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Effect of fast CO2 pressure changes on the yield of lovage (Levisticum officinale Koch.) and celery (Apium graveolens L.) extracts
Journal of Supercritical Fluids 22 (2002) 201– 210 www.elsevier.com/locate/supflu
Effect of fast CO2 pressure changes on the yield of lovage (Le6isticum officinale Koch.) and celery (Apium gra6eolens L.) extracts Egidijus Dauksˇas a, Petras Rimantas Venskutonis a,*, Bjo¨rn Sivik b, Tobias Nillson c a
Department of Food Technology, Kaunas Uni6ersity of Technology, Rad6ile; nu¸ pl. 19, Kaunas 3028, Lithuania b Food Technology, Chemical Center, Uni6ersity of Lund, PO Box 124, S-221 00 Lund, Sweden c Analytical Chemistry, Chemical Center, Uni6ersity of Lund, PO Box 124, S-221 00 Lund, Sweden Received 4 September 2000; received in revised form 22 May 2001; accepted 17 August 2001
Abstract The effect of pressure alterations on the yield of CO2 extracts from different anatomical parts of lovage (Le6isticum officinale Koch.) and celery (Apium gra6eolens L.) was studied. It was found that by applying frequent pressure changes in the extraction vessel it is possible to increase the rate of the isolation of CO2 soluble materials from lovage seeds and leaves, lovage and celery roots. However, after passing a sufficient amount of the supercritical solvent, the yields were similar both for constant and pulsing extraction pressures. The composition of the extracts was analyzed by gas chromatography and mass spectrometry and it was found that the phthalides were very important constituents in the extracts from all the anatomical parts of lovage, while linoleic acid was the most abundant component in the celery root extracts. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Celery; Lovage; Pressure changes; Supercritical CO2
1. Introduction Lovage and celery are important plants in some countries, where they have been used to flavor various foods and also for medicinal purposes. Investigations on lovage flavor and isolation of
* Corresponding author. Tel.: + 370-7-356-426; fax: +3707-456-647. E-mail address: [email protected] (P. Rimantas Venskutonis).
volatile aroma compounds have been reviewed in our previous publications concerning supercritical CO2 extraction [1] and distillation of different anatomical parts of the plant [2,3]. Celery (Apium gra6eolens L.) is native in Eurasia and common with other members of the Umbelliferae family. The commercial essential oil is usually described as possessing an appetizing flavor and it exhibits a strong aroma caused by several phthalides [4–14]. The yield of oil isolated by hydrodistillation or liquid CO2 from seeds has been found to be up to 3% [4,15].
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The process of the isolation of essential oils from various botanical sources depends on the morphological characteristics of the plant material. It is known that the oil ducts or canals in Umbelliferae plants are mainly schizogenous: developing close to vascular strands in the mesophyll of leaves and other organs by the separation of cells rather than by the death and dissolution of cells [16]. The walls of the canals are covered by a soft layer of cells producing the balsam, which is a solution of resin in the essential oil. Such receptacles can be clearly seen on a crosssection of Umbelliferae plant’s stem, root or seeds [17 – 19]. The schizogenous ducts are internal, inconspicuous and much more difficult to study [19]. In other literature sources [20], the secretory system of Umbelliferae family root anatomy is described as possessing two types of schizogenous oil ducts containing essential oils. The primary oil ducts are triangular and located between the pericycle cells of the seedling (they usually form two arcs), while the other two oil ducts lying in the middle are rhomboid. The theoretical basis of the extraction of botanical materials has been extensively described [21,22]. Some studies have shown that the transportation of liquid solutes in a capillary matrix can be enhanced by exposure to very fast pressure alterations [23]. The principles of such an extraction concept have been explained by Kasjanov et al. [24]. The frequency and period of the required pressure fluctuations depend on the properties of capillary matrix (density, porosity, etc.), any bonding between the solute materials and the plant matrix, extent of solvent heating and the intensity of pressure reduction [23]. The carbon dioxide pressure alteration method using subcritical conditions has been used for the extraction of dill seeds [25]. The extract yield obtained by decreasing the pressure of CO2 from 58.46 to 45.95 bar at a rate of 5 bar min − 1 and by periodic replacement of the isolated material, was 12.68% (w/w). This was approximately two times higher than the yield obtained by conventional CO2 extraction at 58.46 bar and 20 °C [25]. This study is an attempt to apply fast pressure fluctuation method for the extraction of lovage
and celery materials, thereby facilitating an increase in the extraction yield, and hence a reduction in the extraction time.
2. Materials and methods
2.1. Materials The lovage (Le6isticum officinale Koch.) and celery (Apium gra6eolens L.) were collected from the experimental garden of the Lithuanian Institute of Horticulture in 1996. The roots, leaves and seeds were harvested manually. Raw material was dried at 30 °C in a ventilated drying oven ‘Vasara’ (Utena, Lithuania) and stored in paper bags at ambient temperature to protect against direct light until further analysis. The samples were ground prior to extraction using a Knifetec 1095 Sample Mill (Tecator AB, Ho¨ gana¨ s, Sweden) for 20 s. Carbon dioxide, 99.99% purity (Aga Gas, Sweden) and hexane (\ 99%) from Merck (Darmstad, Germany) was used in the experiments.
