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Extraction and identification of proanthocyanidins from grape seed (Vitis Vinifera) using supercritical carbon dioxide
J. of Supercritical Fluids 55 (2011) 924–928
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The Journal of Supercritical Fluids journal homepage: www.elsevier.com/locate/supflu
Extraction and identification of proanthocyanidins from grape seed (Vitis Vinifera) using supercritical carbon dioxide Esen Eyiler Yilmaz ∗ , Emin Burcin Özvural, Halil Vural Hacettepe University, Departement of Food Engineering, Beytepe 06800, Ankara, Turkey
a r t i c l e
i n f o
Article history: Received 7 September 2010 Received in revised form 23 October 2010 Accepted 24 October 2010 Keywords: Supercritical carbon dioxide extraction Proantocyanidins Grape seed High performance liquid chromatography
a b s t r a c t In this study, supercritical carbon dioxide extraction of proantocyanidins (PRCs) was performed and the effect of different pressure, temperature and ethanol percentage was investigated. High performance liquid chromatography was used for the analysis of the compounds and it was found that the most effective parameter on the extraction was the amount of the ethanol percentage. Each compound was extracted from grape seeds at their maximum level when different parameters were used which was probably because of their different polarities. Gallic acid (GA), epigallocatechin (EGC) and epigallocatechingallate (EGCG) were extracted at their maximum level when the 300 bar 50 ◦ C and 20% of ethanol was used. The maximum amount of catechin (CT) and epicatechin (ECT) were obtained when 300 bar 30 ◦ C and 20% of ethanol was used for extraction, and 250 bar, 30 ◦ C and 15% of ethanol was needed to extract highest amount of epicatechingallate (ECG). © 2010 Elsevier B.V. All rights reserved.
1. Introduction Grape seed is a residue of wine and grape juice industry and is generally used as an animal feed. The investigation on grape seed has been increasing since its positive effects was shown on human health. Grape seed contains 8–15% oil and this oil contains high amounts of unsaturated fatty acids and antioxidant-rich compounds [1]. These antioxidants are mostly phenolic compounds like cathecins and procyanidins [2–4]. These phenols may act against the in vitro oxidation of low density lipoproteins [5], and also have anticarcinogenic, antiviral and antimutagenic [6] activities, besides these they prevent the thrombosis, reduces cholesterol and regulates the autonomic nerves [7]. The applications of the grape seed oil have been increasing in pharmaceuticals, medical, cosmetic and food industry due its properties. The extraction of seed oil includes many different stages like cleanup of the biomass, drying, crushing and pressing. By the application of pressing most of the oil can be extracted but nevertheless a considerable amount of oil remains in the cake, and this remaining oil can be extracted by hexane; which should be evaporated after the process [8]. The main drawback of this process is the usage of n-hexane at the last stages [1]. The methods for the extraction of phenolic compounds from grape seed include usage of methanol [3,4,9], ethanol [6,10,11] and acetone [12]. Different extraction temperatures are being used with different times that range from minutes to several hours.
Due to the complexity of the procedure to extract oil and phenolic compounds, newer methods are in consideration. Supercritical fluid extraction (SFE) is one of these methods, extensively studied nowadays because of its unique properties. SFE has many advantages, like absence of light and air, over the conventional extraction procedures. Air and light can cause degradation of both the phenolic compounds and unsaturated fatty acids (UFAs). Among many supercritical fluids carbon dioxide is the one that is mostly used owing to its non-toxicity, cheap and non-flammable properties [1]. Besides these properties CO2 has moderate critical pressure (7.28 MPa) and temperature (304.1 K) which in turn prevents the thermal degradation of phenolic compounds. Supercritical carbon dioxide extraction of grape seed oil has been extensively applied [1,7,8,13], however; for the extraction of phenolic compounds a co-solvent like methanol or ethanol should be used, because carbon dioxide is a non-polar substance and polar substances like phenolic compounds cannot be extracted from grape seeds. In this study, the extraction of phenolic compounds from grape seed using supercritical carbon dioxide with ethanol as a co-solvent was performed. The effect of different temperature, 30 and 50 ◦ C, pressure, 250, 275 and 300 bar, and co-solvent percentages, 5, 10, 15 and 20 wt%, to the extraction amount has been investigated. The amounts of the phenolic compounds extracted were analyzed using RP-HPLC with a gradient program. 2. Material and methods 2.1. Material
∗ Corresponding author. Tel.: +90 3122977100; fax: +90 3122992123. E-mail address: [email protected] (E.E. Yilmaz). 0896-8446/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.supflu.2010.10.046
The carignan type of grape seeds used in the study was kindly given by Kavaklidere Winery. The marc given was dried
5.0 32.6ab 16.5ab 6.0ab 2.6ab 0.0b 7.1 32.6b 18.8b 7.1b 6.2b 0.3c
13.6 51.9 25.1 13.3 15.3 6.8
± ± ± ± ± ±
ab
0.0 0.1ab 0.1a 0.0a 0.6a 0.1b
19.6 83.9 44.1 20.9 24.0 8.5
± ± ± ± ± ±
b
5.9 19.0 5.8 4.8 13.5 7.2 2.6 ± 0.0 9.3 ± 0.3a ND ND 11.3 ± 0.0a 1.1 ± 0.0a
± ± ± ± ± ±
a
GA EGC CT ECT EGCG ECG 8.05 19.03 20.57 27.76 28.44 33.08
a
SC-CO2 was modified with ethanol as a co-solvent and the percentages were 5, 10, 15 and 20% and the temperature was 30 ◦ C. The extraction with SCF was performed twice. Extraction of PRCs from the oil extracted with SFE was performed in 2 parallels. Rt = retention time of the components in HPLC (min), ND = not detected. a–c: Means within the same pressure value and same row with different superscript letters are different (p < 0.05).
