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Supercritical extraction of savory oil: study of antioxidant activity and extract characterization
Journal of Supercritical Fluids 14 (1999) 129–138
Supercritical extraction of savory oil: study of antioxidant activity and extract characterization M.M. Esquı´vel *, M.A. Ribeiro, M.G. Bernardo-Gil Centro de Engenharia Biolo´gica e Quı´mica, Departamento de Engenharia Quı´mica, Instituto Superior Te´cnico, Av. Rovisco Pais, 1096 Codex, Lisbon, Portugal Received 6 October 1997; received in revised form 12 May 1998; accepted 2 June 1998
Abstract The experimental results of supercritical CO extraction on summer savory (Satureja hortensis L.) oil at pressures 2 ranging from 12 to 18 MPa and at a temperature of 313 K are presented. The optimum conditions achieved to obtain the maximum extraction yield were 12 MPa, 120 kg CO /h kg solid and 1 h of extraction. The extract was fractionated 2 in three separators, operated in series. The optimum fractionation conditions which minimize the coextraction of unwanted compounds were investigated using a two-level factorial design approach. The extracts obtained were compared with summer savory essential oil isolated by steam distillation and by a modified Clevenger apparatus. A study of the antioxidant activity of summer savory extracts was performed using the Rancimat method. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Antioxidant; Essential oil; Fractionation; Savory; Supercritical extraction
1. Introduction Herbs have a long history of culinary and medicinal use stretching back to biblical times and beyond. Not only are many of the flavours and aromas distinctively pleasant, but they can be used to conceal off-flavours and odours. There is now evidence that their addition, for example to meat, may help to prevent the formation of undesirable oxidation products. In the last 15–20 years, spice research has concentrated on two primary areas: the antioxidant and the microbial activity of spices and their essential oils [1]. * Corresponding author. Tel: +35 11 8417312; Fax: +35 11 8499242; e-mail: [email protected]
The high susceptibility to oxidation of the fat and oil polyunsaturated fatty acids used in human foods or animal feed requires the application of antioxidants. The antioxidants can be either synthetic or natural products, which scavenge oxygen free radicals, thereby inhibiting or delaying oxidation. The search for natural antioxidants has been difficult, not only because of the eventual toxicity of some synthetic antioxidants, especially butylated hydroxyanisole (BHA) which has a component which can eventually cause cancer, but also because most of them are heat-sensitive and volatile in steam [2]. Therefore, the possibility of using natural antioxidants obtained from vegetable and/or micro-organisms is currently being investigated. However, the industrial utilization of natu-
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ral antioxidants is limited, mainly because of the low efficiency/cost ratio observed for the extraction of known natural antioxidants [2]. Some sources of natural antioxidant compounds include spices, herbs and by products from the vegetable oil refining industry. The extraction of flavouring materials with supercritical carbon dioxide has been carried out previously [3,4]. Recently, studies of supercritical carbon dioxide extraction have been reported on rose concrete [5], sage oil [6 ], black pepper [7], orange peel [8], ginger oil [9], and oregano, thyme and rosemary [10]. Savory (S. hortensis L.) is an annual herb of the mint or Labiatae family. It is native to southern Europe and the Mediterranean region, and is often called summer savory. The leaves are narrow, elliptical, dark green, and about 0.5–1.0 cm long. The volatile oil of savory is present at less than 1% of the herb in weight. The major component of the essential oil from summer savory is carvacrol (25–45%). Limonene, c-terpinene, p-cymene and b-cariophylene are also present in appreciable amounts. This paper deals with the extraction of savory oil by means of supercritical carbon dioxide from 10 to 18 MPa and at a temperature of 313 K. The dependence of the extraction rate on the solvent ratio was studied at a pressure of 12 MPa and a temperature of 313 K. Fractional separation of the supercritical extract was performed to minimize the coextraction of unwanted compounds. The composition of the supercritical extracts was compared with those obtained by hydrodistillation methods (steam distillation and the Clevenger method ).
2. Materials and methods Summer savory (S. hortensis, L.) was collected in June in the central region of Portugal and was air-dried. In the extraction experiments, the stalks were removed and the remaining plant was used as received (air-dried and coarsely cut). The particle diameter after size reduction was estimated using sieves (Retsch 5657), and the mean particle diameter was 0.3 mm.