2.2. Apparatus and extraction methodology A schematic diagram of experimental apparatus used in this study is shown in Fig. 1. A Milroyal B-C pump (Dosapro Milton Roy, Pont-SaintPierre, France) was used for extractions at 40 °C, and at pressures up to 350 bar. The temperature in the extractors and separators was maintained
Fig. 1. Schematic drawing of the supercritical extraction equipment. (1) Gas tube; (2) Shut-off valve; (3) Gas filter; (4) Ethanol bath; − 22 °C; (5) Pump; (6) Safety valve; (7) Pressure gauge; (8) Shut-off valve; (9) Extractor; (10) Water bath; (11) Micro metering valve; (12) Separators; (13) Sampling valves; (14) Pressure maintaining valve; (15) Flow meter; (16) Extra pump.
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Table 1 Experimental data No.
Extracted material
Amount of extracted material, g
Extraction vessel size, ml
Amount of CO2, kg
1 2 3 4 5 6
Roots of lovage Roots of lovage Seeds of lovage Leaves of lovage Leaves of lovage Roots of celery
10 100 100 10 50 50
47 200 200 47 200 200
2 10 10 2 7 7
by the thermostatic water jackets. Two different capacity extraction vessels were used: 47 ml for 10 g samples and 200 ml for 50 and/or 100 g of the ground plant material (Table 1). Glass wool was placed on the top and the bottom of the extraction vessels to prevent system contamination by any particles of the raw material, as well as to eliminate free space in the vessel. The CO2 flow rate was kept approximately at 0.025 kg min − 1 (measured by a mass flowmeter). The extract was collected in the first separator (12, Fig. 1). The second separator, which was connected to the first one through a valve, was used in the extraction of leaves, to separate the chlorophyll from the extract. The volume of both separators was 200 ml. The temperature in the first and second separator was maintained at 40 and 35 °C, respectively. The pressure in first separator was 50 bar during the extraction of roots and seeds and 100 bar during extraction of leaves; the pressure in the second separator in both cases was 45– 50 bar. The pump S-216-J with 414 kPa Driving Air Supply (Teledyne Sprague Engineering, CA) was used as an additional pump to create pressure fluctuations. When the piston of the second pump is elevated the pressure made by the main pump immediately drops. The frequency of the piston motion and consequently pressure fluctuations in the extraction system can be increased to 60 cycles min − 1 by changing the air pressure which was supplied from the outer line. All parts of the extraction system were made of stainless steel. Precision analytical balances (Mettler AE 163, 0.01 mg resolution, Mettler Instrument AG, Switzerland) were used for weighing the amounts of extract obtained. Two replicates were analyzed for every sample and the mean value calculated.
2.3. Gas chromatography and mass spectrometry (GS-MS) The extracts were diluted with cyclohexane and examined by GC-MS, using a Hewlett-Packard 5890 series II gas chromatograph equipped with Hewlett-Packard 5972 mass detector with electron impact ion source (Hewlett-Packard, Rockville, MP). The system was run from a computer with the Hewlett-Packard MS Chemstation software, version B.02.05. The column used was a HP-5 M.S. (Crosslinked 5% pH Me Silicone, 30 m × 0.25 mm, 0.25 mm film thickness). Helium was used as carrier gas at a constant flow of 0.84 ml min − 1. In all runs, 2 ml of sample were injected in split mode with a split ratio of 1:14 at 280 °C. The oven temperature was programmed from 35 °C (1 min hold) to 270 °C (10 min hold) at 4 °C min − 1. The MS interface was heated at 320 °C. The compounds were identified by their mass spectra (computerized comparison with reference spectra in the MS Chemstation library) and retention time in the capillary column. Two replicate GC analyses were performed and the results were expressed in GC area % as a mean value.