29.3 173.1 90.3 43.1 27.3 1.9 15.1 59.6 22.8 14.6 25.9 1.5 9.5 32.8 12.0 9.9 25.2 1.1 0.3 0.6a 0.2a 0.2a 0.3a 0.2a
± ± ± ± ± ± 4.9 12.7 1.3 5.2 13.0 0.9 16.7 66.6 48.6 23.5 10.3 2.5 13.6 ± 2.4 45.0 ± 12.5bc 21.5 ± 7.2a 13.1 ± 3.6ab 23.3 ± 3.3b ND 10.4 ± 1.6 33.2 ± 4.5ab 12.6 ± 2.8a 9.0 ± 1.0a 26.3 ± 4.2b ND 4.5 ± 0.3 9.6 ± 0.1a ND ND 12.2 ± 0.1a ND a
5% 20% 15% 10%
P = 250 bar
5% Compound Rt
mg of component extracted/kg of grape seed
Table 1 The amount of the PRCs (ppm) extracted from the grape seeds at 30 ◦ C, 250, 275 and 300 bar.
P = 275 bar
10%
ab
15%
b
20%
± ± ± ± ± ±
b
4.2 19.4c 17.0b 6.9b 0.6a 0.0a
5%
P = 300 bar
a
10%
± ± ± ± ± ±
0.3 2.6a 0.7a 0.8a 3.4b 0.2a
ab
15%
± ± ± ± ± ±
b
1.1 17.7a 7.3a 2.0a 0.2b 0.3ab
20%
± ± ± ± ± ±
7.0c 55.8b 28.5b 11.8b 3.1b 0.4b
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925
at room temperature and the grape seeds were separated from their peels, after removing the peels the seeds were kept at −18 ◦ C until they were used. The standards used for HPLC analysis were (+)-catechin hydrate (96%), (−)-epicatechin (>97%), (−)-epigallocatechin (from green tea > 95%), (−)-epicatechin gallate (from green tea, 98%), (−)-epigallocatechin gallate (97%) and were obtained from Sigma–Aldrich (Steinheim, Germany). Ethanol which was used as a co-solvent and methanol, used for extraction of PRCs from the grape seed oil, were obtained from Riedel de-Haen (Germany). HPLC grade acetic acid (100%) was from Sigma–Aldrich (Germany) and acetonitrile was from Merck (Darmstadt, Germany). 2.2. Methods 2.2.1. Sample preparations Seeds, which were kept at −18 ◦ C were equilibrated to room temperature and then were crushed with a mixer. The seeds were crushed for 2 min, after every 30 s. The samples were waited for 30 s to prevent seeds from warming up. Crashed seeds were then sieved and the ones that were between 425 and 230 m. 30 g of the sieved seeds were weighed and were put into the extraction vessel. 2.2.2. Supercritical fluid extraction of grape seeds The extraction of antioxidant substances from the seed was performed using and analytical supercritical fluid extractor (SFE-100-2-FMC10, Thar Instruments, PA, USA). The instrument was equipped with automated back pressure regulator, 100 ml extraction vessel, 500 ml collection vessel, six zone temperature controller, high pressure P-50 series pump, cooling systems filled with glycol, and a series III pump for co-solvent (cannot operate over 400 bar). The co-solvent pump was purged before each extraction in order to ensure that co-solvent entered the system (Thar Instruments, Series III Pump, Manuel). The carbon dioxide flow rate was fixed to 5 g/min and was same for all the extraction parameters. Low flow rate was selected in order to ensure that the residence time was longer in the supercritical fluid extraction vessel. Due to the fact that PRCs could not be extracted with CO2 , because of its non-polar nature, the CO2 was modified with ethanol at the level of 5, 10, 15 and 20% (wt%). Ethanol was selected because it is a polar solvent is permitted in food industry. Other than the ethanol percentages 3 different pressures (250, 275 and 300 bar) and 2 different temperatures, 30 and 50 ◦ C were used during the extractions. The length of the extraction was for 1 h. After the extraction the collection vessel was washed with 25 ml of ethanol to clean the surfaces of the vessel, and to minimize the losses that can occur. The grape seed oil, PRCs was collected in a 250 ml volumetric flask and the ethanol was removed with rotary evaporator (Büchi, B465, Switzerland) at 45 ◦ C. After removal of the ethanol the oil containing the PRCs were analyzed with HPLC (Agilent 1100). The extractions with SC-CO2 were performed in two replications. 2.2.3. HPLC analysis of the extracts The samples obtained from the SFE process were analyzed according to the methods of [14,15]. According to those methods 1 ml of the oil in the volumetric flask was transferred to a centrifuge tube and 1 ml of methanol was added and was vortexed for 2 min. The vortexed samples were then centrifuged 10 min at 3000 rpm (Nüve, NF 1215, Istanbul, Turkey) and the supernatant was taken to a test tube. The extraction was repeated twice and the supernatants were mixed and were analyzed with Agilent 1100 RP-HPLC. The experiments were performed in 2 parallels. The analysis in HPLC were performed with a nucleosil C18 HPLC column (250*4.6 mm, Supelco Inc., Bellefonte, PA, USA), at 280 nm and 35 ◦ C using a DAD detector. The flow rate was 1 ml/min and the injection volume was 10 l. Gradient flow was used with 2 mobile phases which are 2%
± ± ± ± ± ± 32.90 218.88 78.83 35.15 47.12 6.48
20%
1.7b 3.9a 2.1b 0.9b 0.4ab 0.1a 16.5 65.6 23.1 12.8 38.3 4.2
± ± ± ± ± ± 15% 10%
8.6 ± 0.6a 26.5 ± 1.8a 5.2 ± 0.8a 6.8 ± 0.1ab 27.5 ± 0.5ab ND
5%
5.6 ± 1.0a 17.9 ± 4.0a 2.4 ± 1.0a 2.8 ± 3.9a 21.6 ± 4.8a ND 17.1 57.1 27.0 15.2 34.4 3.6 16.3 ± 1.0b 53.8 ± 4.9b 18.5 ± 1.9b 11.8 ± 0.6a 38.0 ± 1.6c ND
± ± ± ± ± ±
4.01b 23.6b 10.2b 4.0a 4.8c 0.9a
300 bar
6.9 22.2 3.9 5.9 26.1 0.9 GA EGC CT ECT EGCG ECG 8.05 19.03 20.57 27.76 28.44 33.08
3.6 ± 0.1a 10.2 ± 0.0a ND 4.2 ± 0.1a 12.2 ± 0.2a ND
± ± ± ± ± ±
0.1b 0.3b 0.2a 0.4ab 0.3b 0.1a
11.1 29.8 10.8 7.7 30.0 1.0
± ± ± ± ± ±
0.0c 4.5b 2.2b 1.2b 1.2c 0.0a
15.3 43.4 20.1 12.6 35.8 2.1
± ± ± ± ± ±
1.1d 4.0c 2.3c 0.1c 0.4d 0.8a
4.2 ± 0.2a 13.7 ± 0.7a ND ND 18.3 ± 0.0a ND
7.4 ± 0.3a 23.4 ± 2.0ab 3.3 ± 0.2a ND 26.4 ± 2.4b ND
20% 15% 10% 5%
275 bar
20% 15% 10% 250 bar
5% Compound Rt
mg of component extracted/kg of grape seed
Table 2 The amount of the PRCs (ppm) extracted from the grape seeds at 50 ◦ C, 250, 275 and 300 bar.
