A auto-injector, a 5975C triple-axis mass spectrometer detector and GC/MS Solution software. The column used was an Agilent HP-5MS capillary column (30 m × 0.25 mm i.d., 0.25 μm phase thickness). The chromatographic method was as follows. The oven temperature was initially held at 60 ∘ C for 4 min, then increased to 106 ∘ C at 2.5 ∘ C min−1 , from 106 to 130 ∘ C at 1 ∘ C min−1 and from 130 to 250 ∘ C at 20 ∘ C min−1 and finally held at 250 ∘ C for 10 min. Sample injections (1 𝜇L) were performed in split mode (1:10). Chromatographic separation was carried out at a constant pressure of 143.6 kPa and helium (99.996 mass%) was used as carrier gas. The injector temperature was 250 ∘ C and the mass spectrometer ion source and interface temperatures were 230 and 280 ∘ C respectively. The mass spectrometer operated under electron impact mode (70 eV), was used in total ion current (TIC) mode and scanned the mass range from m/z 40 to 500. VO components were identified by comparing their mass spectra with authentic standards, by matching the mass spectral fragmentation patterns with the Wiley 229 mass spectral library and also by comparing their retention indices with literature values measured on columns with identical polarities.15,41,42 A mixture of aliphatic hydrocarbons (C6 –C26 ) in hexane was injected under the above temperature program to calculate the retention indices using the generalized equation of Van den Dool and Kratz.43 A calibration curve was employed for the determination of thymol content.
RESULTS AND DISCUSSION
Extraction temperature (∘ C) Solvent Ethyl lactate Extraction yield (%) Thymol concentration (mg g−1 ) Thymol recovery (mg g−1 ) Ethanol Extraction yield (%) Thymol concentration (mg g−1 ) Thymol recovery (mg g−1 ) Limonene Extraction yield (%) Thymol concentration (mg g−1 ) Thymol recovery (mg g−1 )
60
130
200
6.21 ± 0.03 138.8 ± 1.0
8.02 ± 0.30 117.8 ± 4.3
22.66 ± 0.26 46.5 ± 0.2
8.62 ± 0.03
9.44 ± 0.01
10.53 ± 0.16
10.55 ± 1.97 65.9 ± 8.9
15.91 ± 0.36 68.9 ± 7.8
21.84 ± 2.41 48.8 ± 6.7
7.03 ± 2.23
10.98 ± 1.49
10.58 ± 0.28
4.37 ± 0.15 181.5 ± 2.5
7.16 ± 0.54 125.9 ± 4.9
9.52 ± 1.47 100.6 ± 8.2
7.93 ± 0.39
9.00 ± 0.32
9.52 ± 0.70
relative affinity of thymol in the solvents studied follows the order ethanol > ethyl lactate > D-limonene. Pressurized liquid extraction The influence of the extraction time (10 and 20 min) on the extraction yield and the thymol concentration was assessed using T. vulgaris as model plant matrix and ethyl lactate at 200 ∘ C as solvent. The average values from duplicate experiments and the corresponding standard deviations provide the following results. The extraction yield ((mass of extract/mass of plant matrix) × 100) obtained using 10 min extraction time is 22.66 ± 0.26; when 20 min extraction time is considered, the yield is 27.29 ± 0.90, meaning a 17% yield increase in the latter case. Moreover, the thymol concentration obtained in both experiments was higher at 10 min (46.5 ± 0.2 mg g−1 at 10 min and 32.5 ± 0.3 mg g−1 at 20 min). Considering these results, 10 min was selected as the appropriate extraction time to study the effect of temperature and type of solvent on thymol extraction and recovery. The results obtained from the extraction of thymol from T. vulgaris using the three different pressurized liquid solvents at 60, 130 and 200 ∘ C are shown in Table 2. As expected, regardless of the solvent used, extraction yield increased with temperature; the highest increase was observed when ethyl lactate was employed. The highest concentration of thymol in the extracts was obtained with limonene at the lowest temperature tested (60 ∘ C). Moreover, regardless of the temperature applied, extraction with limonene produced higher concentrations of thymol in the extracts. That is, although the solubility of thymol in limonene is lower than its solubility in either ethyl lactate or ethanol (Table 1), limonene still possesses higher selectivity regarding the extraction of thymol from thyme leaves. The extraction pattern applying either ethyl lactate or limonene is very similar, showing a decrease in thymol concentration with
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Solubility measurements Table 1 shows the solubility of thymol in D-limonene and ethanol (data measured in this work) together with its solubility in ethyl lactate reported in a previous work35 and presented here for comparison purposes only. As expected, regardless of the solvent employed, the solubility of thymol is enhanced by increasing temperature. Furthermore, it was observed that thymol is very soluble in all three solvents, particularly in ethanol and ethyl lactate, with concentrations around 900 mg g−1 at temperatures close to 40 ∘ C. Comparatively, at the same temperature, the solubility of thymol in limonene is somewhat lower (∼730 mg g−1 ).That is, the J Sci Food Agric 2015; 95: 2901–2907
Table 2. Extraction yield, thymol concentration and thymol recovery obtained in PLE of Thymus vulgaris using ethyl lactate, ethanol and limonene (10 min)
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Table 3. Extraction yield, thymol concentration and thymol recovery obtained in PLE of Thymus zygis and Thymus citriodorus using ethyl lactate, ethanol and limonene (60 ∘ C, 10 min)
Table 4. Extraction yield, thymol concentration and thymol recovery obtained in SFE/CO2 (40 ∘ C) of Thymus vulgaris,11 Thymus zygis and Thymus citriodorus (this work)
Solvent
Parameter
Ethyl lactate Extraction yield (%) Thymol concentration (mg g−1 ) Thymol recovery (mg g−1 ) Ethanol Extraction yield (%) Thymol concentration (mg g−1 ) Thymol recovery (mg g−1 ) Limonene Extraction yield (%) Thymol concentration (mg g−1 ) Thymol recovery (mg g−1 )