The CO used in this work was 99.5% pure 2 (w/w), and was supplied by Ar Liquido (Portugal ). 2.1. Supercritical extraction apparatus and procedure The extraction experiments were performed with a fixed-bed tubular extractor with a capacity of 100 cm3 and a cross-sectional area of 3.5 cm2, assembled as described elsewhere [11]. Carbon dioxide from a bottle (6 MPa) was passed through a cold bath (about 275 K ) and was then pumped via an air-driven liquid pump Model MCP-71; (Haskel Inc., Burbank, CA, USA). The pressure was controlled with a back pressure regulator (Model 26-1722-024-043, Tescom Inc., Minnesota, USA). The temperature was measured using three thermocouples connected to a high-performance temperature/process indicator (Model DP-41-TC, Omega) with an accuracy of ±0.1 K. The pressure at the exit of the extractor was measured using a differential manometer within an accuracy of ±1 MPa. After leaving the extractor, the CO loaded with 2 extract flowed through the first separation stage, a steel vessel with a capacity of 30 cm3, and the pressure was reduced to about 9 MPa and 288 K. In the second separation stage, a vertical glass (diameter 6 cm, length 20 cm), the pressure was reduced again to about 0.2 MPa, and the temperature was reduced to 268 K. Finally, to avoid the loss of volatile compounds, a glass coil immersed in a dry ice/acetone bath was placed at the exit of the apparatus at about 203 K and at ambient pressure. Samples were taken during each run. For each sample, the amount of extracts deposited in the collectors was measured by weighing. After each sampling, the pipes around the collectors were washed with acetone and then purged using a vacuum pump. The extracts collected were separated from the acetone using a rotary evaporator (Heidolph, VV2000), weighed, and added to the extracts obtained in the previous separation step. The extractor was discharged and cleaned manually at the end of each run. The amount of CO used was measured using a 2 dry test meter (Model DTM-200A, American
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Meter Company, Philadelphia, USA) within an accuracy of 0.005 L. The pressure and temperature conditions were measured at the end of the apparatus within an accuracy of ±0.01 MPa and ±0.1 K, respectively. A second apparatus was used to perform the extraction in order to allow a better study of the fractionation of collection, and to increase the quantity of the extracts. The main differences in the modified supercritical extraction apparatus were that a 500 cm3 extractor (L/D=5.8), two separation vessels (type SFE 500, Separex, Champigneulles, France), operated in series with a volume of 200 cm3 each were used. A glass coil, at ambient pressure and a temperature of 203 K was placed at the end of the apparatus to avoid loss of the more volatile compounds. A Haskel pump assured CO circulation. The optimum 2 extraction conditions which minimized the coextraction of unwanted compounds were investigated in this new apparatus. 2.2. Hydrodistillation procedure The amount of essential oil in savory was also determined by steam distillation. The apparatus used for the extraction of essential oil was a modified Clevenger apparatus [12] and a steam distillation apparatus. Dried plant matter was steam-distilled for 2 h, which was sufficient to complete the extraction. The oil was collected via a side-arm. The amount of oil recovered was measured gravimetrically. 2.2.1. Clevenger procedure Summer savory (100 g) was placed along with 1 l of distilled water in a 2 l flask and connected to a modified Clevenger apparatus. After 2 h, distillation was stopped. 2.2.2. Steam distillation procedure In each run, a flow of steam vapour at atmospheric pressure was passed through a vertical column containing 200 g of summer savory. During distillation, two fractions were obtained at periods of 1 and 2 h. After 2 h, distillation was stopped.
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2.3. GC analysis of volatile compounds The essential oil and supercritical extracts were analysed by gas chromatography (GC ) on a Hewlett-Packard 5890 Series II chromatograph. GC separation was carried out with nitrogen N45 (Ar Lı´quido, Portugal ) as the carrier gas (5 ml min−1) using a flame ionization detector at 523 K. A DB5 column (5% phenyl, 95% methylpolysiloxane, 0.32 mm i.d., 50 m in length, film thickness 0.17 mm) was used to perform the separation. The column temperature was programmed to hold at 333 K for 10 min, then rise to 453 K at 2 K min−1, with a final isothermal hold at 453 K for 30 min. The sample components were identified by comparing the retention times with those of the chromatographic standard of the compounds (Sigma Aldrich Quimica SA, Madrid, Spain). The peak areas were determined using a Hewlett-Packard 3396 Series II integrator. For quantitative analyses, the peak areas were converted to absolute values using response factors estimated from standard compounds. 2.4. Rancimat method The rancimat method (Metrohm Rancimat 679) was used for to detect antioxidant activity. Samples of essential oil (or supercritical extract), dissolved in sunflower oil (7.0 g) at a concentration of 1350 ppm were heated at 393 K. A continuous air stream (20 l h−1) was passed through the heated samples and the volatile compounds were absorbed in a conductivity cell. The conductivities were monitored continuously until a sudden rise signified the end of the induction period. For the determination of the antioxidant activity of the solid extraction residues (husks), a sample was made of 0.5 g of vegetal material mixed with 7.0 g of sunflower oil.
3. Results and discussion The results of the GC analysis of essential oil obtained from summer savory by hydrodistillation are presented in Fig. 1. The composition of the
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Fig. 1. Percentages by weight (% w/w) of summer savory essential oil obtained by steam distillation (AV ) in the first and second hours and by the Clevenger apparatus.
savory essential oil obtained was different from that described by Deighton et al. [13], mainly because Portuguese savory oil does not contain thymol, a-tujone and b-tujone. In spite of this, there was no major qualitative difference because of the country of origin of the savory essential oil. Sample preparation, drying and storage greatly affected the composition and the quality of the volatile oil [14]. The composition of the essential oil did not change when either of the two hydrodistillation methods were used, the major components of savory essential oil being carvacrol and c-terpinene. The maximum essential oil yield was obtained with the Clevenger apparatus, and was 1.5% as a result of the the different contact mode between the plant material and water. No differences were observed in the components over time when steam distillation was used, the total yield being 1.1%. Fig. 2 shows the percentage of solute collected in supercritical extraction with carbon dioxide
from summer savory as a function of pressure in the range 10–18 MPa. The percentage of solute was calculated by dividing the total mass of solute collected at the end of 5 h of extraction by the initial extractable mass present in the solid bed. In the present experiments, the initial extractable mass in the solid sample was obtained using hexane as a solvent and includes, in addition to the essential oils, some natural oils and other high molecular-weight material. For supercritical extraction, the temperature was 313 K and the superficial velocity of carbon dioxide was 0.01 cm s−1. Above 12 MPa, the increase in the carbon dioxide density did not increase the extraction yield, which remained approximately constant despite the increase in pressure. One explanation for this behaviour could be the fact that savory does not have very high molecular-weight compounds, which are easily extractable at higher pressures. The dependence of the extraction rate on the solvent ratio for summer savory oil at a pressure
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Fig. 2. Percentage of solute collected as a function of pressure in the range 10–18 MPa.
of 12 MPa and a temperature of 313 K are presented in Fig. 3. The solvent ratio is a very important parameter for supercritical extraction since it usually influences the economics of the operation.