SC-CO2 was modified with ethanol as a co-solvent and the percentages was 5, 10, 15 and 20% and the temperature was 50 ◦ C. The extraction with SCF was performed twice. Extraction of PRCs from the oil extracted with SFE was performed in 2 parallels. Rt = retention time of the components in HPLC, ND = not detected. a–d: Means within the same pressure and same row with different superscript letters are different (p < 0.05).
E.E. Yilmaz et al. / J. of Supercritical Fluids 55 (2011) 924–928
3.1c 59.2b 12.4c 4.7c 13.8b 2.3a
926
Fig. 1. The chromatogram of PRCs.
acetic acid (in water mobile phase A) and 100% acetonitrile as phase B, the analysis time was 35 min. The gradient program was as follows: 100% A to 98% in 2 min, 96% A in 6 min, 94% A in 4 min, 92% A in 4 min, 91% A in 5 min, 90% A in 2 min, 88% A in 1 min, 87% A in 3 min, 80% A at 2 min, 78% A at 30th min, and isocratic for last 5 min The chromatogram of the PRCs is given in Fig. 1. 2.2.4. Statistical analysis All data analysis was performed using SPSS for Windows® 16. The statistical significances of the differences between means were determined using Duncan’s multiple range test. 3. Results 3.1. Effect of pressure on the extracts The effect of pressure on the extraction of PRCs can be seen at Tables 1–3. Here Tables 1 and 2 represent the effects of different parameters for each of the PRCs, while Table 3 gives the average value of each parameter for all samples with the same pressure. According to the results obtained, the most abundant material found within the examined grape seeds was EGC and the least one was ECG. The amount of the EGC was increased excessively when temperature, pressure and percentage of ethanol was increased. As can be observed from the results increasing the pressure increased the extracted quantity of PRCs, which was also observed by Murga et al. [16]. Berna et al. [17] also observed in their study, that increasing the pressure cause and increase on amount of CT extracted. This result was due to the increased solvent solubility, occurred according to the increased pressure. Chafer et al. [18] reported that the solubility of GA in supercritical CO2 + ethanol (as co-solvent) was increased, as the pressure was elevated. 3.2. Effect of ethanol percentage on the extracts In a study performed by Murga et al. [16] it was stated that; when less than 5% of ethanol was used only GA was able to be extracted, CT, ECG and ECT could not be extracted. Due to this fact we have used 5, 10 15 and 20% in our study. Effect of different ethanol percentages on the extraction of PRCs from grape seed is given in Tables 1 and 2. CT was not able to be extracted from the grape seeds at 5% of ethanol until the pressure was increased to 300bar, which could be effect of increased solvent solubility at higher pressures. A similar case was observed by Berna et al. [17]; they stated that it was not possible to measure the solubility of CT until 110 bar and 5% of ethanol. Increasing the percentage of ethanol increased the amount of the extracted PRCs generally, however, at 250 bar and 30 ◦ C increasing the percentage of ethanol to 20% did not cause an increase at the amounts of PRCs, which in fact lead to a decrease. The reason for that could be that the polarity
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Table 3 Effect of pressure on proanthocyanidin amount. Main factors
Gallic acid (GA)
Epigallocatechin (EGC)
Catechin (CT)
Epicatechin (ECT)
Epigallo catechingallat (EGCG)
Epicatechin gallat (ECG)
Pressure (bar)c 250 275 300
9.8 ± 6.3a 11.3 ± 5.5a 15.3 ± 15.3a
33.7 ± 27.2a 37.8 ± 22.2a 75.9 ± 77.8b
13.8 ± 16.1a 16.4 ± 16.9a 29.5 ± 34.9a
8.7 ± 6.8a 9.1 ± 8.6a 16.3 ± 14.7a
21.0 ± 9.1a 23.6 ± 9.7a 28.2 ± 10.6a
3.4 ± 3.3a 0.9 ± 1.4ab 2.0 ± 2.3b
The extraction with SCF was performed twice. Extraction of PRCs from the oil extracted with SFE was performed in 2 parallels. a and b: Means within the same factor and the same column with different superscript letter are different (p < 0.05). c: Each number represents the average value of each parameter for all samples with the same pressure.