3. Results and discussion
3.1. Effect of extraction conditions on the extract yield from lo6age root The first series of experiments were carried out with lovage root (Table 1, no.1) to determine the effect of extraction conditions on the extract yield. In our previous study [1] it was found that 250
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bar and 40 °C were preferable parameters for obtaining a high extraction yield from lovage roots. Therefore, the same conditions (Table 2, Experiment c 1) were used to extract lovage roots without using the extra pump (16, Fig. 1). The total yield of the extract was 1.589 0.03 g from 100 g of raw material (Table 2). This amount was used as a target figure for comparison purposes with results obtained during subsequent extractions where pressure fluctuations were applied. Extraction pressure was changed by using an extra pump (16, Fig. 1); the results obtained are presented in Table 2. It was found that pressure changes and their frequency effect the extract yield. By fluctuating the pressure in the range from 200 to 140 bar at a frequency of 10 cycles/ min the yield was increased up to 1.73 g per 100 g of raw material (i. e. approximately a 10% increase compared to the yield obtained at constant pressure of 250 bar). However, when the maximal pressure was increased up to 250 bar (in this case the minimal pressure was 180 bar) and altered at the same frequency (10 min − 1), the yield decreased to 1.56 g/100 g, similar to the yield obtained at the constant pressure of 250 bar. A further increase in pressure to 350 bar was not efficient; the extract yield was 1.7090.04 g/100 g, i.e. similar to the yield obtained during Experiment c 2 (Table 2). Finally, the frequency of pressure (maximal=250; minimal= 220) change was increased to 60 min − 1, the highest extract yield (2.18 g 100 g − 1) was obtained. The parameters applied allowed to increase the yield by 30% compared to that obtained at constant pressure of 250 bar. The results obtained suggest that by using an
extra pump and fluctuating the pressure, the frequency of pressure change has a greater effect on the yield than the effect of maximal and minimal pressure magnitude. This phenomenon can be explained by the theory [23] suggesting that by applying intensive and very fast pressure increase the mass transport is performed by the molar filtration, which creates the formation of an intensive flow of the substances resulting in an increase of the total yield. The second series of the experiments were performed with lovage roots by using a larger extractor (Table 1, no. 2). In this case two identical separators (50 bar pressure; 40 and 35 °C temperature, respectively) were used. In the beginning of the extraction (until 3 kg of CO2 was passed) extract yields were measured after passing each 0.5 kg; afterwards the yields were measured at the intervals of 1 kg of the used CO2. The yield obtained at 250 bar and 40 °C without the extra pump was used as a control. Another two extractions were performed at the same optimal pressure and temperature parameters using the extra pump (16, Fig. 1), which created pressure fluctuations from 250 to 180 bar at a frequency of 10 min − 1, and from 250 to 220 bar at a frequency of 60 min − 1. These results are presented in Fig. 2. In general, at the end of the extractions (10 kg CO2 used) there were no considerable differences between the yields obtained at all three sets of conditions applied. However, when the pressure was changed to a frequency of 60 min − 1, the CO2-soluble substances were extracted more extensively at the beginning of the extraction procedure than at the end. The effect of pressure fluctuation at a frequency of 10 min − 1 was negligible during all extraction cycles.
Table 2 The yield of the extract from lovage root at different extraction pressure parameters, n = 2 (47 ml extraction vessel) Experiment No.
Maximal pressure, bar
Minimal pressure, bar
Frequency of pressure change, min−1
Extract yield, g 100 g−1
1 2 3 4 5
250 200 250 350 250
250 140 180 290 220
– 10 91 10 91 10 91 60 9 5
1.58 90.03 1.73 9 0.03 1.56 90.04 1.70 9 0.04 2.23 90.05
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Fig. 2. The kinetics of the extraction of lovage root with CO2: ", constant pressure; , pressure fluctuation in the range of 250 l 180 bar at a frequency of 10 min − 1; , pressure fluctuation in the range of 250 l220 bar at a frequency of 60 min − 1.
3.2. Extraction of lo6age seeds The experiments were continued with lovage seeds (Table 1, no. 3) using the same extraction parameters used for roots. In this case the frequency of a pressure change was 60 min − 1. The extraction kinetic curves for lovage seed are presented in Fig. 3. These curves show that the process of the isolation of soluble substances from the seeds was quite similar for both extraction methods. In general, the extraction kinetics from the seeds was quite different when compared to roots. Approximately 50% of the extract obtained from seeds was isolated after initially passing 1 kg of CO2, while the remaining soluble substances were extracted at a considerably lower rate. In the case of the roots (Fig. 2) 50% of the extract was obtained after passing 2– 3 kg of CO2. Finally, the total extract yield from the seeds ( 6 g/100 g) was almost 3 times higher than that found from the roots ( 2 g/100 g). These differences in extraction rate and yield can be explained by the different structural properties and chemical composition of the seeds and roots. For instance, seeds contain significant amount of lipids,
whereas roots consist mainly of a polysaccharide matrix.
3.3. Extraction of lo6age lea6es The third set of experiments on the extraction of lovage was conducted using plant leaves. The aerial parts of the plant contain high amounts of chlorophyll. It was reported that the content of chlorophyll in the extract increases by increasing extraction pressure [26]. Hence, several initial extractions were performed to determine the optimal parameters for the separation of chlorophyll from the other extract components. These experiments were started without using the second pump (Table 1, no. 4), and by maintaining the pressure in the first separator at 175, 150 or 100 bar and a temperature of 40 °C, to prevent chlorophyll contamination of the extract collected in the second separator. The conditions in the second separator were 50 bar and 35 °C. The highest yield of the extract with the lowest chlorophyll content (relatively light yellow color based on the visual assessment of the extracts), in the second separator (1.09 g 100 g − 1) was obtained
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by separation at 100 bar. The yield of the green colored extract in the first separator was 0.29 g/100 g. After determining the optimal pressure for the first separator, next experiment (Table 1, no. 5) was continued alternating the fluctuation of pressure over the range of 250− 220 bar at the frequency of 60 min − 1. The yield of the extract was 0.17 g/100 g and 1.82 g/100 g in the first and second separators, respectively. Consequently the yield of the chlorophyll-reduced extract increased 1.8 times when compared to that obtained at constant pressure. This result is also in a good agreement with data obtained during the extraction of lovage root. The extraction kinetics curves (Fig. 4) show that pressure fluctuation intensifies extraction and that extracts are isolated from the lovage leaves faster. However, the total extract yield at the end of the process was close for both extraction methods: 2.39 g/100 g with pressure fluctuation and 2.71 g/100 g at constant pressure. Extraction kinetics curves for lovage leaves were quite similar to those obtained during the extraction of seeds, i.e. 50% of the extract was isolated after passing 1 kg of CO2. Total yield
of the extract from the leaves was slightly higher as compared with lovage roots.