Fig. 3 shows that the optimum values were achieved for 1 h of extraction and a solvent ratio of about 120 kg CO /h kg solid, which corresponds 2 to a solvent superficial velocity of 0.09–
Fig. 3. Dependence of the extraction rate on the solvent ratio at 12 MPa and 313 K at different extraction times: %, 1 h; n, 2 h; ×, 3 h; 6, 4 h.
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0.1 cm s−1 in our experimental conditions. The extraction rate increased with the solvent ratio until a concentration of 120 kg CO /h kg solid was 2 reached, but for higher solvent ratios the total amount of extract per unit of time declined. Similar results have been obtained for the extraction of caffeine from coffee beans [15]. The protection factor (PF ) was calculated by dividing the induction time of the sample by the induction time of the sunflower oil. When materials do not have antioxidant activity, the induction time of their dispersion is equal to the induction time of the control sample (sunflower oil ), and PF is equal to 1. A PF of greater than 1 indicates antioxidant activity, while a PF of less than 1 indicates pro-oxidant activity. The protection factor of the commercial antioxidant Herbor at a concentration of 400 ppm was used for comparison. Fig. 4 shows the protection factors for the dry plant, the essential oil, the solid distillation residues and the solid extraction residues with CO at 2 12 MPa and 313 K for different extraction times and extracts obtained in the three collectors. The essential oil of summer savory did not show any antioxidant effect. The extracts from the three collectors also did not exhibit a high antioxidant effect, which could be expected, because qualitatively, the chromatograms of the SFE extracts were very similar to those of the steam distillation extract (essential oil ). In general, the GC-FID chromatograms of the SFE and hydrodistillation extracts from savory show the same major components as would be expected from previous reports [16 ]. The residues of supercritical extraction show a protection factor which is close to that of Herbor. The antioxidant activity of these residues increases with extraction time. In fact, in these study conditions, CO only extracts the more volatile com2 pounds and some additional species like plant waxes and odd-numbered n-alkanes [16 ], and in this case the compound concentrations responsible for the antioxidant activity of savory (not identified in the present work) will increase in the extraction residue. These results indicate that supercritical extrac-
tion could be an effective way of concentrating antioxidants from solid materials at low pressures. To optimize the pressure and temperature conditions of the collector system (composed of two cyclones and a glass coil, as discussed above), we used a two-level factorial design ( TLFD) approach [17]. This statistically based method involves the simultaneous adjustment of experimental variables (or factors) at only two values ( levels), their high level designated by + and their low level designated by −. The number of variables studied was four, i.e. P , P , T and T , P and P being the 1 2 1 2 1 2 pressure in the first and in the second cyclones and T and T the temperature in the first and 1 2 second cyclones, respectively. The first dependent variable studied was the total extraction yield (Y ), defined as the ratio of the total mass recovered in 2 h to the initial mass of extractable material in the bed. We chose not to run the full combination of high and low levels but a half fraction, and since we had four factors, we needed 2(4−1)=8 experiments. To select the levels which were expected to produce significant changes in the yield, we based our work on the pressure and temperature conditions indicated in the literature [18]. In general, to collect the waxes in the first cyclone, the carbon dioxide should be liquified and at low temperature. To collect the oxygenated and more volatile compounds, the carbon dioxide should be gaseous in the second collector. Therefore, we chose 6 MPa (−) and 8 MPa (+) for the low and high level of P , 1.5 MPa (−) and 2.5 MPa (+) for P , 263 K 1 2 (−) and 283 K (+) for T , and 268 K (−) and 1 282 K (+) for T . The effect of the factors 2 P , P , T and T were calculated by averaging 1 2 1 2 the responses at the plus level and subtracting the average at the minus level: Effect=Mean Y −Mean Y . (1) + − The data and results obtained for TLFD for the fractionation of the SFE extract are presented in Table 1. The yields obtained for the two cyclones and for the glass coil are also reported. The runs were performed in a random order, and in duplicate. To determine the most significant factor, we chose those which had the largest effect, i.e.
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Fig. 4. Antioxidant protection factors for different products obtained by supercritical extraction and hydrodistillation.
T , T and P , as can be seen in Table 2. P was 2 1 1 2 not a significant factor. According to the results listed in Table 1, to obtain the maximum total
yield, the factors T and P need to be set at their 2 1 high levels, T needs to be set at its low level. The 1 results obtained are only valid under the collecting
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Table 1 Factors, levels and extraction yields (%) obtained for factorial design Run
Factors 1
P 2
T 1
T 2
Total
1st collector (cyclone)
2nd collector (cyclone)
3rd collector (glass coil )
− + − + − + − + + +
− − + + − − + + + +
− − − − + + + + − −
− + + − + − − + a b
4.6 43,4 33.9 8.9 3.8 20.8 11.9 23.3 32.6 28.8
2.5 1.6 6.1 1.4 1.4 0.7 2.0 0.6 2.0 2.0
1.7 17.1 2.2 7.2 1.3 6.2 0.4 21.3 19.2 23.4
0.4 24.7 25.6 0.3 1.1 13.9 9.5 1.4 11.4 2.4
P
1 2 3 4 5 6 7 8 9 10
Yields (%)
a268 K, b269 K. Table 2 Estimates of effects based on total yield for fractionation conditions Effect P (pressure in the 1 P (pressure in the 2 T (temperature in 1 T (temperature in 2 PP 1 2 PT 1 1 PT 1 2 PT 2 1 PT 2 2 TT 1 2
first cyclone) second cyclone) the first cyclone) the second cyclone)
9.1 2.8 −9.2 15.9 −15.9 2.2 5.3 5.3 2.2 −15.9
conditions described, i.e. if a trap is used at the end of the apparatus. Actually, the yields are only increased because the major volatile components are retained in the third collector, which in normal conditions is not used. Chemical characterization of summer savory oil provides some indication of the conditions which should be applied during the supercritical fluid extraction process. Since summer savory oil does not contain high molecular-weight compounds, we assumed that the optimum separation conditions were obtained when the maximum content of carvacrol was detected in the oil collected in the second stage. Based on the results presented in Fig. 5, the oil obtained in the second and third
collectors had a similar composition, containing a high content of carvacrol (about 45%) and c-terpinene (about 30%). Several experiments were carried out to determine the best operating parameters to perform fractionation. Maximizing the yield in the second cyclone was tried by performing a run using the optimized P , T and T conditions, reducing the 1 2 1 second cyclone temperature (runs 9 and 10), obtaining for these set of conditions a lower total yield and a higher yield in the second cyclone. However, the conditions at which a richer extract in carvacrol was obtained in the second cyclone were P =8 MPa and T =273 K for the first sepa1 1 rator and P =1.5 MPa and T =268 K for the 2 2 second separator. This being one of the main objectives of this paper, these conditions were chosen as the optimal conditions. In the first separator cuticular waxes were selectively precipitated, which is normal because alkanes are extracted more rapidly with pure CO than the components of essential oils, which 2 might be expected since plant waxes are found on the tissue surface. Deighton et al. [13] studied the antioxidant properties of the volatile oil fraction of several plants using electron paramagnetic ressonance ( EPR) spectroscopy. They found that carvacrol was the major antioxidant present in the essential oil of summer savory, which has been demonstrated to have fungicidal and bactericidal
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Fig. 5. Percentages by weight (% w/w) of savory extracts obtained by supercritical extraction at 12 MPa and 313 K.