Table 4 The effect of temperature on the extracted amount of PRCs. Gallic acid (GA) Temperature (◦ C)a 30 12.2 ± 1.4 50 12.1 ± 0.4 a
Epigallocatechin (EGC)
Catechin (CT)
Epicatechin (ECT)
Epigallo catechingallat (EGCG)
Epicatechin gallat (ECG)
49.7 ± 10.8 48.6 ± 4.8
23.7 ± 4.9 16.1 ± 1.0
13.1 ± 2.0 9.6 ± 0.1
18.9 ± 1.2 29.6 ± 1.2
2.6 ± 0.04 1.6 ± 0.2
Each number represents the average value of each parameter for all samples with the same temperature.
of the supercritical solvent was not favorable for the extraction of the PRCs. The increased amount of the PRCs due to the increased ethanol percentage was expected because the phenolic compounds can be extracted by a co-solvent like ethanol due to increased polarity [16]. Also Chafer et al. [18] reported that the solubility of GA was increased as the percentage of the ethanol used was increased. ECG showed very different behavior from the other substances, it was not possible to extract when 275 bar and 5, 10 and 15% of ethanol was used. Although the highest amounts were extracted at 250 bar and 30 ◦ C, when the temperature was increased to 50 ◦ C at 250 bar the amount of ECG extracted was lowered which is probably due to the degradation of the compound.
3.3. Effect of temperature on the extracts Proanthocyanidins are unstable at high temperatures and can easily undergo oxidation reactions and lose their antioxidant activity [16]. Besides that Palma and Taylor [6] stated that in the presence of air antioxidants like CT can be degraded in several weeks at 55 ◦ C, on the other hand if the temperature is higher, degradation occurs within a few days. According to this knowledge we did not exceeded 50 ◦ C in our experiments. When the temperature was increased to 50 ◦ C the amount of GA and EGC extracted was not changed very much, however; CT, ECT and ECG amounts were decreased (Table 4). Although Chafer et al. [18] found that the solubility of GA decreases when the extraction temperature was increased; Palma and Taylor [6] reported that higher temperatures are required to overcome analyte–matrix interactions. The decrease in the amount of CT, ECT and ECG due to increased temperature could be according to their sensitivity to temperature. The only substance that has an increased amount was EGCG. This could be because at higher temperatures the analyte–matrix interaction within the seed could be accomplished.
4. Discussion Temperature and pressure defines the density of the SCF which consequently affects the solubility of the solute [19]. The solubility of low volatility substances in supercritical and near critical solvent decreases as the temperature increases due to the decrease in the density and as a result decrease in the solubility at low pressures (up to 200 MPa). When higher pressures are used for extraction, the effect of temperature on density is less pronounced and the vapor pressure becomes the dominating factor [20,21].
The HPLC results showed us that GA, EGC and EGCG were extracted at their maximum level when the parameters were 300 bar 50 ◦ C and 20% of ethanol (Table 2). The maximum amount of CT and ECT were obtained when 300 bar 30 ◦ C and 20% of ethanol was used for extraction (Table 1). It was ECG that had its maximum amount at the lowest pressure which was 250 bar, the temperature was 30 ◦ C and the ethanol percentage was 15%. Since GA, EGC and EGCG have higher melting points they were extracted at a higher temperature than other substances. Also they were extracted at the highest pressure used, which was due to the increased solubility of CO2 due to the increased density at high pressures. Although CT and ECT are isomers, CT has a lower melting point and therefore the amount of extracted CT was higher than that of ECT [22]. Nevertheless their maximum amounts were obtained at the same temperature and pressure. The extraction of ECG at low pressure and temperature could be due to its polarity, and the parameters used were not adequate to overcome the analyte matrix interactions, since it has the lowest amount extracted. Since different PRCs could be extracted at their maximum levels at different parameters, a selective extraction might be applied using different temperature, pressure and percentage of ethanol, in order to obtain the desired component. Polyphenols were able to be extracted from the grape seeds by mixing supercritical CO2 with ethanol at different levels. It would be possible to extract higher molecular weight polyphenols from the matrix by increasing the parameters used. It would not be feasible to increase the temperature higher than 55 ◦ C, which could cause rapid degradation of the antioxidant substances. 5. Conclusion Supercritical fluid extraction is a promising new method for the extraction of the compounds which could be easily degraded. Extraction of polyphenols from grape seeds by solvent extraction has been used for a long time, but those methods require high amount of chemical substances. SC-CO2 extraction provides some advantages like, using less or none of the chemical substances, free of oxygen, can be operated at low temperatures and by varying the parameters like temperature, pressure and/or percentage of ethanol different fractions can be extracted. Acknowledgments We would like to thank Prof. Ismail Hakki Boyaci and research assistant F. Ceyda Dudak for their kind help and support with
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the analysis in HPLC. This research was financially supported by Hacettepe University, Research Center Office, project no.: 06 01 602 014. References [1] C.P. Passos, R.M. Silva, F.A. Da Silva, M.A. Coimbra, C.M. Silva, Enhancement of the supercritical fluid extraction of grape seed oil by using enzymatically pre-treated seed, Journal of Supercritical Fluids 48 (2009) 225–229. [2] K. Kantz, V. Singleton, Isolation and determination of polymeric polyphenols using sephadex LH-20 and analysis of grape tissue extracts, American Journal of Enology and Viticulture 41 (1990) 223–228. [3] T. Escribano-Bailh, Y. Gutibrrez-Fernhdez, J. Rivas-Gonzalo, C. Santos-Buelga, Characterization of procyanidins of Vitis vinifera variety Tinta del Pais grape seeds, Journal of Agricultural and Food Chemistry 40 (1992) 1794–1799. [4] T. Fuleki, J. Da Silva, Catechin and procyanidin composition of seeds from grape cultivars grown in ontario, Journal of Agricultural and Food Chemistry 45 (1997) 1156–1160. [5] A. Meyer, O.S. Yi, D. Pearson, A. Waterhouse, E. Frankel, Inhibition of human low-density lipoprotein oxidation in relation to composition of phenolic antioxidants in grapes (Vitis vinifera), Journal of Agricultual and Food Chemistry 45 (1997) 1638–1643. [6] M. Palma, L. Taylor, Extraction of polyphenolic compounds from grape seeds with near critical carbon dioxide, Journal of Chromatography A 849 (1999) 117–124. [7] X. Cao, Y. Ito, Supercritical fluid extraction of grape seed oil and subsequent separation of free fatty acids by high-speed counter-current chromatography, Journal of Chromatography A 1021 (2003) 117–124. [8] L. Fiori, Grape seed oil supercritical extraction kinetic and solubility data: critical approach and modeling, Journal of Supercritical Fluids 43 (2007) 43–54. [9] M. Ashraf-Khorassani, L.T. Taylor, Sequential fractionation of grape seeds into oils, polyphenols and procyanidins via a single system employing CO2 -based fluids, Journal of Agricultural and Food Chemistry 52 (2004) 2440–2444. [10] L. Fiori, D. De Faveri, A. Casazza, P. Perego, Grape by-products: extraction of polyphenolic compounds using supercritical CO2 and liquid organic solvent – a preliminary investigationSubproductos de la uva: extracción de compuestos polifenólicos usando CO2 supercrítico y disolventes orgánicos líquidos, CyTA – Journal of Food 7 (2009) 163–171.