T. zygis
T. citriodorus
9.25 ± 0.48 67.7 ± 1.0 6.26 ± 0.23
8.32 ± 0.21 – –
12.09 ± 0.01 54.0 ± 0.2 6.53 ± 0.02
11.10 ± 1.56 – –
9.36 ± 0.17 89.6 ± 1.3 8.39 ± 0.03
11.35 ± 0.17 – –
an increase in temperature in both cases, while with pressurized ethanol as extraction solvent a slightly higher concentration of thymol was obtained on increasing the temperature from 60 to 130 ∘ C. Nevertheless, when the temperature was increased from 130 to 200 ∘ C, a final decrease in concentration was observed, probably due to a larger co-extraction of other compounds with increasing temperature. Thymol recoveries (mass of thymol in the extract/mass of thymol in the plant matrix) increase somewhat with increasing extraction temperature. At 200 ∘ C they are higher than those obtained by traditional extraction methods such as Soxhlet (∼6.8 mg g−1 ) or steam distillation (∼8.2 mg g−1 )23,24 and just slightly lower than those obtained by PLE with non-green solvents such as hexane (∼10.7 mg g−1 ), dichloromethane (∼12.2 mg g−1 ) or ethyl acetate (∼12.8 mg g−1 ). Finally, thymol recoveries achieved considering the three pressurized solvents selected in this study were higher than those obtained with subcritical water extraction (∼7 mg g−1 ). Table 3 shows the results obtained applying PLE of thymol from the other two thyme species using the three different solvents. The extraction temperature was set at 60 ∘ C since the higher concentration (and just slightly lower recovery) of thymol in the extracts was obtained at this temperature. In the case of T. zygis, although the extraction yields obtained were higher than those achieved for T. vulgaris, the thymol concentrations were lower, presuming a higher content of thymol in T. vulgaris in comparison with T. zygis. These data are in agreement with previously reported results comparing the concentration of thymol obtained by SFE in T. vulgaris26,27 (up to 370 mg g−1 thymol) and T. zygis14 (less than 240 mg g−1 thymol). As in the case of T. vulgaris, limonene was the solvent which recovered the highest amounts of thymol from T. zygis, followed by ethyl lactate. In the case of T. citriodorus extraction, regardless of the solvent employed, thymol was detected in very low amounts and its concentration could not be quantified. This result agrees with a previous report which demonstrated that significantly lower amounts of thymol were detected in the VO of T. citriodorus in comparison with T. zygis or T. vulgaris.44
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Supercritical fluid extraction As mentioned above, SFE assays were carried out at 40 ∘ C for 4 h. The different extraction pressures and CO2 /thyme ratios employed
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Extraction yield (%) Ext. 1: 30 MPa, 26 CO2 /thyme Ext. 2: 15 MPa, 35 CO2 /thyme Ext. 3: 40 MPa, 35 CO2 /thyme Ext. 4: 40 MPa, 26 CO2 /thyme Concentration of thymol (mg g−1 ) Ext. 1: 30 MPa, 26 CO2 /thyme Ext. 2: 15 MPa, 35 CO2 /thyme Ext. 3: 40 MPa, 35 CO2 /thyme Ext. 4: 40 MPa, 26 CO2 /thyme Thymol recovery (mg g−1 ) Ext. 1: 30 MPa, 26 CO2 /thyme Ext. 2: 15 MPa, 35 CO2 /thyme Ext. 3: 40 MPa, 35 CO2 /thyme Ext. 4: 40 MPa, 26 CO2 /thyme
T. vulgaris
4.14 3.31 – 4.68 165.9 310.0 – 171.3 6.87 10.27 – 8.00
T. zygis
3.52 – 3.57 – 198.7 – 160.8 – 6.99 – 5.84 –
T. citriodorus
4.82 – – 5.25 82.6 – – 71.7 3.98 – – 3.77
are given in Table 4, together with the extraction yields (%), concentrations (mg g−1 ) and recoveries (mg g−1 ) of thymol in the extracts. Extraction yields were rather similar for the three thyme species (in the range 3.3–5.3%) and were in the order T. citriodorus > T. vulgaris > T. zygis. In complete analogy with the general trend in SFE of the plant matrix observed, our results show that extraction yield increases when higher extraction pressures are applied (see Ext. 1 and 4 for T. vulgaris and T. citriodorus). On the one hand, the concentrations of thymol in the different extracts collected were significantly lower for T. citriodorus (see Ext.1 and 4), and thus also the corresponding recoveries (∼4 mg g−1 ).44 On the other hand, the highest thymol recoveries were obtained in the case of T. vulgaris extractions,11 with values of 6.87, 10.27 and 8.00 mg g−1 in Ext. 1, 2 and 4 respectively. These extractions reveal that for this thyme species the increase in CO2 flow resulted in higher recoveries than those produced by increasing extraction pressure. In contrast, similar recoveries were obtained in the case of T. zygis and T. citriodorus regardless of the extraction pressure or CO2 flow employed, indicating that thymol would be exhausted from the plant matrix. SFE of T. vulgaris was also studied using the three green liquid solvents as cosolvents. In this case, three extractions were carried out at 15 MPa and 40 ∘ C (best conditions assessed with pure CO2 ) using ∼10% (w/w) ethyl lactate, ethanol or limonene as CO2 cosolvent. However, no improvement in thymol extraction was obtained, with recoveries in the range 7–9 mg g−1 . Volatile oil composition The VO composition of the different samples obtained from PLE and SFE of T. vulgaris is given in Tables 5 and 6 (% peak area). In general, the VO composition of SFE/cosolvent follows the same trend as that of PLE carried out using the corresponding cosolvent of the supercritical extraction (Table 6). Monoterpene phenols (MP) and their derivatives were the most abundant compounds obtained from both PLE and SFE/cosolvent when ethyl lactate and ethanol were employed, thymol being the main compound. Ethanol produced thymol concentrations of 600 mg g−1 (PLE/ethanol) and 740 mg g−1