properties. Stable free radicals are formed readily in the essential oils of summer savory by the oneelectron oxidation of the phenol carvacrol. It has been suggested that these free radicals may supplement a-tocopherol in the control of lipid peroxidation in the plant membrane [13]. But, as can be seen in Fig. 4, our essential oil had no antioxidant activity under the conditions used in the Rancimat method, which may be explained by the high temperature used, which could cause the degradation of the essential oil [16 ].
4. Conclusions The influence of pressure and the solvent ratio involved in the supercritical extraction of summer savory oil with carbon dioxide was analysed at a constant temperature of 313 K. The extraction experiments were performed with a fixed-bed tubular extractor of a capacity of 100 cm3. To study multistage separation, we used an extraction apparatus with a fixed-bed tubular extractor (500 cm3
capacity) and three separator vessels (i.e. two cyclones and a glass coil as the third collector). The extraction yield increases with increasing pressure at a constant temperature of 313 K and pressures of up to 12 MPa (corresponding to a solvent density of 662 kg m−3). For pressures higher than 12 MPa, no improvement in yield was obtained. The extraction rate increased with the solvent ratio until a concentration of 120 kg CO /h kg 2 solid was reached. For higher solvent ratios, the total amount of extract per unit of time declined. The yields of all products were measured by direct weighing of the precipitates. The conditions chosen in the separation stages were very important, not only in terms of the separation components, but also because of the poor collection efficiencies, which result in low recoveries and could, mistakenly, be attributed to poor extraction efficiencies. The results indicate that it is possible to isolate the main constituent of savory oil, i.e. carvacrol, by optimizing the separation parameters. The opti-
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mum conditions were P =8 MPa and T =273 K, 1 1 and P =1.5 MPa and T =268 K for the first and 2 2 second separators, respectively, the third collector being set at atmospheric pressure and T=203 K to avoid the loss of volatile compounds in the gaseous stream of CO at the exit of the apparatus. 2 The determination of the antioxidant activity of the extracts and extraction residues indicates that supercritical extraction could be an effective way to concentrate the antioxidants of solid materials at low pressures.
[7]
[8]
[9]
[10]
Acknowledgment [11]
This work was sponsored by JNICT (Junta Nacional de Investigac¸a˜o Cientı´fica e Tecnolo´gica) through project PBIC/C/QUI/2354/95.
[12]
[13]
References [14] [1] D.R. Tainter, A.T. Grenis, Recent spice research, in: D.R. Tainter, A.T Grenis ( Eds.), Spices and Seasonings, VCH, New York, 1993, p. 140. [2] A.B. Valenzuela, S.K. Nieto, Los antioxidants: protectores da la calidade en la industria alimentaria, Aceites e grasas, Sept. 1995, p. 310. [3] E. Stahl, D. Gerard, Solubility behaviour and fractionation of essential oils in dense carbon dioxide, Perf. Flavor. 10 (1985) 29. [4] K. Rajaraman, C.S. Narayanan, A.G. Mathew, Extraction of natural products with liquid and supercritical carbon dioxide, Indian Food Ind. 3 (1984) 48. [5] E. Reverchon, G. Della Porta, Rose concrete fractionation by supercritical CO , J. Supercrit. Fluids 9 (1996) 199. 2 [6 ] E. Reverchon, R. Taddeo, G. Della Porta, Extraction of
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sage oil by supercritical CO : influence of some process 2 parameters, J. Supercrit. Fluids 8 (1995) 302. H. Sovova´, J. Jez, M. Ba´rtlova´, J. St’astova´, Supercritical carbon dioxide extraction of black pepper, J. Supercrit. Fluids 8 (1995) 295. B. Mira, M. Blasco, S. Subirats, Supercritical CO extrac2 tion of essential oils from orange peel, J. Supercrit. Fluids 9 (1996) 238. B.C. Roy, M. Goto, T. Hirose, Extraction of ginger oil with supercritical carbon dioxide: experiments and modeling, Ind. Eng. Chem. Res. 35 (1996) 607. M.M. Esquı´vel, R. Santos, C. Sousa, M.F. Rodrigues, M. Molda˜o-Martins, M.G. Bernardo-Gil, M.L. Beira˜o da Costa, J. Empis, T. Lu, L. Schlieper, M.B. King, J. Bastos, M. Rouzet, Evaluation du CO comprime comme solvant 2 de substitution pour l’extraction des composes aromatiques de plantes, in: P. Pellerin, M. Perrut (Coordinateurs), Proceedings of the 3e`me Colloque sur les Fluides Supercritiques, AIPFS, 1996, p. 155. M.M. Esquı´vel, M.G. Bernardo-Gil, Extraction of olive husk oil with compressed carbon dioxide, J. Supercrit. Fluids 6 (1993) 91. J.D. Miquel, H.M.J. Richard, F.G. Sandret, Volatile constituents of Moroccan thyme oil, J. Agric. Food Chem. 24 (1976) 833. N. Deighton, S.M. Glidewell, S.G. Deans, B.A. Goodman, Identification by EPR spectroscopy of carvacrol and thymol as the major sources of free radicals in the oxidation of plant essential oils, J. Sci. Food Agric. 45 (1993) 1. D.R. Tainter, A.T. Grenis, Quality issues dealing with spices, in: D.R. Tainter and A.T Grenis ( Eds.), Spices and Seasonings, VCH, New York, 1993, p. 24. G. Brunner, Gas Extraction, Steinkopff, Darmstadt, 1994, p. 196. S.B. Hawthorne, M.L. Riekkola, K. Serenius, Y. Holm, R. Hiltunen, K. Hartonen, Comparison of hydrodistillation and supercritical fluid extraction for the determination of essential oils in aromatic plants, J. Chromatogr. 634 (1993) 297. M.J. Anderson, P.J. Whitcomb, Optimize your processoptimization efforts, Chem. Eng. Prog. December (1996) 51. E. Reverchon, Supercritical fluid extraction and fractionation of essential oils and related products, J. Supercrit. Fluids 10 (1997) 1.