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Extraction and identification of proanthocyanidins from grape seed (Vitis Vinifera) using supercritical carbon dioxide Esen Eyiler Yilmaz ∗ , Emin Burcin Özvural, Halil Vural Hacettepe University, Departement of Food Engineering, Beytepe 06800, Ankara, Turkey
a r t i c l e
i n f o
Article history: Received 7 September 2010 Received in revised form 23 October 2010 Accepted 24 October 2010 Keywords: Supercritical carbon dioxide extraction Proantocyanidins Grape seed High performance liquid chromatography
a b s t r a c t In this study, supercritical carbon dioxide extraction of proantocyanidins (PRCs) was performed and the effect of different pressure, temperature and ethanol percentage was investigated. High performance liquid chromatography was used for the analysis of the compounds and it was found that the most effective parameter on the extraction was the amount of the ethanol percentage. Each compound was extracted from grape seeds at their maximum level when different parameters were used which was probably because of their different polarities. Gallic acid (GA), epigallocatechin (EGC) and epigallocatechingallate (EGCG) were extracted at their maximum level when the 300 bar 50 ◦ C and 20% of ethanol was used. The maximum amount of catechin (CT) and epicatechin (ECT) were obtained when 300 bar 30 ◦ C and 20% of ethanol was used for extraction, and 250 bar, 30 ◦ C and 15% of ethanol was needed to extract highest amount of epicatechingallate (ECG). © 2010 Elsevier B.V. All rights reserved.
1. Introduction Grape seed is a residue of wine and grape juice industry and is generally used as an animal feed. The investigation on grape seed has been increasing since its positive effects was shown on human health. Grape seed contains 8–15% oil and this oil contains high amounts of unsaturated fatty acids and antioxidant-rich compounds [1]. These antioxidants are mostly phenolic compounds like cathecins and procyanidins [2–4]. These phenols may act against the in vitro oxidation of low density lipoproteins [5], and also have anticarcinogenic, antiviral and antimutagenic [6] activities, besides these they prevent the thrombosis, reduces cholesterol and regulates the autonomic nerves [7]. The applications of the grape seed oil have been increasing in pharmaceuticals, medical, cosmetic and food industry due its properties. The extraction of seed oil includes many different stages like cleanup of the biomass, drying, crushing and pressing. By the application of pressing most of the oil can be extracted but nevertheless a considerable amount of oil remains in the cake, and this remaining oil can be extracted by hexane; which should be evaporated after the process [8]. The main drawback of this process is the usage of n-hexane at the last stages [1]. The methods for the extraction of phenolic compounds from grape seed include usage of methanol [3,4,9], ethanol [6,10,11] and acetone [12]. Different extraction temperatures are being used with different times that range from minutes to several hours.
Due to the complexity of the procedure to extract oil and phenolic compounds, newer methods are in consideration. Supercritical fluid extraction (SFE) is one of these methods, extensively studied nowadays because of its unique properties. SFE has many advantages, like absence of light and air, over the conventional extraction procedures. Air and light can cause degradation of both the phenolic compounds and unsaturated fatty acids (UFAs). Among many supercritical fluids carbon dioxide is the one that is mostly used owing to its non-toxicity, cheap and non-flammable properties [1]. Besides these properties CO2 has moderate critical pressure (7.28 MPa) and temperature (304.1 K) which in turn prevents the thermal degradation of phenolic compounds. Supercritical carbon dioxide extraction of grape seed oil has been extensively applied [1,7,8,13], however; for the extraction of phenolic compounds a co-solvent like methanol or ethanol should be used, because carbon dioxide is a non-polar substance and polar substances like phenolic compounds cannot be extracted from grape seeds. In this study, the extraction of phenolic compounds from grape seed using supercritical carbon dioxide with ethanol as a co-solvent was performed. The effect of different temperature, 30 and 50 ◦ C, pressure, 250, 275 and 300 bar, and co-solvent percentages, 5, 10, 15 and 20 wt%, to the extraction amount has been investigated. The amounts of the phenolic compounds extracted were analyzed using RP-HPLC with a gradient program. 2. Material and methods 2.1. Material
∗ Corresponding author. Tel.: +90 3122977100; fax: +90 3122992123. E-mail address: [email protected] (E.E. Yilmaz). 0896-8446/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.supflu.2010.10.046
The carignan type of grape seeds used in the study was kindly given by Kavaklidere Winery. The marc given was dried
5.0 32.6ab 16.5ab 6.0ab 2.6ab 0.0b 7.1 32.6b 18.8b 7.1b 6.2b 0.3c
13.6 51.9 25.1 13.3 15.3 6.8
± ± ± ± ± ±
ab
0.0 0.1ab 0.1a 0.0a 0.6a 0.1b
19.6 83.9 44.1 20.9 24.0 8.5
± ± ± ± ± ±
b
5.9 19.0 5.8 4.8 13.5 7.2 2.6 ± 0.0 9.3 ± 0.3a ND ND 11.3 ± 0.0a 1.1 ± 0.0a
± ± ± ± ± ±
a
GA EGC CT ECT EGCG ECG 8.05 19.03 20.57 27.76 28.44 33.08
a
SC-CO2 was modified with ethanol as a co-solvent and the percentages were 5, 10, 15 and 20% and the temperature was 30 ◦ C. The extraction with SCF was performed twice. Extraction of PRCs from the oil extracted with SFE was performed in 2 parallels. Rt = retention time of the components in HPLC (min), ND = not detected. a–c: Means within the same pressure value and same row with different superscript letters are different (p < 0.05).