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Table 5. Composition ((peak area of compound/total area) × 100) of volatile oil obtained from SFE using pure CO2 ,11 CO2 with cosolvents and PLE (60 ∘ C) (this work) of Thymus vulgaris PLE Compound 𝛼-Pinene Sabinene 𝛽-Pinene 3-Octenol 𝛿-3-Carene p-Cymene Limonene 1,8-Cineole 𝛾-Terpinene Terpinolene 1-Octanol 𝛽-Linalool Camphor 𝛽-Citronellal Borneol Terpinen-4-ol 𝛼-Terpineol Cymen-8-ol Thymol methyl ether Thymoquinone Perillaldehyde Unknown thymol derivative Thymol Carvacrol Unknown thymol derivative 𝛽-Caryophyllene 𝛾-Cadinene Caryophyllene oxide 1-Octen-3-ol trans-Sabinene hydrate Carvacrol methyl ether Non-identified
RI
Ethyl lactate
933 967 970 980 1010 1026 1029 1031 1058 1087 1088 1099 1146 1158 1169 1174 1190 1193 1244 1266 1268 1283 1301 1307 1309 1420 1520 1590
Ethanol
– – – – – 37.2 – – – – – 4.7 2.2
– – – – – 13.5 6.3 0.7 – – – 2.3 0.5
2.1 – – – – – – 3.6 32.5 2.5 – – – 0.8 – – – 8.2
1.8 0.6 – 0.4 0.9 – – – 60.0 0.8 4.9 1.3
SFE Limonene 13.9 7.9 46.8 – – –
0.9 0.8 3.3 18.3 0.1 1.1 0.1 – – – 0.1 – 0.3 – 3.8 0.2 – – – – – – – 2.5
1.6 – – – 4.5
CO2 – 1.4 – – – 1.8 7.2 – – – 2.0 9.5 3.4 0.7 1.9 – 0.5 – – 57.5 3.4 – 2.5 0.8 0.48 0.6 0.4 5.6
CO2 /ethyl lactate
CO2 /ethanol
– 9.5 18.1 – – – – – –
– – – 0.5 – 6.0 3.1 0.3 0.1
7.8 1.8 0.4 2.0 – – – – 0.7 – – 42.9 3.7 – – – 1.3 – – – 11.8
1.4 0.4
CO2 /limonene 17.3 9.2 43.7 – 0.1 – – – 0.4 0.5 2.6 13.6 0.1 0.7 0.1 – – – – – 0.7 – 4.9 – – – – – – – – 6.1
1.8 0.4 0.2 0.4 0.4 0.5 – – 74.1 6.1 0.7 0.4 2.4 – – – 0.8
Table 6. Composition ((peak area of compound/total area) × 100) of volatile oil obtained from SFE using pure CO2 ,11 CO2 with cosolvents and PLE (60 ∘ C) (this work) of Thymus vulgaris Extraction method SFE/CO2 SFE/CO2 /limonene (10%) SFE/CO2 /ethyl lactate (10%) SFE/CO2 /ethanol (10%) PLE/limonene PLE/ethyl lactate PLE/ethanol
MAL 9.2 16.3 9.9 4.7 21.7 6.9 5.0
MH 3.2 71.2 27.6 9.3 70.2 37.2 19.7
ME 7.2 – – 0.3 – – 0.7
MK 9.6 0.1 1.8 0.4 0.1 2.2 0.5
OS 0.9 – 1.3 2.4 – 0.8 1.6
MA – 1.5 0.4 – 1.3 – –
MP
SH
61.8 4.9 47.3 81.1 4.1 44.7 66.7
2.5 – – 1.0 – – 1.3
MAL, monoterpene alcohols; MH, monoterpene hydrocarbons; ME, monoterpene ethers; MK, monoterpene ketones; OS, oxygenated sesquiterpenes; MA, monoterpene aldehydes; MP, monoterpene phenols (thymol, carvacrol) and derivatives; SH, sesquiterpene hydrocarbons.
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main MH in PLE/ethanol and SFE/ethanol (135 and 60 mg g−1 respectively). In the case of limonene, MH were the main group of compounds identified, with concentrations around 700 mg g−1 . Most likely, the nonpolar character of limonene promoted the extraction of this type of compound. In this respect, 𝛼- and 𝛽-pinene
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(SFE/ethanol), while, using ethyl lactate, concentrations of thymol were around 320–430 mg g−1 . Also, high concentrations of monoterpene hydrocarbons (MH) were obtained in these extracts. The main MH identified in PLE/ethyl lactate and SFE/ethyl lactate were p-cymene (372 mg g−1 ) and 𝛽-pinene (181 mg g−1 ) respectively. In the same way, p-cymene was the
www.soci.org were the main MH identified. Also, considerable concentrations of monoterpene alcohols (∼200 mg g−1 ) were obtained using limonene (130–180 mg g−1 linalool). In the case of SFE using pure CO2 ,11 similarly to PLE and SFE/cosolvent with either ethanol or ethyl lactate, a high concentration of thymol is obtained (575 mg g−1 ). Also, MP and their derivatives, followed by MH, were the most abundant compounds identified in SFE/CO2 essential oil. Nevertheless, the SFE/CO2 extracts presented higher extraction of oxygenated monoterpenes (ethers and ketones) than the rest of the extracts, with 1,8-cineole (72 mg g−1 ) and camphor (95 mg g−1 ) respectively being the main ether and ketone identified. The higher concentrations of these types of compounds may be attributed to the higher selectivity of CO2 toward extraction of volatile terpene compounds.
6
7 8 9 10 11 12
CONCLUSIONS The recovery of thymol from different thyme species has been reported using PLE with green solvents (ethyl lactate, ethanol and limonene) and SFE with pure CO2 and CO2 plus each of the three green solvents as cosolvent. The highest recoveries were attained with T. vulgaris (7–11 mg thymol g−1 leaves), followed by T. zygis (6–8 mg g−1 ) and T. citriodorus (∼4 mg g−1 ). The three green solvents studied in PLE show good capacity to extract thymol from T. vulgaris and T. zygis, but no thymol could be quantified in the PLE samples of T. citriodorus. Furthermore, limonene was the solvent that produced the highest concentrations of thymol in the extracts owing to its lipophilic character. Although PLE proved to be a suitable technology to extract thymol from thyme plants, the highest concentrations of thymol were obtained by SFE owing to the selective extraction of lipophilic volatile compounds that could be attained with supercritical CO2 . Around 310 mg g−1 of thymol was obtained for T. vulgaris extract (15 MPa, 40 ∘ C and CO2 /thyme ratio of 35), with a thymol recovery very similar to those obtained at 200 ∘ C with any of the three liquid solvents in PLE. Also for T. zygis, concentrations were higher and recoveries were similar to PLE; in particular, SFE samples of T. citriodorus contain 70–80 mg g−1 of thymol, while very low amounts of thymol were obtained in PLE samples.
13 14 15
16
17 18 19 20 21 22
ACKNOWLEDGEMENTS The authors gratefully acknowledge the financial support from Ministerio de Ciencia e Innovación (project 25506 FUN-C-FOOD) and Comunidad Autónoma de Madrid (ALIBIRD-CM, project S2013/ABI-2728). DVB thanks Consejo Superior de Investigaciones Científicas (CSIC) of Spain for a JAE-pre fellowship.