3.4. Chemical composition of 6arious lo6age extracts Chemical composition of lovage extracts obtained from various anatomical parts at different pressure parameters is presented in Table 3. The main constituent in roots was cis-ligustilide, which constituted \ 50 area % of the extract. Ligustilides were also important constituents in the extracts of leaves and seeds, however, other compounds such as a-terpinyl acetate and b-phellandrene were also present in large quantities in leaves and seeds, respectively. In general, the composition of CO2 extracts in terms of major compounds was similar to the steam distilled essential oil composition obtained from the same anatomical parts of lovage cultivated in the same location [2]. However, CO2 extracts of leaves and seeds contained considerably higher amounts of phthalides compared to essential oils as well as lower amounts of a-terpinyl acetate (leaves) and b-phel-
Fig. 3. The kinetics of the extraction of lovage seed with CO2: ", constant pressure; , pressure changed in the range of 250 l 220 bar at a frequency of 60 min − 1.
b-phellandrene a- terpinyl acetate 3n-butylidene phthalide (E) 3n-butylidene phthalide (Z) cis-ligustilide trans-ligustilide Palmitic acid Phytol Linoleic Stigmasterol b-sitosterol Total
Compound
2.91 26.09 1.86 1.26 20.39 16.64 3.01 2.29 2.93 1.94 0.88 80.20
1.28 22.12 18.82 3.48 1.72 2.18 1.92 0.97 81.45
fluctuation 60 min−1
3.03 25.93 –
Constant pressure
Leaves
52.00 3.95 2.81 2.62 3.52 1.11 1.28 70.13
0.73
0.28 0.08 1.75
Constant pressure
51.36 4.28 2.86 2.39 3.63 1.05 1.44 70.26
0.87
0.26 0.06 2.06
fluctuation 10 min−1
Root
55.06 4.01 2.09 2.55 3.39 1.07 1.21 72.64
0.78
0.44 0.09 1.95
fluctuation 60 min−1
Table 3 Effect of pressure parameters (bar) on the composition of lovage extracts (200 ml extractor) in GC area %
40.29 20.77 0.69 0.55 3.21 5.08 – 89.14
1.11
15.32 0.77 1.35
Constant pressure
32.59 23.37 – – 2.39 7.02 – 87.51
1.69
19.37 1.07 –
fluctuation 60 min−1
Seed
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Fig. 4. The kinetics of the extraction of lovage leaves with CO2: ", constant pressure; , pressure changed in the range of 250 l 220 bar at a frequency of 60 min − 1.
landrene (seeds). Some compounds of lower volatility, such as higher fatty acids and sterols were also found in the CO2 extracts. There are some differences in the content of the constituents in the extracts obtained by a constant and fluctuating pressures (e.g. 3-butylidene phthalide), however the significance of these differences was not assessed in the present study. Actually, by using a 200 ml extraction vessel similar amount of the CO2-soluble substances can be isolated by using the same amount of solvent both at a constant and fluctuating pressure.
3.5. Extraction of celery root and chemical composition of the extracts Experiments on another Umbelliferae plant material, celery root (Table 1, no. 6), were performed at the same extraction conditions as with lovage root, except that 50 g of celery roots were extracted using 7 kg of CO2. The results are presented in Fig. 5. In this case, the extraction rate was also enhanced by the pressure fluctuation at the beginning of the process, however when 3 kg of CO2 were passed through the material the
amount of the extract became almost equal both at a constant and fluctuating pressure. The chemical composition of celery extracts obtained at constant and fluctuating pressure is presented in Table 4. Linoleic (9,12-octadecanoic) acid was the main constituent in celery extracts. Other important constituents were palmitic acid, stigmasterol, b-sitosterol and sedanolide. The content of the identified compounds was slightly different between the extracts obtained at constant and fluctuating pressures, however the significance of these differences was not evaluated. Most likely, pressure fluctuations had little if any effect on the chemical extract composition.
4. Conclusions The use of pressure fluctuation increased the extraction rate of CO2-soluble substances from all the plant materials studied at the initial stages of extraction. It seems that an increase in the pressure pulsation frequency also has a positive effect on the rate of extraction. However, at later stages of extraction the differences in the total extract
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Fig. 5. The kinetics of the extraction of celery roots with CO2: ", constant pressure; , pressure changed in the range of 250 l 220 bar at a frequency of 60 min − 1.
Table 4 Effect of pressure parameters (bar) on the composition of celery root extracts (200 ml extractor) Compound
Constant pressure
Pressure changed at 60 min−1
Sedanenolide Sedanolide Cis-ligustilide Margaric Palmitic Linoleic Stearic acid Docosane Stigmasterol b-sitosterol Total
3.51 0.87 0.91 0.71 18.24 48.61 0.96 0.94 4.33 4.36 83.44
3.48 0.77 0.81 0.72 15.17 52.37 1.03 1.21 3.71 4.14 83.41
yield between constant pressure and the pressure fluctuation method were not considerable. The fluctuation of the extraction pressure was found to have no considerable influence on the composition of both the lovage and celery extracts.
Acknowledgements The authors wish to thank Johnson Foundation (Royal Institute of Technology, Sweden) for financial support to perform experimental part of the work; The Lithuanian Institute of Horticulture for plant material, and The Lithuanian State Foundation of Science and Studies for aid in preparing this paper.