29.3 173.1 90.3 43.1 27.3 1.9 15.1 59.6 22.8 14.6 25.9 1.5 9.5 32.8 12.0 9.9 25.2 1.1 0.3 0.6a 0.2a 0.2a 0.3a 0.2a
± ± ± ± ± ± 4.9 12.7 1.3 5.2 13.0 0.9 16.7 66.6 48.6 23.5 10.3 2.5 13.6 ± 2.4 45.0 ± 12.5bc 21.5 ± 7.2a 13.1 ± 3.6ab 23.3 ± 3.3b ND 10.4 ± 1.6 33.2 ± 4.5ab 12.6 ± 2.8a 9.0 ± 1.0a 26.3 ± 4.2b ND 4.5 ± 0.3 9.6 ± 0.1a ND ND 12.2 ± 0.1a ND a
5% 20% 15% 10%
P = 250 bar
5% Compound Rt
mg of component extracted/kg of grape seed
Table 1 The amount of the PRCs (ppm) extracted from the grape seeds at 30 ◦ C, 250, 275 and 300 bar.
P = 275 bar
10%
ab
15%
b
20%
± ± ± ± ± ±
b
4.2 19.4c 17.0b 6.9b 0.6a 0.0a
5%
P = 300 bar
a
10%
± ± ± ± ± ±
0.3 2.6a 0.7a 0.8a 3.4b 0.2a
ab
15%
± ± ± ± ± ±
b
1.1 17.7a 7.3a 2.0a 0.2b 0.3ab
20%
± ± ± ± ± ±
7.0c 55.8b 28.5b 11.8b 3.1b 0.4b
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925
at room temperature and the grape seeds were separated from their peels, after removing the peels the seeds were kept at −18 ◦ C until they were used. The standards used for HPLC analysis were (+)-catechin hydrate (96%), (−)-epicatechin (>97%), (−)-epigallocatechin (from green tea > 95%), (−)-epicatechin gallate (from green tea, 98%), (−)-epigallocatechin gallate (97%) and were obtained from Sigma–Aldrich (Steinheim, Germany). Ethanol which was used as a co-solvent and methanol, used for extraction of PRCs from the grape seed oil, were obtained from Riedel de-Haen (Germany). HPLC grade acetic acid (100%) was from Sigma–Aldrich (Germany) and acetonitrile was from Merck (Darmstadt, Germany). 2.2. Methods 2.2.1. Sample preparations Seeds, which were kept at −18 ◦ C were equilibrated to room temperature and then were crushed with a mixer. The seeds were crushed for 2 min, after every 30 s. The samples were waited for 30 s to prevent seeds from warming up. Crashed seeds were then sieved and the ones that were between 425 and 230 m. 30 g of the sieved seeds were weighed and were put into the extraction vessel. 2.2.2. Supercritical fluid extraction of grape seeds The extraction of antioxidant substances from the seed was performed using and analytical supercritical fluid extractor (SFE-100-2-FMC10, Thar Instruments, PA, USA). The instrument was equipped with automated back pressure regulator, 100 ml extraction vessel, 500 ml collection vessel, six zone temperature controller, high pressure P-50 series pump, cooling systems filled with glycol, and a series III pump for co-solvent (cannot operate over 400 bar). The co-solvent pump was purged before each extraction in order to ensure that co-solvent entered the system (Thar Instruments, Series III Pump, Manuel). The carbon dioxide flow rate was fixed to 5 g/min and was same for all the extraction parameters. Low flow rate was selected in order to ensure that the residence time was longer in the supercritical fluid extraction vessel. Due to the fact that PRCs could not be extracted with CO2 , because of its non-polar nature, the CO2 was modified with ethanol at the level of 5, 10, 15 and 20% (wt%). Ethanol was selected because it is a polar solvent is permitted in food industry. Other than the ethanol percentages 3 different pressures (250, 275 and 300 bar) and 2 different temperatures, 30 and 50 ◦ C were used during the extractions. The length of the extraction was for 1 h. After the extraction the collection vessel was washed with 25 ml of ethanol to clean the surfaces of the vessel, and to minimize the losses that can occur. The grape seed oil, PRCs was collected in a 250 ml volumetric flask and the ethanol was removed with rotary evaporator (Büchi, B465, Switzerland) at 45 ◦ C. After removal of the ethanol the oil containing the PRCs were analyzed with HPLC (Agilent 1100). The extractions with SC-CO2 were performed in two replications. 2.2.3. HPLC analysis of the extracts The samples obtained from the SFE process were analyzed according to the methods of [14,15]. According to those methods 1 ml of the oil in the volumetric flask was transferred to a centrifuge tube and 1 ml of methanol was added and was vortexed for 2 min. The vortexed samples were then centrifuged 10 min at 3000 rpm (Nüve, NF 1215, Istanbul, Turkey) and the supernatant was taken to a test tube. The extraction was repeated twice and the supernatants were mixed and were analyzed with Agilent 1100 RP-HPLC. The experiments were performed in 2 parallels. The analysis in HPLC were performed with a nucleosil C18 HPLC column (250*4.6 mm, Supelco Inc., Bellefonte, PA, USA), at 280 nm and 35 ◦ C using a DAD detector. The flow rate was 1 ml/min and the injection volume was 10 l. Gradient flow was used with 2 mobile phases which are 2%
± ± ± ± ± ± 32.90 218.88 78.83 35.15 47.12 6.48
20%
1.7b 3.9a 2.1b 0.9b 0.4ab 0.1a 16.5 65.6 23.1 12.8 38.3 4.2
± ± ± ± ± ± 15% 10%
8.6 ± 0.6a 26.5 ± 1.8a 5.2 ± 0.8a 6.8 ± 0.1ab 27.5 ± 0.5ab ND
5%