Supercritical extraction of savory oil: study of antioxidant activity and extract characterization M.M. Esquı´vel *, M.A. Ribeiro, M.G. Bernardo-Gil Centro de Engenharia Biolo´gica e Quı´mica, Departamento de Engenharia Quı´mica, Instituto Superior Te´cnico, Av. Rovisco Pais, 1096 Codex, Lisbon, Portugal Received 6 October 1997; received in revised form 12 May 1998; accepted 2 June 1998
Abstract The experimental results of supercritical CO extraction on summer savory (Satureja hortensis L.) oil at pressures 2 ranging from 12 to 18 MPa and at a temperature of 313 K are presented. The optimum conditions achieved to obtain the maximum extraction yield were 12 MPa, 120 kg CO /h kg solid and 1 h of extraction. The extract was fractionated 2 in three separators, operated in series. The optimum fractionation conditions which minimize the coextraction of unwanted compounds were investigated using a two-level factorial design approach. The extracts obtained were compared with summer savory essential oil isolated by steam distillation and by a modified Clevenger apparatus. A study of the antioxidant activity of summer savory extracts was performed using the Rancimat method. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Antioxidant; Essential oil; Fractionation; Savory; Supercritical extraction
1. Introduction Herbs have a long history of culinary and medicinal use stretching back to biblical times and beyond. Not only are many of the flavours and aromas distinctively pleasant, but they can be used to conceal off-flavours and odours. There is now evidence that their addition, for example to meat, may help to prevent the formation of undesirable oxidation products. In the last 15–20 years, spice research has concentrated on two primary areas: the antioxidant and the microbial activity of spices and their essential oils [1]. * Corresponding author. Tel: +35 11 8417312; Fax: +35 11 8499242; e-mail: [email protected]
The high susceptibility to oxidation of the fat and oil polyunsaturated fatty acids used in human foods or animal feed requires the application of antioxidants. The antioxidants can be either synthetic or natural products, which scavenge oxygen free radicals, thereby inhibiting or delaying oxidation. The search for natural antioxidants has been difficult, not only because of the eventual toxicity of some synthetic antioxidants, especially butylated hydroxyanisole (BHA) which has a component which can eventually cause cancer, but also because most of them are heat-sensitive and volatile in steam [2]. Therefore, the possibility of using natural antioxidants obtained from vegetable and/or micro-organisms is currently being investigated. However, the industrial utilization of natu-
0896-8446/99/$ – see front matter © 1999 Elsevier Science B.V. All rights reserved. PII S 08 9 6 -8 4 4 6 ( 9 8 ) 0 0 1 15 - 6
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ral antioxidants is limited, mainly because of the low efficiency/cost ratio observed for the extraction of known natural antioxidants [2]. Some sources of natural antioxidant compounds include spices, herbs and by products from the vegetable oil refining industry. The extraction of flavouring materials with supercritical carbon dioxide has been carried out previously [3,4]. Recently, studies of supercritical carbon dioxide extraction have been reported on rose concrete [5], sage oil [6 ], black pepper [7], orange peel [8], ginger oil [9], and oregano, thyme and rosemary [10]. Savory (S. hortensis L.) is an annual herb of the mint or Labiatae family. It is native to southern Europe and the Mediterranean region, and is often called summer savory. The leaves are narrow, elliptical, dark green, and about 0.5–1.0 cm long. The volatile oil of savory is present at less than 1% of the herb in weight. The major component of the essential oil from summer savory is carvacrol (25–45%). Limonene, c-terpinene, p-cymene and b-cariophylene are also present in appreciable amounts. This paper deals with the extraction of savory oil by means of supercritical carbon dioxide from 10 to 18 MPa and at a temperature of 313 K. The dependence of the extraction rate on the solvent ratio was studied at a pressure of 12 MPa and a temperature of 313 K. Fractional separation of the supercritical extract was performed to minimize the coextraction of unwanted compounds. The composition of the supercritical extracts was compared with those obtained by hydrodistillation methods (steam distillation and the Clevenger method ).