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1 Adzet T, Granger R, Passet J and San Martin R, Le polymorphisme chimique dans le genre Thymus: sa signification taxonomique. Biochem Syst Ecol 5:269–272 (1977). 2 Pourmortazavi SM and Hajimirsadeghi SS, Supercritical fluid extraction in plant essential and volatile oil analysis. J Chromatogr A 1163:2–24 (2007). 3 Prakash V, Leafy Spices. CRC Press, Boca Raton, FL (1990). 4 Mookherjee BD, Wilson RA, Trenkle RW, Zampino MJ and Sands KP, New dimensions in flavor research. ACS Symp Ser 388:176–187 (1989). 5 Ferley J, Poutignat N, Azzopard Y and Balducci F, Aromathérapie préventive des surinfections chez les bronchiteux chroniques:
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integrated Soxhlet using limonene followed by microwave Clevenger distillation. J Chromatogr A 1196/1197:147–152 (2008). Toplisek T and Gustafson R, Cleaning with D-limonene: a substitute for chlorinated solvents? Precision Cleaning 17–22 (1995). Mamidipally PK and Liu SX, First approach on rice bran oil extraction using limonene. Eur J Lipid Sci Technol 106:122–125 (2004). Chemat-Djenni Z, Ferhat MA, Tomao V and Chemat F, Carotenoid extraction from tomato using a green solvent resulting from orange processing waste. J Essent Oil Bearing Plants 2:139–147 (2010). Fornari T, Ruiz-Rodriguez A, Vicente G, Vázquez E, García-Risco MR and Reglero G, Kinetic study of the supercritical CO2 extraction of different plants from Lamiaceae family. J Supercrit Fluids 64:1–8 (2012). Baranauskiené R, Venskutonis PR, Viskelis P and Dambrauskiené E, Influence of nitrogen fertilizers on the yield and composition of thyme (Thymus vulgaris). J Agric Food Chem 51:7751–7758 (2003). Hudaib M, Speroni E, Di Pietra AM and Cavrini V, GC/MS evaluation of thyme (Thymus vulgaris L.) oil composition and variations during the vegetative cycle. J Pharmaceut Biomed Anal 129:691–700 (2002). Van den Dool H and Kratz PD, A generalization of the retention index system including linear temperature programmed gas–liquid partition chromatography. J Chromatogr A 11:463–471 (1963). Omidbaigi R, Sefidkon F and Hejazi M, Essential oil composition of Thymus citriodorus L. cultivated in Iran. Flav Fragr J 20:237–238 (2005).
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Revised: 25 November 2014
Accepted article published: 1 December 2014
Published online in Wiley Online Library: 30 December 2014
(wileyonlinelibrary.com) DOI 10.1002/jsfa.7031
Extraction of thymol from different varieties of thyme plants using green solvents David Villanueva Bermejo,a Ivan Angelov,b Gonzalo Vicente,a Roumiana P Stateva,b* Mónica Rodriguez García-Risco,a Guillermo Reglero,a Elena Ibañeza and Tiziana Fornaria Abstract BACKGROUND: Thymol (2-isopropyl-5-methylphenol) is the main monoterpene phenol found in thyme essential oil. This compound has revealed several biological properties, including antibacterial, anti-inflammatory and antioxidant activities. In this work, a comparison was made between the performance of different green solvents (ethanol, limonene and ethyl lactate), by pressurized liquid extraction (PLE) and supercritical fluid extraction (SFE) at different conditions, to extract thymol from three different varieties of thyme (Thymus vulgaris, Thymus zygis and Thymus citriodorus). Additionally, new solubility data of thymol in limonene and ethanol at ambient pressure and temperatures in the range 30–43 ∘ C are reported. RESULTS: The highest thymol recoveries were attained with T. vulgaris (7–11 mg g−1 ). No thymol could be quantified in the PLE samples of T. citriodorus. The highest concentrations of thymol in the extracts were obtained with limonene. Thymol is very soluble in both solvents, particularly in ethanol (∼900 mg g−1 at ∼40 ∘ C), and is the main compound (in terms of peak area) present in the essential oil extracts obtained. CONCLUSION: The three solvents show good capacity to extract thymol from T. vulgaris and T. zygis by PLE. Although PLE proved to be a suitable technology to extract thymol from thyme plants, the highest concentrations of thymol were obtained by SFE with supercritical CO2 . © 2014 Society of Chemical Industry Keywords: pressurized liquid extraction (PLE); supercritical fluid extraction (SFE); thyme; ethyl lactate; limonene
INTRODUCTION
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∗
Correspondence to: Roumiana P. Stateva, Institute of Chemical Engineering, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria. E-mail: [email protected]
a Instituto de Investigación en Ciencias de la Alimentación CIAL (CSIC-UAM), CEI UAM + CSIC, Universidad Autónoma de Madrid, C/Nicolás Cabrera 9, E-28049 Madrid, Spain b Institute of Chemical Engineering, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
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The genus Thymus (family Lamiaceae) comprises over 350 aromatic plant species with quite different botanical characteristics and broad chemical heterogeneity.1 One of the most valued constituents of thyme (especially of the leaves) is the volatile oil (VO), which is formed by terpenes, sesquiterpenes and several oxygenated derivative compounds (alcohols, aldehydes, ketones, acids, phenols, ethers, esters, etc.), all of which are responsible for the characteristic plant odor and flavor.2 Thyme VO is appreciated for food flavoring,3 in cosmetics and perfumery4 and in the pharmaceutical industries.5 The main biological properties that have been recognized as its pharmacological actions are antimicrobial, antitussive, antispasmodic and antioxidant activities.6,7 Thymol and its isomer carvacrol are among the most biologically active constituents of thyme VO. Thymol (2-isopropyl-5methylphenol) is the main monoterpene phenol found in the VO of several varieties of thyme. Thymol and carvacrol have shown anti-inflammatory, immunomodulatory, antioxidant, antibacterial and antifungal properties.8 – 10 The variety most studied is Thymus vulgaris,11 – 13 while attention has also been focused on Thymus zygis,14 a thyme variety widespread over Portugal and Spain, as well as on Thymus citriodorus, a plant cultivated throughout the Mediterranean region.15 The oil of the herb thyme has usually been obtained by either steam distillation (in which case, according to the European
Pharmacopoeia, it is referred to as the essential oil of the herb) or traditional solid–liquid extraction methods such as Soxhlet, maceration or extraction under reflux. Although these methods are relatively simple, they suffer from several shortcomings, such as a long extraction time and a relatively high solvent consumption, to name just two. Pressurized liquid extraction (PLE), a relatively recent solvent extraction technique, can, in principle, eliminate some of the drawbacks of classical solvent extraction methods. PLE is based on the use of solvents at temperatures above their normal boiling points and at pressures high enough to keep the extraction fluid in the liquid state during the whole extraction process. Thus faster extraction processes can be realized, with higher extraction yields and lower volumes of organic solvents,16 which are essential principles of ‘green extraction’.17 Although several studies have been reported on PLE of VOs from a wide range of raw herbal materials,18 – 22 only a few