5.6 ± 1.0a 17.9 ± 4.0a 2.4 ± 1.0a 2.8 ± 3.9a 21.6 ± 4.8a ND 17.1 57.1 27.0 15.2 34.4 3.6 16.3 ± 1.0b 53.8 ± 4.9b 18.5 ± 1.9b 11.8 ± 0.6a 38.0 ± 1.6c ND
± ± ± ± ± ±
4.01b 23.6b 10.2b 4.0a 4.8c 0.9a
300 bar
6.9 22.2 3.9 5.9 26.1 0.9 GA EGC CT ECT EGCG ECG 8.05 19.03 20.57 27.76 28.44 33.08
3.6 ± 0.1a 10.2 ± 0.0a ND 4.2 ± 0.1a 12.2 ± 0.2a ND
± ± ± ± ± ±
0.1b 0.3b 0.2a 0.4ab 0.3b 0.1a
11.1 29.8 10.8 7.7 30.0 1.0
± ± ± ± ± ±
0.0c 4.5b 2.2b 1.2b 1.2c 0.0a
15.3 43.4 20.1 12.6 35.8 2.1
± ± ± ± ± ±
1.1d 4.0c 2.3c 0.1c 0.4d 0.8a
4.2 ± 0.2a 13.7 ± 0.7a ND ND 18.3 ± 0.0a ND
7.4 ± 0.3a 23.4 ± 2.0ab 3.3 ± 0.2a ND 26.4 ± 2.4b ND
20% 15% 10% 5%
275 bar
20% 15% 10% 250 bar
5% Compound Rt
mg of component extracted/kg of grape seed
Table 2 The amount of the PRCs (ppm) extracted from the grape seeds at 50 ◦ C, 250, 275 and 300 bar.
SC-CO2 was modified with ethanol as a co-solvent and the percentages was 5, 10, 15 and 20% and the temperature was 50 ◦ C. The extraction with SCF was performed twice. Extraction of PRCs from the oil extracted with SFE was performed in 2 parallels. Rt = retention time of the components in HPLC, ND = not detected. a–d: Means within the same pressure and same row with different superscript letters are different (p < 0.05).
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3.1c 59.2b 12.4c 4.7c 13.8b 2.3a
926
Fig. 1. The chromatogram of PRCs.
acetic acid (in water mobile phase A) and 100% acetonitrile as phase B, the analysis time was 35 min. The gradient program was as follows: 100% A to 98% in 2 min, 96% A in 6 min, 94% A in 4 min, 92% A in 4 min, 91% A in 5 min, 90% A in 2 min, 88% A in 1 min, 87% A in 3 min, 80% A at 2 min, 78% A at 30th min, and isocratic for last 5 min The chromatogram of the PRCs is given in Fig. 1. 2.2.4. Statistical analysis All data analysis was performed using SPSS for Windows® 16. The statistical significances of the differences between means were determined using Duncan’s multiple range test. 3. Results 3.1. Effect of pressure on the extracts The effect of pressure on the extraction of PRCs can be seen at Tables 1–3. Here Tables 1 and 2 represent the effects of different parameters for each of the PRCs, while Table 3 gives the average value of each parameter for all samples with the same pressure. According to the results obtained, the most abundant material found within the examined grape seeds was EGC and the least one was ECG. The amount of the EGC was increased excessively when temperature, pressure and percentage of ethanol was increased. As can be observed from the results increasing the pressure increased the extracted quantity of PRCs, which was also observed by Murga et al. [16]. Berna et al. [17] also observed in their study, that increasing the pressure cause and increase on amount of CT extracted. This result was due to the increased solvent solubility, occurred according to the increased pressure. Chafer et al. [18] reported that the solubility of GA in supercritical CO2 + ethanol (as co-solvent) was increased, as the pressure was elevated. 3.2. Effect of ethanol percentage on the extracts In a study performed by Murga et al. [16] it was stated that; when less than 5% of ethanol was used only GA was able to be extracted, CT, ECG and ECT could not be extracted. Due to this fact we have used 5, 10 15 and 20% in our study. Effect of different ethanol percentages on the extraction of PRCs from grape seed is given in Tables 1 and 2. CT was not able to be extracted from the grape seeds at 5% of ethanol until the pressure was increased to 300bar, which could be effect of increased solvent solubility at higher pressures. A similar case was observed by Berna et al. [17]; they stated that it was not possible to measure the solubility of CT until 110 bar and 5% of ethanol. Increasing the percentage of ethanol increased the amount of the extracted PRCs generally, however, at 250 bar and 30 ◦ C increasing the percentage of ethanol to 20% did not cause an increase at the amounts of PRCs, which in fact lead to a decrease. The reason for that could be that the polarity
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Table 3 Effect of pressure on proanthocyanidin amount. Main factors
Gallic acid (GA)
Epigallocatechin (EGC)
Catechin (CT)
Epicatechin (ECT)
Epigallo catechingallat (EGCG)
Epicatechin gallat (ECG)
Pressure (bar)c 250 275 300
9.8 ± 6.3a 11.3 ± 5.5a 15.3 ± 15.3a
33.7 ± 27.2a 37.8 ± 22.2a 75.9 ± 77.8b
13.8 ± 16.1a 16.4 ± 16.9a 29.5 ± 34.9a
8.7 ± 6.8a 9.1 ± 8.6a 16.3 ± 14.7a
21.0 ± 9.1a 23.6 ± 9.7a 28.2 ± 10.6a
3.4 ± 3.3a 0.9 ± 1.4ab 2.0 ± 2.3b
The extraction with SCF was performed twice. Extraction of PRCs from the oil extracted with SFE was performed in 2 parallels. a and b: Means within the same factor and the same column with different superscript letter are different (p < 0.05). c: Each number represents the average value of each parameter for all samples with the same pressure.