2. Materials and methods Summer savory (S. hortensis, L.) was collected in June in the central region of Portugal and was air-dried. In the extraction experiments, the stalks were removed and the remaining plant was used as received (air-dried and coarsely cut). The particle diameter after size reduction was estimated using sieves (Retsch 5657), and the mean particle diameter was 0.3 mm.
The CO used in this work was 99.5% pure 2 (w/w), and was supplied by Ar Liquido (Portugal ). 2.1. Supercritical extraction apparatus and procedure The extraction experiments were performed with a fixed-bed tubular extractor with a capacity of 100 cm3 and a cross-sectional area of 3.5 cm2, assembled as described elsewhere [11]. Carbon dioxide from a bottle (6 MPa) was passed through a cold bath (about 275 K ) and was then pumped via an air-driven liquid pump Model MCP-71; (Haskel Inc., Burbank, CA, USA). The pressure was controlled with a back pressure regulator (Model 26-1722-024-043, Tescom Inc., Minnesota, USA). The temperature was measured using three thermocouples connected to a high-performance temperature/process indicator (Model DP-41-TC, Omega) with an accuracy of ±0.1 K. The pressure at the exit of the extractor was measured using a differential manometer within an accuracy of ±1 MPa. After leaving the extractor, the CO loaded with 2 extract flowed through the first separation stage, a steel vessel with a capacity of 30 cm3, and the pressure was reduced to about 9 MPa and 288 K. In the second separation stage, a vertical glass (diameter 6 cm, length 20 cm), the pressure was reduced again to about 0.2 MPa, and the temperature was reduced to 268 K. Finally, to avoid the loss of volatile compounds, a glass coil immersed in a dry ice/acetone bath was placed at the exit of the apparatus at about 203 K and at ambient pressure. Samples were taken during each run. For each sample, the amount of extracts deposited in the collectors was measured by weighing. After each sampling, the pipes around the collectors were washed with acetone and then purged using a vacuum pump. The extracts collected were separated from the acetone using a rotary evaporator (Heidolph, VV2000), weighed, and added to the extracts obtained in the previous separation step. The extractor was discharged and cleaned manually at the end of each run. The amount of CO used was measured using a 2 dry test meter (Model DTM-200A, American
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Meter Company, Philadelphia, USA) within an accuracy of 0.005 L. The pressure and temperature conditions were measured at the end of the apparatus within an accuracy of ±0.01 MPa and ±0.1 K, respectively. A second apparatus was used to perform the extraction in order to allow a better study of the fractionation of collection, and to increase the quantity of the extracts. The main differences in the modified supercritical extraction apparatus were that a 500 cm3 extractor (L/D=5.8), two separation vessels (type SFE 500, Separex, Champigneulles, France), operated in series with a volume of 200 cm3 each were used. A glass coil, at ambient pressure and a temperature of 203 K was placed at the end of the apparatus to avoid loss of the more volatile compounds. A Haskel pump assured CO circulation. The optimum 2 extraction conditions which minimized the coextraction of unwanted compounds were investigated in this new apparatus. 2.2. Hydrodistillation procedure The amount of essential oil in savory was also determined by steam distillation. The apparatus used for the extraction of essential oil was a modified Clevenger apparatus [12] and a steam distillation apparatus. Dried plant matter was steam-distilled for 2 h, which was sufficient to complete the extraction. The oil was collected via a side-arm. The amount of oil recovered was measured gravimetrically. 2.2.1. Clevenger procedure Summer savory (100 g) was placed along with 1 l of distilled water in a 2 l flask and connected to a modified Clevenger apparatus. After 2 h, distillation was stopped. 2.2.2. Steam distillation procedure In each run, a flow of steam vapour at atmospheric pressure was passed through a vertical column containing 200 g of summer savory. During distillation, two fractions were obtained at periods of 1 and 2 h. After 2 h, distillation was stopped.
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2.3. GC analysis of volatile compounds The essential oil and supercritical extracts were analysed by gas chromatography (GC ) on a Hewlett-Packard 5890 Series II chromatograph. GC separation was carried out with nitrogen N45 (Ar Lı´quido, Portugal ) as the carrier gas (5 ml min−1) using a flame ionization detector at 523 K. A DB5 column (5% phenyl, 95% methylpolysiloxane, 0.32 mm i.d., 50 m in length, film thickness 0.17 mm) was used to perform the separation. The column temperature was programmed to hold at 333 K for 10 min, then rise to 453 K at 2 K min−1, with a final isothermal hold at 453 K for 30 min. The sample components were identified by comparing the retention times with those of the chromatographic standard of the compounds (Sigma Aldrich Quimica SA, Madrid, Spain). The peak areas were determined using a Hewlett-Packard 3396 Series II integrator. For quantitative analyses, the peak areas were converted to absolute values using response factors estimated from standard compounds. 2.4. Rancimat method The rancimat method (Metrohm Rancimat 679) was used for to detect antioxidant activity. Samples of essential oil (or supercritical extract), dissolved in sunflower oil (7.0 g) at a concentration of 1350 ppm were heated at 393 K. A continuous air stream (20 l h−1) was passed through the heated samples and the volatile compounds were absorbed in a conductivity cell. The conductivities were monitored continuously until a sudden rise signified the end of the induction period. For the determination of the antioxidant activity of the solid extraction residues (husks), a sample was made of 0.5 g of vegetal material mixed with 7.0 g of sunflower oil.