www.soci.org communications can be found on PLE of VO from thyme. For instance, Dawidowicz et al.23,24 studied the use of hexane, dichloromethane, ethyl acetate and water at pressurized liquid conditions to extract the VO from T. vulgaris. Higher recoveries of thymol were obtained using the non-green solvents hexane (∼10.7 mg g−1 ), dichloromethane (∼12.2 mg g−1 ) and ethyl acetate (∼12.8 mg g−1 ) in comparison with those obtained using subcritical water extraction (∼7 mg g−1 ). On the other hand, supercritical fluid extraction (SFE) has also been suggested as a valuable alternative to classical extraction methods to recover essential oils from plants and herbs. Several studies related to SFE of thyme leaves have already been published.11,14,25 – 28 In all of them, pure CO2 was employed as extraction solvent and, depending on the extraction conditions, different concentrations of thymol were achieved. In the ongoing search for potential new green extraction solvents that might replace toxic organic solvents, two solvents, namely ethyl lactate and D-limonene, have recently been suggested.17 Ethyl lactate (ethyl 2-hydroxypropanoate) is an agrochemical which is an economically viable alternative to traditional liquid solvents; it is fully biodegradable, non-corrosive, non-carcinogenic and non-ozone-depleting. It was self-affirmed as GRAS (‘generally recognized as safe’) and, owing to its low toxicity, approved by the US Food and Drug Administration (FDA) as a pharmaceutical and food additive. These characteristics have increased the interest of researchers in its potential as a green solvent for food applications.29 – 34 D-Limonene (1-methyl-4-(1-methylethenyl)cyclohexene) is also a non-toxic, non-corrosive, non-carcinogenic and fully biodegradable solvent approved by the FDA as GRAS. This monoterpenic molecule is the major component of essential oils extracted from citrus peels and is a major by-product of the citrus fruit industry.36,37 It has been demonstrated that D-limonene is a valuable alternative to petroleum solvents and it has been used to remove oil from rice bran38 and to extract lycopene from tomato.39 Although high solubility of thymol in ethyl lactate at different temperatures has been reported in a recent contribution,35 to the best of our knowledge, there are still no data published in the open literature related to the extraction of thymol from thyme using either pressurized ethyl lactate or D-limonene. Therefore the aim of the present work is to provide new insights into the potential of several green compressed solvents to extract thymol from different thyme species.
MATERIALS AND METHODS Chemicals Ethanol was HPLC grade from Panreac (Barcelona, Spain). Standards employed were thymol (>99%), carvacrol (98%), camphor (>95%), 𝛼-pinene (>99%), 𝛽-linalool (>97%), verbenone (>97%), D-limonene (90%) and ethyl lactate (>98%) from Sigma-Aldrich (St. Louis, MO, USA). Borneol (>99%) and 1,8-cineole (>98%) were purchased from Fluka (Buch, Switzerland). Eugenol (>99%) and 𝛼-terpineol (>96%) were supplied by Extrasynthèse (Lyon, France). CO2 for SFE (99.999%) was provided by Carburos Metálicos (Madrid, Spain).
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Thyme leaf preparation The thyme (T. vulgaris, T. zygis and T. citriodorus) samples consisted of dried leaves obtained from a herbalist’s producer (Murcia,
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Spain). The leaves were ground in a cooled mill. Particles of size smaller than 250 μm were separated using sieves and employed in the experiments. The whole sample was stored at −20 ∘ C until used. Solubility measurements Solubility measurements of thymol in the liquid solvents were made at atmospheric pressure as a function of temperature over the range 30–43 ∘ C. For all solutions studied, the liquid solvent (either D-limonene or ethanol) and thymol in excess were placed in glass vessels (10 mL) with magnetic stir bars. The vessels were put inside a water bath heated by a magnetic hotplate stirrer (RCT classic IKAMAG® safety control, IKA Works GmbH & Co. KG, Staufen, Germany), which was used to agitate the samples for 24 h at a fixed temperature, controlled by an electronic contact thermometer with probe (VWR VT-5, VWR International, LLC, West Chester, PA, USA) having an accuracy of 0.2 K. After equilibrium had been reached, the stirring was stopped and the vessels were left still for more than 48 h to allow complete phase separation. Samples of clear saturated liquid solution (100 𝜇L) were taken using a micropipette and placed in glass vials. Samples were appropriately diluted for gas chromatography/mass spectrometry (GC/MS) analysis. Pressurized liquid extraction Thymol extractions were carried out in an Accelerated Solvent Extraction system ASE 350 (Dionex Corporation, Sunnyvale, CA, USA) equipped with a solvent controller unit. The cells employed (10 mL capacity) were placed in an oven; each cell was filled with ∼1 g of solid sample. After loading the sample into the extraction cell, the cell was filled with the corresponding solvent up to a pressure of 10 MPa (which ensured the liquid state of the three solvents employed at the three temperatures studied) and heated to the selected temperature. In order to prevent over-pressurization of the cell, a static valve opened and closed automatically when the cell pressure exceeded the set point. The solvent that escaped during this venting was collected in the collection vial. Then a static extraction continued in which all system valves were closed. After extraction, the cell was washed with solvent and subsequently the solvent was purged from the cell using nitrogen gas until complete depressurization was accomplished. Supercritical fluid extraction Extractions were carried out using a pilot-plant supercritical fluid extractor (SF2000, Thar Technology, Pittsburgh, PA, USA) comprising a 2 L cylindrical extraction cell and two separators (S1 and S2), each of 0.5 L capacity, with independent control of temperature (±2 K) and pressure (±0.1 MPa). The extraction device also includes a recirculation system, where CO2 is condensed and pumped up to the extraction pressure desired. For each experiment, the extraction vessel was packed with 0.55 kg of thyme leaves. Following the results of previous kinetic studies on T. vulgaris, the overall extraction time was set at 4 h.40 The SFE assays for the three thyme species considered were performed at 40 ∘ C, pressures in the range 15–40 MPa and CO2 /thyme ratios of 25–35 kg kg−1 (CO2 flow rate of 60–70 g min−1 ). The temperature was set to 30 ∘ C in the separators, where decompression was accomplished to 5 MPa (CO2 recirculation system pressure). The SFE assays of T. vulgaris correspond to previously reported data,11 while those of T. zygis and T. citriodorus were accomplished in this work.