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Effect of fast CO2 pressure changes on the yield of lovage (Le6isticum officinale Koch.) and celery (Apium gra6eolens L.) extracts Egidijus Dauksˇas a, Petras Rimantas Venskutonis a,*, Bjo¨rn Sivik b, Tobias Nillson c a
Department of Food Technology, Kaunas Uni6ersity of Technology, Rad6ile; nu¸ pl. 19, Kaunas 3028, Lithuania b Food Technology, Chemical Center, Uni6ersity of Lund, PO Box 124, S-221 00 Lund, Sweden c Analytical Chemistry, Chemical Center, Uni6ersity of Lund, PO Box 124, S-221 00 Lund, Sweden Received 4 September 2000; received in revised form 22 May 2001; accepted 17 August 2001
Abstract The effect of pressure alterations on the yield of CO2 extracts from different anatomical parts of lovage (Le6isticum officinale Koch.) and celery (Apium gra6eolens L.) was studied. It was found that by applying frequent pressure changes in the extraction vessel it is possible to increase the rate of the isolation of CO2 soluble materials from lovage seeds and leaves, lovage and celery roots. However, after passing a sufficient amount of the supercritical solvent, the yields were similar both for constant and pulsing extraction pressures. The composition of the extracts was analyzed by gas chromatography and mass spectrometry and it was found that the phthalides were very important constituents in the extracts from all the anatomical parts of lovage, while linoleic acid was the most abundant component in the celery root extracts. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Celery; Lovage; Pressure changes; Supercritical CO2
1. Introduction Lovage and celery are important plants in some countries, where they have been used to flavor various foods and also for medicinal purposes. Investigations on lovage flavor and isolation of
* Corresponding author. Tel.: + 370-7-356-426; fax: +3707-456-647. E-mail address: [email protected] (P. Rimantas Venskutonis).
volatile aroma compounds have been reviewed in our previous publications concerning supercritical CO2 extraction [1] and distillation of different anatomical parts of the plant [2,3]. Celery (Apium gra6eolens L.) is native in Eurasia and common with other members of the Umbelliferae family. The commercial essential oil is usually described as possessing an appetizing flavor and it exhibits a strong aroma caused by several phthalides [4–14]. The yield of oil isolated by hydrodistillation or liquid CO2 from seeds has been found to be up to 3% [4,15].
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The process of the isolation of essential oils from various botanical sources depends on the morphological characteristics of the plant material. It is known that the oil ducts or canals in Umbelliferae plants are mainly schizogenous: developing close to vascular strands in the mesophyll of leaves and other organs by the separation of cells rather than by the death and dissolution of cells [16]. The walls of the canals are covered by a soft layer of cells producing the balsam, which is a solution of resin in the essential oil. Such receptacles can be clearly seen on a crosssection of Umbelliferae plant’s stem, root or seeds [17 – 19]. The schizogenous ducts are internal, inconspicuous and much more difficult to study [19]. In other literature sources [20], the secretory system of Umbelliferae family root anatomy is described as possessing two types of schizogenous oil ducts containing essential oils. The primary oil ducts are triangular and located between the pericycle cells of the seedling (they usually form two arcs), while the other two oil ducts lying in the middle are rhomboid. The theoretical basis of the extraction of botanical materials has been extensively described [21,22]. Some studies have shown that the transportation of liquid solutes in a capillary matrix can be enhanced by exposure to very fast pressure alterations [23]. The principles of such an extraction concept have been explained by Kasjanov et al. [24]. The frequency and period of the required pressure fluctuations depend on the properties of capillary matrix (density, porosity, etc.), any bonding between the solute materials and the plant matrix, extent of solvent heating and the intensity of pressure reduction [23]. The carbon dioxide pressure alteration method using subcritical conditions has been used for the extraction of dill seeds [25]. The extract yield obtained by decreasing the pressure of CO2 from 58.46 to 45.95 bar at a rate of 5 bar min − 1 and by periodic replacement of the isolated material, was 12.68% (w/w). This was approximately two times higher than the yield obtained by conventional CO2 extraction at 58.46 bar and 20 °C [25]. This study is an attempt to apply fast pressure fluctuation method for the extraction of lovage
and celery materials, thereby facilitating an increase in the extraction yield, and hence a reduction in the extraction time.
2. Materials and methods
2.1. Materials The lovage (Le6isticum officinale Koch.) and celery (Apium gra6eolens L.) were collected from the experimental garden of the Lithuanian Institute of Horticulture in 1996. The roots, leaves and seeds were harvested manually. Raw material was dried at 30 °C in a ventilated drying oven ‘Vasara’ (Utena, Lithuania) and stored in paper bags at ambient temperature to protect against direct light until further analysis. The samples were ground prior to extraction using a Knifetec 1095 Sample Mill (Tecator AB, Ho¨ gana¨ s, Sweden) for 20 s. Carbon dioxide, 99.99% purity (Aga Gas, Sweden) and hexane (\ 99%) from Merck (Darmstad, Germany) was used in the experiments.