3. Results and discussion The results of the GC analysis of essential oil obtained from summer savory by hydrodistillation are presented in Fig. 1. The composition of the
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Fig. 1. Percentages by weight (% w/w) of summer savory essential oil obtained by steam distillation (AV ) in the first and second hours and by the Clevenger apparatus.
savory essential oil obtained was different from that described by Deighton et al. [13], mainly because Portuguese savory oil does not contain thymol, a-tujone and b-tujone. In spite of this, there was no major qualitative difference because of the country of origin of the savory essential oil. Sample preparation, drying and storage greatly affected the composition and the quality of the volatile oil [14]. The composition of the essential oil did not change when either of the two hydrodistillation methods were used, the major components of savory essential oil being carvacrol and c-terpinene. The maximum essential oil yield was obtained with the Clevenger apparatus, and was 1.5% as a result of the the different contact mode between the plant material and water. No differences were observed in the components over time when steam distillation was used, the total yield being 1.1%. Fig. 2 shows the percentage of solute collected in supercritical extraction with carbon dioxide
from summer savory as a function of pressure in the range 10–18 MPa. The percentage of solute was calculated by dividing the total mass of solute collected at the end of 5 h of extraction by the initial extractable mass present in the solid bed. In the present experiments, the initial extractable mass in the solid sample was obtained using hexane as a solvent and includes, in addition to the essential oils, some natural oils and other high molecular-weight material. For supercritical extraction, the temperature was 313 K and the superficial velocity of carbon dioxide was 0.01 cm s−1. Above 12 MPa, the increase in the carbon dioxide density did not increase the extraction yield, which remained approximately constant despite the increase in pressure. One explanation for this behaviour could be the fact that savory does not have very high molecular-weight compounds, which are easily extractable at higher pressures. The dependence of the extraction rate on the solvent ratio for summer savory oil at a pressure
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Fig. 2. Percentage of solute collected as a function of pressure in the range 10–18 MPa.
of 12 MPa and a temperature of 313 K are presented in Fig. 3. The solvent ratio is a very important parameter for supercritical extraction since it usually influences the economics of the operation.
Fig. 3 shows that the optimum values were achieved for 1 h of extraction and a solvent ratio of about 120 kg CO /h kg solid, which corresponds 2 to a solvent superficial velocity of 0.09–
Fig. 3. Dependence of the extraction rate on the solvent ratio at 12 MPa and 313 K at different extraction times: %, 1 h; n, 2 h; ×, 3 h; 6, 4 h.
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0.1 cm s−1 in our experimental conditions. The extraction rate increased with the solvent ratio until a concentration of 120 kg CO /h kg solid was 2 reached, but for higher solvent ratios the total amount of extract per unit of time declined. Similar results have been obtained for the extraction of caffeine from coffee beans [15]. The protection factor (PF ) was calculated by dividing the induction time of the sample by the induction time of the sunflower oil. When materials do not have antioxidant activity, the induction time of their dispersion is equal to the induction time of the control sample (sunflower oil ), and PF is equal to 1. A PF of greater than 1 indicates antioxidant activity, while a PF of less than 1 indicates pro-oxidant activity. The protection factor of the commercial antioxidant Herbor at a concentration of 400 ppm was used for comparison. Fig. 4 shows the protection factors for the dry plant, the essential oil, the solid distillation residues and the solid extraction residues with CO at 2 12 MPa and 313 K for different extraction times and extracts obtained in the three collectors. The essential oil of summer savory did not show any antioxidant effect. The extracts from the three collectors also did not exhibit a high antioxidant effect, which could be expected, because qualitatively, the chromatograms of the SFE extracts were very similar to those of the steam distillation extract (essential oil ). In general, the GC-FID chromatograms of the SFE and hydrodistillation extracts from savory show the same major components as would be expected from previous reports [16 ]. The residues of supercritical extraction show a protection factor which is close to that of Herbor. The antioxidant activity of these residues increases with extraction time. In fact, in these study conditions, CO only extracts the more volatile com2 pounds and some additional species like plant waxes and odd-numbered n-alkanes [16 ], and in this case the compound concentrations responsible for the antioxidant activity of savory (not identified in the present work) will increase in the extraction residue. These results indicate that supercritical extrac-
tion could be an effective way of concentrating antioxidants from solid materials at low pressures. To optimize the pressure and temperature conditions of the collector system (composed of two cyclones and a glass coil, as discussed above), we used a two-level factorial design ( TLFD) approach [17]. This statistically based method involves the simultaneous adjustment of experimental variables (or factors) at only two values ( levels), their high level designated by + and their low level designated by −. The number of variables studied was four, i.e. P , P , T and T , P and P being the 1 2 1 2 1 2 pressure in the first and in the second cyclones and T and T the temperature in the first and 1 2 second cyclones, respectively. The first dependent variable studied was the total extraction yield (Y ), defined as the ratio of the total mass recovered in 2 h to the initial mass of extractable material in the bed. We chose not to run the full combination of high and low levels but a half fraction, and since we had four factors, we needed 2(4−1)=8 experiments. To select the levels which were expected to produce significant changes in the yield, we based our work on the pressure and temperature conditions indicated in the literature [18]. In general, to collect the waxes in the first cyclone, the carbon dioxide should be liquified and at low temperature. To collect the oxygenated and more volatile compounds, the carbon dioxide should be gaseous in the second collector. Therefore, we chose 6 MPa (−) and 8 MPa (+) for the low and high level of P , 1.5 MPa (−) and 2.5 MPa (+) for P , 263 K 1 2 (−) and 283 K (+) for T , and 268 K (−) and 1 282 K (+) for T . The effect of the factors 2 P , P , T and T were calculated by averaging 1 2 1 2 the responses at the plus level and subtracting the average at the minus level: Effect=Mean Y −Mean Y . (1) + − The data and results obtained for TLFD for the fractionation of the SFE extract are presented in Table 1. The yields obtained for the two cyclones and for the glass coil are also reported. The runs were performed in a random order, and in duplicate. To determine the most significant factor, we chose those which had the largest effect, i.e.