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Table 1. Experimental solubilities of thymol in ethanol and limonene (this work) and ethyl lactate35 Ethanol T (∘ C)
30.9 33.8 37.2 40.4 43.0
Limonene
Solubility (mg g−1 )
T (∘ C)
744 821 885 946 977
31.1 34.5 37.0 40.3
Ethyl lactate
Solubility (mg g−1 )
T (∘ C)
527 593 674 726
28.0 30.5 34.5 35.4 36.3 38.0 40.3 44.8
Solubility (mg g−1 ) 746 766 804 817 830 843 872 918
GC/MS analysis PLE and SFE samples and samples corresponding to the solid–liquid solubility measurements were analyzed in an Agilent 7890A system (Agilent Technologies, Santa Clara, CA, USA) comprising a split/splitless injector, a flame ionization detector, an electronic pressure control, a G4513A auto-injector, a 5975C triple-axis mass spectrometer detector and GC/MS Solution software. The column used was an Agilent HP-5MS capillary column (30 m × 0.25 mm i.d., 0.25 μm phase thickness). The chromatographic method was as follows. The oven temperature was initially held at 60 ∘ C for 4 min, then increased to 106 ∘ C at 2.5 ∘ C min−1 , from 106 to 130 ∘ C at 1 ∘ C min−1 and from 130 to 250 ∘ C at 20 ∘ C min−1 and finally held at 250 ∘ C for 10 min. Sample injections (1 𝜇L) were performed in split mode (1:10). Chromatographic separation was carried out at a constant pressure of 143.6 kPa and helium (99.996 mass%) was used as carrier gas. The injector temperature was 250 ∘ C and the mass spectrometer ion source and interface temperatures were 230 and 280 ∘ C respectively. The mass spectrometer operated under electron impact mode (70 eV), was used in total ion current (TIC) mode and scanned the mass range from m/z 40 to 500. VO components were identified by comparing their mass spectra with authentic standards, by matching the mass spectral fragmentation patterns with the Wiley 229 mass spectral library and also by comparing their retention indices with literature values measured on columns with identical polarities.15,41,42 A mixture of aliphatic hydrocarbons (C6 –C26 ) in hexane was injected under the above temperature program to calculate the retention indices using the generalized equation of Van den Dool and Kratz.43 A calibration curve was employed for the determination of thymol content.
RESULTS AND DISCUSSION
Extraction temperature (∘ C) Solvent Ethyl lactate Extraction yield (%) Thymol concentration (mg g−1 ) Thymol recovery (mg g−1 ) Ethanol Extraction yield (%) Thymol concentration (mg g−1 ) Thymol recovery (mg g−1 ) Limonene Extraction yield (%) Thymol concentration (mg g−1 ) Thymol recovery (mg g−1 )
60
130
200
6.21 ± 0.03 138.8 ± 1.0
8.02 ± 0.30 117.8 ± 4.3
22.66 ± 0.26 46.5 ± 0.2
8.62 ± 0.03
9.44 ± 0.01
10.53 ± 0.16
10.55 ± 1.97 65.9 ± 8.9
15.91 ± 0.36 68.9 ± 7.8
21.84 ± 2.41 48.8 ± 6.7
7.03 ± 2.23
10.98 ± 1.49
10.58 ± 0.28
4.37 ± 0.15 181.5 ± 2.5
7.16 ± 0.54 125.9 ± 4.9
9.52 ± 1.47 100.6 ± 8.2
7.93 ± 0.39
9.00 ± 0.32
9.52 ± 0.70
relative affinity of thymol in the solvents studied follows the order ethanol > ethyl lactate > D-limonene. Pressurized liquid extraction The influence of the extraction time (10 and 20 min) on the extraction yield and the thymol concentration was assessed using T. vulgaris as model plant matrix and ethyl lactate at 200 ∘ C as solvent. The average values from duplicate experiments and the corresponding standard deviations provide the following results. The extraction yield ((mass of extract/mass of plant matrix) × 100) obtained using 10 min extraction time is 22.66 ± 0.26; when 20 min extraction time is considered, the yield is 27.29 ± 0.90, meaning a 17% yield increase in the latter case. Moreover, the thymol concentration obtained in both experiments was higher at 10 min (46.5 ± 0.2 mg g−1 at 10 min and 32.5 ± 0.3 mg g−1 at 20 min). Considering these results, 10 min was selected as the appropriate extraction time to study the effect of temperature and type of solvent on thymol extraction and recovery. The results obtained from the extraction of thymol from T. vulgaris using the three different pressurized liquid solvents at 60, 130 and 200 ∘ C are shown in Table 2. As expected, regardless of the solvent used, extraction yield increased with temperature; the highest increase was observed when ethyl lactate was employed. The highest concentration of thymol in the extracts was obtained with limonene at the lowest temperature tested (60 ∘ C). Moreover, regardless of the temperature applied, extraction with limonene produced higher concentrations of thymol in the extracts. That is, although the solubility of thymol in limonene is lower than its solubility in either ethyl lactate or ethanol (Table 1), limonene still possesses higher selectivity regarding the extraction of thymol from thyme leaves. The extraction pattern applying either ethyl lactate or limonene is very similar, showing a decrease in thymol concentration with
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Solubility measurements Table 1 shows the solubility of thymol in D-limonene and ethanol (data measured in this work) together with its solubility in ethyl lactate reported in a previous work35 and presented here for comparison purposes only. As expected, regardless of the solvent employed, the solubility of thymol is enhanced by increasing temperature. Furthermore, it was observed that thymol is very soluble in all three solvents, particularly in ethanol and ethyl lactate, with concentrations around 900 mg g−1 at temperatures close to 40 ∘ C. Comparatively, at the same temperature, the solubility of thymol in limonene is somewhat lower (∼730 mg g−1 ).That is, the J Sci Food Agric 2015; 95: 2901–2907
Table 2. Extraction yield, thymol concentration and thymol recovery obtained in PLE of Thymus vulgaris using ethyl lactate, ethanol and limonene (10 min)
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Table 3. Extraction yield, thymol concentration and thymol recovery obtained in PLE of Thymus zygis and Thymus citriodorus using ethyl lactate, ethanol and limonene (60 ∘ C, 10 min)