2.2. Apparatus and extraction methodology A schematic diagram of experimental apparatus used in this study is shown in Fig. 1. A Milroyal B-C pump (Dosapro Milton Roy, Pont-SaintPierre, France) was used for extractions at 40 °C, and at pressures up to 350 bar. The temperature in the extractors and separators was maintained
Fig. 1. Schematic drawing of the supercritical extraction equipment. (1) Gas tube; (2) Shut-off valve; (3) Gas filter; (4) Ethanol bath; − 22 °C; (5) Pump; (6) Safety valve; (7) Pressure gauge; (8) Shut-off valve; (9) Extractor; (10) Water bath; (11) Micro metering valve; (12) Separators; (13) Sampling valves; (14) Pressure maintaining valve; (15) Flow meter; (16) Extra pump.
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Table 1 Experimental data No.
Extracted material
Amount of extracted material, g
Extraction vessel size, ml
Amount of CO2, kg
1 2 3 4 5 6
Roots of lovage Roots of lovage Seeds of lovage Leaves of lovage Leaves of lovage Roots of celery
10 100 100 10 50 50
47 200 200 47 200 200
2 10 10 2 7 7
by the thermostatic water jackets. Two different capacity extraction vessels were used: 47 ml for 10 g samples and 200 ml for 50 and/or 100 g of the ground plant material (Table 1). Glass wool was placed on the top and the bottom of the extraction vessels to prevent system contamination by any particles of the raw material, as well as to eliminate free space in the vessel. The CO2 flow rate was kept approximately at 0.025 kg min − 1 (measured by a mass flowmeter). The extract was collected in the first separator (12, Fig. 1). The second separator, which was connected to the first one through a valve, was used in the extraction of leaves, to separate the chlorophyll from the extract. The volume of both separators was 200 ml. The temperature in the first and second separator was maintained at 40 and 35 °C, respectively. The pressure in first separator was 50 bar during the extraction of roots and seeds and 100 bar during extraction of leaves; the pressure in the second separator in both cases was 45– 50 bar. The pump S-216-J with 414 kPa Driving Air Supply (Teledyne Sprague Engineering, CA) was used as an additional pump to create pressure fluctuations. When the piston of the second pump is elevated the pressure made by the main pump immediately drops. The frequency of the piston motion and consequently pressure fluctuations in the extraction system can be increased to 60 cycles min − 1 by changing the air pressure which was supplied from the outer line. All parts of the extraction system were made of stainless steel. Precision analytical balances (Mettler AE 163, 0.01 mg resolution, Mettler Instrument AG, Switzerland) were used for weighing the amounts of extract obtained. Two replicates were analyzed for every sample and the mean value calculated.
2.3. Gas chromatography and mass spectrometry (GS-MS) The extracts were diluted with cyclohexane and examined by GC-MS, using a Hewlett-Packard 5890 series II gas chromatograph equipped with Hewlett-Packard 5972 mass detector with electron impact ion source (Hewlett-Packard, Rockville, MP). The system was run from a computer with the Hewlett-Packard MS Chemstation software, version B.02.05. The column used was a HP-5 M.S. (Crosslinked 5% pH Me Silicone, 30 m × 0.25 mm, 0.25 mm film thickness). Helium was used as carrier gas at a constant flow of 0.84 ml min − 1. In all runs, 2 ml of sample were injected in split mode with a split ratio of 1:14 at 280 °C. The oven temperature was programmed from 35 °C (1 min hold) to 270 °C (10 min hold) at 4 °C min − 1. The MS interface was heated at 320 °C. The compounds were identified by their mass spectra (computerized comparison with reference spectra in the MS Chemstation library) and retention time in the capillary column. Two replicate GC analyses were performed and the results were expressed in GC area % as a mean value.
3. Results and discussion
3.1. Effect of extraction conditions on the extract yield from lo6age root The first series of experiments were carried out with lovage root (Table 1, no.1) to determine the effect of extraction conditions on the extract yield. In our previous study [1] it was found that 250
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bar and 40 °C were preferable parameters for obtaining a high extraction yield from lovage roots. Therefore, the same conditions (Table 2, Experiment c 1) were used to extract lovage roots without using the extra pump (16, Fig. 1). The total yield of the extract was 1.589 0.03 g from 100 g of raw material (Table 2). This amount was used as a target figure for comparison purposes with results obtained during subsequent extractions where pressure fluctuations were applied. Extraction pressure was changed by using an extra pump (16, Fig. 1); the results obtained are presented in Table 2. It was found that pressure changes and their frequency effect the extract yield. By fluctuating the pressure in the range from 200 to 140 bar at a frequency of 10 cycles/ min the yield was increased up to 1.73 g per 100 g of raw material (i. e. approximately a 10% increase compared to the yield obtained at constant pressure of 250 bar). However, when the maximal pressure was increased up to 250 bar (in this case the minimal pressure was 180 bar) and altered at the same frequency (10 min − 1), the yield decreased to 1.56 g/100 g, similar to the yield obtained at the constant pressure of 250 bar. A further increase in pressure to 350 bar was not efficient; the extract yield was 1.7090.04 g/100 g, i.e. similar to the yield obtained during Experiment c 2 (Table 2). Finally, the frequency of pressure (maximal=250; minimal= 220) change was increased to 60 min − 1, the highest extract yield (2.18 g 100 g − 1) was obtained. The parameters applied allowed to increase the yield by 30% compared to that obtained at constant pressure of 250 bar. The results obtained suggest that by using an
extra pump and fluctuating the pressure, the frequency of pressure change has a greater effect on the yield than the effect of maximal and minimal pressure magnitude. This phenomenon can be explained by the theory [23] suggesting that by applying intensive and very fast pressure increase the mass transport is performed by the molar filtration, which creates the formation of an intensive flow of the substances resulting in an increase of the total yield. The second series of the experiments were performed with lovage roots by using a larger extractor (Table 1, no. 2). In this case two identical separators (50 bar pressure; 40 and 35 °C temperature, respectively) were used. In the beginning of the extraction (until 3 kg of CO2 was passed) extract yields were measured after passing each 0.5 kg; afterwards the yields were measured at the intervals of 1 kg of the used CO2. The yield obtained at 250 bar and 40 °C without the extra pump was used as a control. Another two extractions were performed at the same optimal pressure and temperature parameters using the extra pump (16, Fig. 1), which created pressure fluctuations from 250 to 180 bar at a frequency of 10 min − 1, and from 250 to 220 bar at a frequency of 60 min − 1. These results are presented in Fig. 2. In general, at the end of the extractions (10 kg CO2 used) there were no considerable differences between the yields obtained at all three sets of conditions applied. However, when the pressure was changed to a frequency of 60 min − 1, the CO2-soluble substances were extracted more extensively at the beginning of the extraction procedure than at the end. The effect of pressure fluctuation at a frequency of 10 min − 1 was negligible during all extraction cycles.