Table 4 The effect of temperature on the extracted amount of PRCs. Gallic acid (GA) Temperature (◦ C)a 30 12.2 ± 1.4 50 12.1 ± 0.4 a
Epigallocatechin (EGC)
Catechin (CT)
Epicatechin (ECT)
Epigallo catechingallat (EGCG)
Epicatechin gallat (ECG)
49.7 ± 10.8 48.6 ± 4.8
23.7 ± 4.9 16.1 ± 1.0
13.1 ± 2.0 9.6 ± 0.1
18.9 ± 1.2 29.6 ± 1.2
2.6 ± 0.04 1.6 ± 0.2
Each number represents the average value of each parameter for all samples with the same temperature.
of the supercritical solvent was not favorable for the extraction of the PRCs. The increased amount of the PRCs due to the increased ethanol percentage was expected because the phenolic compounds can be extracted by a co-solvent like ethanol due to increased polarity [16]. Also Chafer et al. [18] reported that the solubility of GA was increased as the percentage of the ethanol used was increased. ECG showed very different behavior from the other substances, it was not possible to extract when 275 bar and 5, 10 and 15% of ethanol was used. Although the highest amounts were extracted at 250 bar and 30 ◦ C, when the temperature was increased to 50 ◦ C at 250 bar the amount of ECG extracted was lowered which is probably due to the degradation of the compound.
3.3. Effect of temperature on the extracts Proanthocyanidins are unstable at high temperatures and can easily undergo oxidation reactions and lose their antioxidant activity [16]. Besides that Palma and Taylor [6] stated that in the presence of air antioxidants like CT can be degraded in several weeks at 55 ◦ C, on the other hand if the temperature is higher, degradation occurs within a few days. According to this knowledge we did not exceeded 50 ◦ C in our experiments. When the temperature was increased to 50 ◦ C the amount of GA and EGC extracted was not changed very much, however; CT, ECT and ECG amounts were decreased (Table 4). Although Chafer et al. [18] found that the solubility of GA decreases when the extraction temperature was increased; Palma and Taylor [6] reported that higher temperatures are required to overcome analyte–matrix interactions. The decrease in the amount of CT, ECT and ECG due to increased temperature could be according to their sensitivity to temperature. The only substance that has an increased amount was EGCG. This could be because at higher temperatures the analyte–matrix interaction within the seed could be accomplished.
4. Discussion Temperature and pressure defines the density of the SCF which consequently affects the solubility of the solute [19]. The solubility of low volatility substances in supercritical and near critical solvent decreases as the temperature increases due to the decrease in the density and as a result decrease in the solubility at low pressures (up to 200 MPa). When higher pressures are used for extraction, the effect of temperature on density is less pronounced and the vapor pressure becomes the dominating factor [20,21].
The HPLC results showed us that GA, EGC and EGCG were extracted at their maximum level when the parameters were 300 bar 50 ◦ C and 20% of ethanol (Table 2). The maximum amount of CT and ECT were obtained when 300 bar 30 ◦ C and 20% of ethanol was used for extraction (Table 1). It was ECG that had its maximum amount at the lowest pressure which was 250 bar, the temperature was 30 ◦ C and the ethanol percentage was 15%. Since GA, EGC and EGCG have higher melting points they were extracted at a higher temperature than other substances. Also they were extracted at the highest pressure used, which was due to the increased solubility of CO2 due to the increased density at high pressures. Although CT and ECT are isomers, CT has a lower melting point and therefore the amount of extracted CT was higher than that of ECT [22]. Nevertheless their maximum amounts were obtained at the same temperature and pressure. The extraction of ECG at low pressure and temperature could be due to its polarity, and the parameters used were not adequate to overcome the analyte matrix interactions, since it has the lowest amount extracted. Since different PRCs could be extracted at their maximum levels at different parameters, a selective extraction might be applied using different temperature, pressure and percentage of ethanol, in order to obtain the desired component. Polyphenols were able to be extracted from the grape seeds by mixing supercritical CO2 with ethanol at different levels. It would be possible to extract higher molecular weight polyphenols from the matrix by increasing the parameters used. It would not be feasible to increase the temperature higher than 55 ◦ C, which could cause rapid degradation of the antioxidant substances. 5. Conclusion Supercritical fluid extraction is a promising new method for the extraction of the compounds which could be easily degraded. Extraction of polyphenols from grape seeds by solvent extraction has been used for a long time, but those methods require high amount of chemical substances. SC-CO2 extraction provides some advantages like, using less or none of the chemical substances, free of oxygen, can be operated at low temperatures and by varying the parameters like temperature, pressure and/or percentage of ethanol different fractions can be extracted. Acknowledgments We would like to thank Prof. Ismail Hakki Boyaci and research assistant F. Ceyda Dudak for their kind help and support with
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