Table 4. Extraction yield, thymol concentration and thymol recovery obtained in SFE/CO2 (40 ∘ C) of Thymus vulgaris,11 Thymus zygis and Thymus citriodorus (this work)
Solvent
Parameter
Ethyl lactate Extraction yield (%) Thymol concentration (mg g−1 ) Thymol recovery (mg g−1 ) Ethanol Extraction yield (%) Thymol concentration (mg g−1 ) Thymol recovery (mg g−1 ) Limonene Extraction yield (%) Thymol concentration (mg g−1 ) Thymol recovery (mg g−1 )
T. zygis
T. citriodorus
9.25 ± 0.48 67.7 ± 1.0 6.26 ± 0.23
8.32 ± 0.21 – –
12.09 ± 0.01 54.0 ± 0.2 6.53 ± 0.02
11.10 ± 1.56 – –
9.36 ± 0.17 89.6 ± 1.3 8.39 ± 0.03
11.35 ± 0.17 – –
an increase in temperature in both cases, while with pressurized ethanol as extraction solvent a slightly higher concentration of thymol was obtained on increasing the temperature from 60 to 130 ∘ C. Nevertheless, when the temperature was increased from 130 to 200 ∘ C, a final decrease in concentration was observed, probably due to a larger co-extraction of other compounds with increasing temperature. Thymol recoveries (mass of thymol in the extract/mass of thymol in the plant matrix) increase somewhat with increasing extraction temperature. At 200 ∘ C they are higher than those obtained by traditional extraction methods such as Soxhlet (∼6.8 mg g−1 ) or steam distillation (∼8.2 mg g−1 )23,24 and just slightly lower than those obtained by PLE with non-green solvents such as hexane (∼10.7 mg g−1 ), dichloromethane (∼12.2 mg g−1 ) or ethyl acetate (∼12.8 mg g−1 ). Finally, thymol recoveries achieved considering the three pressurized solvents selected in this study were higher than those obtained with subcritical water extraction (∼7 mg g−1 ). Table 3 shows the results obtained applying PLE of thymol from the other two thyme species using the three different solvents. The extraction temperature was set at 60 ∘ C since the higher concentration (and just slightly lower recovery) of thymol in the extracts was obtained at this temperature. In the case of T. zygis, although the extraction yields obtained were higher than those achieved for T. vulgaris, the thymol concentrations were lower, presuming a higher content of thymol in T. vulgaris in comparison with T. zygis. These data are in agreement with previously reported results comparing the concentration of thymol obtained by SFE in T. vulgaris26,27 (up to 370 mg g−1 thymol) and T. zygis14 (less than 240 mg g−1 thymol). As in the case of T. vulgaris, limonene was the solvent which recovered the highest amounts of thymol from T. zygis, followed by ethyl lactate. In the case of T. citriodorus extraction, regardless of the solvent employed, thymol was detected in very low amounts and its concentration could not be quantified. This result agrees with a previous report which demonstrated that significantly lower amounts of thymol were detected in the VO of T. citriodorus in comparison with T. zygis or T. vulgaris.44
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Supercritical fluid extraction As mentioned above, SFE assays were carried out at 40 ∘ C for 4 h. The different extraction pressures and CO2 /thyme ratios employed
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Extraction yield (%) Ext. 1: 30 MPa, 26 CO2 /thyme Ext. 2: 15 MPa, 35 CO2 /thyme Ext. 3: 40 MPa, 35 CO2 /thyme Ext. 4: 40 MPa, 26 CO2 /thyme Concentration of thymol (mg g−1 ) Ext. 1: 30 MPa, 26 CO2 /thyme Ext. 2: 15 MPa, 35 CO2 /thyme Ext. 3: 40 MPa, 35 CO2 /thyme Ext. 4: 40 MPa, 26 CO2 /thyme Thymol recovery (mg g−1 ) Ext. 1: 30 MPa, 26 CO2 /thyme Ext. 2: 15 MPa, 35 CO2 /thyme Ext. 3: 40 MPa, 35 CO2 /thyme Ext. 4: 40 MPa, 26 CO2 /thyme
T. vulgaris
4.14 3.31 – 4.68 165.9 310.0 – 171.3 6.87 10.27 – 8.00
T. zygis
3.52 – 3.57 – 198.7 – 160.8 – 6.99 – 5.84 –
T. citriodorus
4.82 – – 5.25 82.6 – – 71.7 3.98 – – 3.77
are given in Table 4, together with the extraction yields (%), concentrations (mg g−1 ) and recoveries (mg g−1 ) of thymol in the extracts. Extraction yields were rather similar for the three thyme species (in the range 3.3–5.3%) and were in the order T. citriodorus > T. vulgaris > T. zygis. In complete analogy with the general trend in SFE of the plant matrix observed, our results show that extraction yield increases when higher extraction pressures are applied (see Ext. 1 and 4 for T. vulgaris and T. citriodorus). On the one hand, the concentrations of thymol in the different extracts collected were significantly lower for T. citriodorus (see Ext.1 and 4), and thus also the corresponding recoveries (∼4 mg g−1 ).44 On the other hand, the highest thymol recoveries were obtained in the case of T. vulgaris extractions,11 with values of 6.87, 10.27 and 8.00 mg g−1 in Ext. 1, 2 and 4 respectively. These extractions reveal that for this thyme species the increase in CO2 flow resulted in higher recoveries than those produced by increasing extraction pressure. In contrast, similar recoveries were obtained in the case of T. zygis and T. citriodorus regardless of the extraction pressure or CO2 flow employed, indicating that thymol would be exhausted from the plant matrix. SFE of T. vulgaris was also studied using the three green liquid solvents as cosolvents. In this case, three extractions were carried out at 15 MPa and 40 ∘ C (best conditions assessed with pure CO2 ) using ∼10% (w/w) ethyl lactate, ethanol or limonene as CO2 cosolvent. However, no improvement in thymol extraction was obtained, with recoveries in the range 7–9 mg g−1 . Volatile oil composition The VO composition of the different samples obtained from PLE and SFE of T. vulgaris is given in Tables 5 and 6 (% peak area). In general, the VO composition of SFE/cosolvent follows the same trend as that of PLE carried out using the corresponding cosolvent of the supercritical extraction (Table 6). Monoterpene phenols (MP) and their derivatives were the most abundant compounds obtained from both PLE and SFE/cosolvent when ethyl lactate and ethanol were employed, thymol being the main compound. Ethanol produced thymol concentrations of 600 mg g−1 (PLE/ethanol) and 740 mg g−1