Table 2 The yield of the extract from lovage root at different extraction pressure parameters, n = 2 (47 ml extraction vessel) Experiment No.
Maximal pressure, bar
Minimal pressure, bar
Frequency of pressure change, min−1
Extract yield, g 100 g−1
1 2 3 4 5
250 200 250 350 250
250 140 180 290 220
– 10 91 10 91 10 91 60 9 5
1.58 90.03 1.73 9 0.03 1.56 90.04 1.70 9 0.04 2.23 90.05
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Fig. 2. The kinetics of the extraction of lovage root with CO2: ", constant pressure; , pressure fluctuation in the range of 250 l 180 bar at a frequency of 10 min − 1; , pressure fluctuation in the range of 250 l220 bar at a frequency of 60 min − 1.
3.2. Extraction of lo6age seeds The experiments were continued with lovage seeds (Table 1, no. 3) using the same extraction parameters used for roots. In this case the frequency of a pressure change was 60 min − 1. The extraction kinetic curves for lovage seed are presented in Fig. 3. These curves show that the process of the isolation of soluble substances from the seeds was quite similar for both extraction methods. In general, the extraction kinetics from the seeds was quite different when compared to roots. Approximately 50% of the extract obtained from seeds was isolated after initially passing 1 kg of CO2, while the remaining soluble substances were extracted at a considerably lower rate. In the case of the roots (Fig. 2) 50% of the extract was obtained after passing 2– 3 kg of CO2. Finally, the total extract yield from the seeds ( 6 g/100 g) was almost 3 times higher than that found from the roots ( 2 g/100 g). These differences in extraction rate and yield can be explained by the different structural properties and chemical composition of the seeds and roots. For instance, seeds contain significant amount of lipids,
whereas roots consist mainly of a polysaccharide matrix.
3.3. Extraction of lo6age lea6es The third set of experiments on the extraction of lovage was conducted using plant leaves. The aerial parts of the plant contain high amounts of chlorophyll. It was reported that the content of chlorophyll in the extract increases by increasing extraction pressure [26]. Hence, several initial extractions were performed to determine the optimal parameters for the separation of chlorophyll from the other extract components. These experiments were started without using the second pump (Table 1, no. 4), and by maintaining the pressure in the first separator at 175, 150 or 100 bar and a temperature of 40 °C, to prevent chlorophyll contamination of the extract collected in the second separator. The conditions in the second separator were 50 bar and 35 °C. The highest yield of the extract with the lowest chlorophyll content (relatively light yellow color based on the visual assessment of the extracts), in the second separator (1.09 g 100 g − 1) was obtained
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by separation at 100 bar. The yield of the green colored extract in the first separator was 0.29 g/100 g. After determining the optimal pressure for the first separator, next experiment (Table 1, no. 5) was continued alternating the fluctuation of pressure over the range of 250− 220 bar at the frequency of 60 min − 1. The yield of the extract was 0.17 g/100 g and 1.82 g/100 g in the first and second separators, respectively. Consequently the yield of the chlorophyll-reduced extract increased 1.8 times when compared to that obtained at constant pressure. This result is also in a good agreement with data obtained during the extraction of lovage root. The extraction kinetics curves (Fig. 4) show that pressure fluctuation intensifies extraction and that extracts are isolated from the lovage leaves faster. However, the total extract yield at the end of the process was close for both extraction methods: 2.39 g/100 g with pressure fluctuation and 2.71 g/100 g at constant pressure. Extraction kinetics curves for lovage leaves were quite similar to those obtained during the extraction of seeds, i.e. 50% of the extract was isolated after passing 1 kg of CO2. Total yield
of the extract from the leaves was slightly higher as compared with lovage roots.