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Fig. 4. Antioxidant protection factors for different products obtained by supercritical extraction and hydrodistillation.
T , T and P , as can be seen in Table 2. P was 2 1 1 2 not a significant factor. According to the results listed in Table 1, to obtain the maximum total
yield, the factors T and P need to be set at their 2 1 high levels, T needs to be set at its low level. The 1 results obtained are only valid under the collecting
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Table 1 Factors, levels and extraction yields (%) obtained for factorial design Run
Factors 1
P 2
T 1
T 2
Total
1st collector (cyclone)
2nd collector (cyclone)
3rd collector (glass coil )
− + − + − + − + + +
− − + + − − + + + +
− − − − + + + + − −
− + + − + − − + a b
4.6 43,4 33.9 8.9 3.8 20.8 11.9 23.3 32.6 28.8
2.5 1.6 6.1 1.4 1.4 0.7 2.0 0.6 2.0 2.0
1.7 17.1 2.2 7.2 1.3 6.2 0.4 21.3 19.2 23.4
0.4 24.7 25.6 0.3 1.1 13.9 9.5 1.4 11.4 2.4
P
1 2 3 4 5 6 7 8 9 10
Yields (%)
a268 K, b269 K. Table 2 Estimates of effects based on total yield for fractionation conditions Effect P (pressure in the 1 P (pressure in the 2 T (temperature in 1 T (temperature in 2 PP 1 2 PT 1 1 PT 1 2 PT 2 1 PT 2 2 TT 1 2
first cyclone) second cyclone) the first cyclone) the second cyclone)
9.1 2.8 −9.2 15.9 −15.9 2.2 5.3 5.3 2.2 −15.9
conditions described, i.e. if a trap is used at the end of the apparatus. Actually, the yields are only increased because the major volatile components are retained in the third collector, which in normal conditions is not used. Chemical characterization of summer savory oil provides some indication of the conditions which should be applied during the supercritical fluid extraction process. Since summer savory oil does not contain high molecular-weight compounds, we assumed that the optimum separation conditions were obtained when the maximum content of carvacrol was detected in the oil collected in the second stage. Based on the results presented in Fig. 5, the oil obtained in the second and third
collectors had a similar composition, containing a high content of carvacrol (about 45%) and c-terpinene (about 30%). Several experiments were carried out to determine the best operating parameters to perform fractionation. Maximizing the yield in the second cyclone was tried by performing a run using the optimized P , T and T conditions, reducing the 1 2 1 second cyclone temperature (runs 9 and 10), obtaining for these set of conditions a lower total yield and a higher yield in the second cyclone. However, the conditions at which a richer extract in carvacrol was obtained in the second cyclone were P =8 MPa and T =273 K for the first sepa1 1 rator and P =1.5 MPa and T =268 K for the 2 2 second separator. This being one of the main objectives of this paper, these conditions were chosen as the optimal conditions. In the first separator cuticular waxes were selectively precipitated, which is normal because alkanes are extracted more rapidly with pure CO than the components of essential oils, which 2 might be expected since plant waxes are found on the tissue surface. Deighton et al. [13] studied the antioxidant properties of the volatile oil fraction of several plants using electron paramagnetic ressonance ( EPR) spectroscopy. They found that carvacrol was the major antioxidant present in the essential oil of summer savory, which has been demonstrated to have fungicidal and bactericidal
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Fig. 5. Percentages by weight (% w/w) of savory extracts obtained by supercritical extraction at 12 MPa and 313 K.
properties. Stable free radicals are formed readily in the essential oils of summer savory by the oneelectron oxidation of the phenol carvacrol. It has been suggested that these free radicals may supplement a-tocopherol in the control of lipid peroxidation in the plant membrane [13]. But, as can be seen in Fig. 4, our essential oil had no antioxidant activity under the conditions used in the Rancimat method, which may be explained by the high temperature used, which could cause the degradation of the essential oil [16 ].
4. Conclusions The influence of pressure and the solvent ratio involved in the supercritical extraction of summer savory oil with carbon dioxide was analysed at a constant temperature of 313 K. The extraction experiments were performed with a fixed-bed tubular extractor of a capacity of 100 cm3. To study multistage separation, we used an extraction apparatus with a fixed-bed tubular extractor (500 cm3
capacity) and three separator vessels (i.e. two cyclones and a glass coil as the third collector). The extraction yield increases with increasing pressure at a constant temperature of 313 K and pressures of up to 12 MPa (corresponding to a solvent density of 662 kg m−3). For pressures higher than 12 MPa, no improvement in yield was obtained. The extraction rate increased with the solvent ratio until a concentration of 120 kg CO /h kg 2 solid was reached. For higher solvent ratios, the total amount of extract per unit of time declined. The yields of all products were measured by direct weighing of the precipitates. The conditions chosen in the separation stages were very important, not only in terms of the separation components, but also because of the poor collection efficiencies, which result in low recoveries and could, mistakenly, be attributed to poor extraction efficiencies. The results indicate that it is possible to isolate the main constituent of savory oil, i.e. carvacrol, by optimizing the separation parameters. The opti-
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mum conditions were P =8 MPa and T =273 K, 1 1 and P =1.5 MPa and T =268 K for the first and 2 2 second separators, respectively, the third collector being set at atmospheric pressure and T=203 K to avoid the loss of volatile compounds in the gaseous stream of CO at the exit of the apparatus. 2 The determination of the antioxidant activity of the extracts and extraction residues indicates that supercritical extraction could be an effective way to concentrate the antioxidants of solid materials at low pressures.
[7]
[8]
[9]
[10]
Acknowledgment [11]
This work was sponsored by JNICT (Junta Nacional de Investigac¸a˜o Cientı´fica e Tecnolo´gica) through project PBIC/C/QUI/2354/95.
[12]
[13]
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