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Extraction of thymol from thyme using green solvents
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Table 5. Composition ((peak area of compound/total area) × 100) of volatile oil obtained from SFE using pure CO2 ,11 CO2 with cosolvents and PLE (60 ∘ C) (this work) of Thymus vulgaris PLE Compound 𝛼-Pinene Sabinene 𝛽-Pinene 3-Octenol 𝛿-3-Carene p-Cymene Limonene 1,8-Cineole 𝛾-Terpinene Terpinolene 1-Octanol 𝛽-Linalool Camphor 𝛽-Citronellal Borneol Terpinen-4-ol 𝛼-Terpineol Cymen-8-ol Thymol methyl ether Thymoquinone Perillaldehyde Unknown thymol derivative Thymol Carvacrol Unknown thymol derivative 𝛽-Caryophyllene 𝛾-Cadinene Caryophyllene oxide 1-Octen-3-ol trans-Sabinene hydrate Carvacrol methyl ether Non-identified
RI
Ethyl lactate
933 967 970 980 1010 1026 1029 1031 1058 1087 1088 1099 1146 1158 1169 1174 1190 1193 1244 1266 1268 1283 1301 1307 1309 1420 1520 1590
Ethanol
– – – – – 37.2 – – – – – 4.7 2.2
– – – – – 13.5 6.3 0.7 – – – 2.3 0.5
2.1 – – – – – – 3.6 32.5 2.5 – – – 0.8 – – – 8.2
1.8 0.6 – 0.4 0.9 – – – 60.0 0.8 4.9 1.3
SFE Limonene 13.9 7.9 46.8 – – –
0.9 0.8 3.3 18.3 0.1 1.1 0.1 – – – 0.1 – 0.3 – 3.8 0.2 – – – – – – – 2.5
1.6 – – – 4.5
CO2 – 1.4 – – – 1.8 7.2 – – – 2.0 9.5 3.4 0.7 1.9 – 0.5 – – 57.5 3.4 – 2.5 0.8 0.48 0.6 0.4 5.6
CO2 /ethyl lactate
CO2 /ethanol
– 9.5 18.1 – – – – – –
– – – 0.5 – 6.0 3.1 0.3 0.1
7.8 1.8 0.4 2.0 – – – – 0.7 – – 42.9 3.7 – – – 1.3 – – – 11.8
1.4 0.4
CO2 /limonene 17.3 9.2 43.7 – 0.1 – – – 0.4 0.5 2.6 13.6 0.1 0.7 0.1 – – – – – 0.7 – 4.9 – – – – – – – – 6.1
1.8 0.4 0.2 0.4 0.4 0.5 – – 74.1 6.1 0.7 0.4 2.4 – – – 0.8
Table 6. Composition ((peak area of compound/total area) × 100) of volatile oil obtained from SFE using pure CO2 ,11 CO2 with cosolvents and PLE (60 ∘ C) (this work) of Thymus vulgaris Extraction method SFE/CO2 SFE/CO2 /limonene (10%) SFE/CO2 /ethyl lactate (10%) SFE/CO2 /ethanol (10%) PLE/limonene PLE/ethyl lactate PLE/ethanol
MAL 9.2 16.3 9.9 4.7 21.7 6.9 5.0
MH 3.2 71.2 27.6 9.3 70.2 37.2 19.7
ME 7.2 – – 0.3 – – 0.7
MK 9.6 0.1 1.8 0.4 0.1 2.2 0.5
OS 0.9 – 1.3 2.4 – 0.8 1.6
MA – 1.5 0.4 – 1.3 – –
MP
SH
61.8 4.9 47.3 81.1 4.1 44.7 66.7
2.5 – – 1.0 – – 1.3
MAL, monoterpene alcohols; MH, monoterpene hydrocarbons; ME, monoterpene ethers; MK, monoterpene ketones; OS, oxygenated sesquiterpenes; MA, monoterpene aldehydes; MP, monoterpene phenols (thymol, carvacrol) and derivatives; SH, sesquiterpene hydrocarbons.
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main MH in PLE/ethanol and SFE/ethanol (135 and 60 mg g−1 respectively). In the case of limonene, MH were the main group of compounds identified, with concentrations around 700 mg g−1 . Most likely, the nonpolar character of limonene promoted the extraction of this type of compound. In this respect, 𝛼- and 𝛽-pinene
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(SFE/ethanol), while, using ethyl lactate, concentrations of thymol were around 320–430 mg g−1 . Also, high concentrations of monoterpene hydrocarbons (MH) were obtained in these extracts. The main MH identified in PLE/ethyl lactate and SFE/ethyl lactate were p-cymene (372 mg g−1 ) and 𝛽-pinene (181 mg g−1 ) respectively. In the same way, p-cymene was the
www.soci.org were the main MH identified. Also, considerable concentrations of monoterpene alcohols (∼200 mg g−1 ) were obtained using limonene (130–180 mg g−1 linalool). In the case of SFE using pure CO2 ,11 similarly to PLE and SFE/cosolvent with either ethanol or ethyl lactate, a high concentration of thymol is obtained (575 mg g−1 ). Also, MP and their derivatives, followed by MH, were the most abundant compounds identified in SFE/CO2 essential oil. Nevertheless, the SFE/CO2 extracts presented higher extraction of oxygenated monoterpenes (ethers and ketones) than the rest of the extracts, with 1,8-cineole (72 mg g−1 ) and camphor (95 mg g−1 ) respectively being the main ether and ketone identified. The higher concentrations of these types of compounds may be attributed to the higher selectivity of CO2 toward extraction of volatile terpene compounds.
6
7 8 9 10 11 12
CONCLUSIONS The recovery of thymol from different thyme species has been reported using PLE with green solvents (ethyl lactate, ethanol and limonene) and SFE with pure CO2 and CO2 plus each of the three green solvents as cosolvent. The highest recoveries were attained with T. vulgaris (7–11 mg thymol g−1 leaves), followed by T. zygis (6–8 mg g−1 ) and T. citriodorus (∼4 mg g−1 ). The three green solvents studied in PLE show good capacity to extract thymol from T. vulgaris and T. zygis, but no thymol could be quantified in the PLE samples of T. citriodorus. Furthermore, limonene was the solvent that produced the highest concentrations of thymol in the extracts owing to its lipophilic character. Although PLE proved to be a suitable technology to extract thymol from thyme plants, the highest concentrations of thymol were obtained by SFE owing to the selective extraction of lipophilic volatile compounds that could be attained with supercritical CO2 . Around 310 mg g−1 of thymol was obtained for T. vulgaris extract (15 MPa, 40 ∘ C and CO2 /thyme ratio of 35), with a thymol recovery very similar to those obtained at 200 ∘ C with any of the three liquid solvents in PLE. Also for T. zygis, concentrations were higher and recoveries were similar to PLE; in particular, SFE samples of T. citriodorus contain 70–80 mg g−1 of thymol, while very low amounts of thymol were obtained in PLE samples.
13 14 15
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17 18 19 20 21 22
ACKNOWLEDGEMENTS The authors gratefully acknowledge the financial support from Ministerio de Ciencia e Innovación (project 25506 FUN-C-FOOD) and Comunidad Autónoma de Madrid (ALIBIRD-CM, project S2013/ABI-2728). DVB thanks Consejo Superior de Investigaciones Científicas (CSIC) of Spain for a JAE-pre fellowship.
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