3.4. Chemical composition of 6arious lo6age extracts Chemical composition of lovage extracts obtained from various anatomical parts at different pressure parameters is presented in Table 3. The main constituent in roots was cis-ligustilide, which constituted \ 50 area % of the extract. Ligustilides were also important constituents in the extracts of leaves and seeds, however, other compounds such as a-terpinyl acetate and b-phellandrene were also present in large quantities in leaves and seeds, respectively. In general, the composition of CO2 extracts in terms of major compounds was similar to the steam distilled essential oil composition obtained from the same anatomical parts of lovage cultivated in the same location [2]. However, CO2 extracts of leaves and seeds contained considerably higher amounts of phthalides compared to essential oils as well as lower amounts of a-terpinyl acetate (leaves) and b-phel-
Fig. 3. The kinetics of the extraction of lovage seed with CO2: ", constant pressure; , pressure changed in the range of 250 l 220 bar at a frequency of 60 min − 1.
b-phellandrene a- terpinyl acetate 3n-butylidene phthalide (E) 3n-butylidene phthalide (Z) cis-ligustilide trans-ligustilide Palmitic acid Phytol Linoleic Stigmasterol b-sitosterol Total
Compound
2.91 26.09 1.86 1.26 20.39 16.64 3.01 2.29 2.93 1.94 0.88 80.20
1.28 22.12 18.82 3.48 1.72 2.18 1.92 0.97 81.45
fluctuation 60 min−1
3.03 25.93 –
Constant pressure
Leaves
52.00 3.95 2.81 2.62 3.52 1.11 1.28 70.13
0.73
0.28 0.08 1.75
Constant pressure
51.36 4.28 2.86 2.39 3.63 1.05 1.44 70.26
0.87
0.26 0.06 2.06
fluctuation 10 min−1
Root
55.06 4.01 2.09 2.55 3.39 1.07 1.21 72.64
0.78
0.44 0.09 1.95
fluctuation 60 min−1
Table 3 Effect of pressure parameters (bar) on the composition of lovage extracts (200 ml extractor) in GC area %
40.29 20.77 0.69 0.55 3.21 5.08 – 89.14
1.11
15.32 0.77 1.35
Constant pressure
32.59 23.37 – – 2.39 7.02 – 87.51
1.69
19.37 1.07 –
fluctuation 60 min−1
Seed
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Fig. 4. The kinetics of the extraction of lovage leaves with CO2: ", constant pressure; , pressure changed in the range of 250 l 220 bar at a frequency of 60 min − 1.
landrene (seeds). Some compounds of lower volatility, such as higher fatty acids and sterols were also found in the CO2 extracts. There are some differences in the content of the constituents in the extracts obtained by a constant and fluctuating pressures (e.g. 3-butylidene phthalide), however the significance of these differences was not assessed in the present study. Actually, by using a 200 ml extraction vessel similar amount of the CO2-soluble substances can be isolated by using the same amount of solvent both at a constant and fluctuating pressure.
3.5. Extraction of celery root and chemical composition of the extracts Experiments on another Umbelliferae plant material, celery root (Table 1, no. 6), were performed at the same extraction conditions as with lovage root, except that 50 g of celery roots were extracted using 7 kg of CO2. The results are presented in Fig. 5. In this case, the extraction rate was also enhanced by the pressure fluctuation at the beginning of the process, however when 3 kg of CO2 were passed through the material the
amount of the extract became almost equal both at a constant and fluctuating pressure. The chemical composition of celery extracts obtained at constant and fluctuating pressure is presented in Table 4. Linoleic (9,12-octadecanoic) acid was the main constituent in celery extracts. Other important constituents were palmitic acid, stigmasterol, b-sitosterol and sedanolide. The content of the identified compounds was slightly different between the extracts obtained at constant and fluctuating pressures, however the significance of these differences was not evaluated. Most likely, pressure fluctuations had little if any effect on the chemical extract composition.
4. Conclusions The use of pressure fluctuation increased the extraction rate of CO2-soluble substances from all the plant materials studied at the initial stages of extraction. It seems that an increase in the pressure pulsation frequency also has a positive effect on the rate of extraction. However, at later stages of extraction the differences in the total extract
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Fig. 5. The kinetics of the extraction of celery roots with CO2: ", constant pressure; , pressure changed in the range of 250 l 220 bar at a frequency of 60 min − 1.
Table 4 Effect of pressure parameters (bar) on the composition of celery root extracts (200 ml extractor) Compound
Constant pressure
Pressure changed at 60 min−1
Sedanenolide Sedanolide Cis-ligustilide Margaric Palmitic Linoleic Stearic acid Docosane Stigmasterol b-sitosterol Total
3.51 0.87 0.91 0.71 18.24 48.61 0.96 0.94 4.33 4.36 83.44
3.48 0.77 0.81 0.72 15.17 52.37 1.03 1.21 3.71 4.14 83.41
yield between constant pressure and the pressure fluctuation method were not considerable. The fluctuation of the extraction pressure was found to have no considerable influence on the composition of both the lovage and celery extracts.
Acknowledgements The authors wish to thank Johnson Foundation (Royal Institute of Technology, Sweden) for financial support to perform experimental part of the work; The Lithuanian Institute of Horticulture for plant material, and The Lithuanian State Foundation of Science and Studies for aid in preparing this paper.
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