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Improvement of supercritical CO2 and n-hexane extraction of wild growing pomegranate seed oil by microwave pretreatment
Industrial Crops & Products 104 (2017) 21–27
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Improvement of supercritical CO2 and n-hexane extraction of wild growing pomegranate seed oil by microwave pretreatment
MARK
Sanja Đurđevića, Stoja Milovanovića, Katarina Šavikinb, Mihailo Ristićb, Nebojša Menkovićb, ⁎ Dejan Pljevljakušićb, , Slobodan Petrovića, Aleksandra Bogdanovića a b
University of Belgrade, Faculty of Technology and Metallurgy, Karnegijeva 4, 11000 Belgrade, Serbia Institute for Medicinal Plants Research “Dr Josif Pančić”, Tadeuša Koćuška 1, 11000 Belgrade, Serbia
A R T I C L E I N F O
A B S T R A C T
Keywords: Pomegranate seed oil Microwave pretreatment Supercritical fluid extraction Fatty acid composition Punicic acid
Microwave radiation was suggested as a pretreatment technique to increase the yield of pomegranate seed oil. Seeds were pretreated at 100, 250 and 600 W during 2 and 6 min and then extracted by supercritical carbon dioxide (scCO2) in high pressure unit as well as by n-hexane in Soxhlet apparatus. Even at the lowest microwave pretreatment parameters applied (100 W for 2 min) increased the yield of seed oil obtained by both extraction techniques compared with untreated seeds (from 27.7 to 34.0% and from 21.6 to 25.5% for Soxhlet and scCO2 extractions, respectively). Maximal oil yield in Soxhlet extraction (36.3%) was obtained with microwave radiation of 600 W for 6 min while for scCO2 extraction maximal oil yield (27.2%) was with 250 W for 6 min microwave radiation pretreatment. The qualitative and quantitative composition of fatty acids of the obtained oils was determined by gas chromatography/flame ionization detection and gas chromatography/mass spectrometry. Punicic acid was the most abundant fatty acid in pomegranate seed oil (> 60%). Microwave pretreatment of seeds showed negligible influence on profile and the amount of fatty acids in obtained extracts, compared with its significant influence on extraction yield. Our results recognize microwave pretreatment as a promising technique for intensification of oil extraction from pomegranate seeds.
1. Introduction Punica granatum L., known as pomegranate, is deep red colored fruit, with leathery skin, grenade-shaped and crowned by the pointed calyx. The fruit can be divided into three parts: the seeds (about 3% of the fruit weight), juice (about 30% of the fruit weight) and the peels, that also include the interior network of membranes (Lansky and Newman, 2007). Pomegranate seeds, usually considered as a waste of juice industry, comprise 12–20% of fatty oil (Lansky and Newman, 2007; Al-Maiman and Ahmad, 2002). According to previously published results, pomegranate seed oil (PSO) consists of 65–80% conjugated linolenic acids (CLnAs), among which the most important is punicic acid (Abbasi et al., 2008). Fatty acids are important components of human cell membranes and are known as precursors to many substances in the body. Preventive role of fatty acids in development of cardiovascular diseases and in alleviation of some other health
problem have been reported (Wijendran and Hayes, 2004). Also, they promote the reduction of both, total and HDL cholesterol (Dubois et al., 2007; FAO, 2010). According to their content of phytoestrogens, it is recommended that women in menopause could employ pomegranate seed oil as external and internal phytoestrogen medicaments, as a possible alternative or supplement to conventional hormone replacement therapy (HRT) (Lansky, 1999). Moreover, it has been reported that CLnA can up-regulate the tumor suppressor gene PTPRG, and may have anti-cancer properties (Amarù and Field, 2009). Also, because of low toxicity and confirmed effectiveness in lowering skin irritations, revitalizing dull or mature skin and wrinkles reduction, PSO are usually used in cosmetic industry products. Pomegranate oil inhibits two inflammatory enzymes, cyclooxygenase and lipoxygenase, which may help protect the skin against the age-accelerating threats of ultraviolet light and inflammation, which can help result in younger-looking skin (Ashoori et al., 1994; Schubert et al., 1999).
Abbreviations: ANOVA, analysis of variance; CLnAs, conjugated linolenic acids; FAME, fatty acid methyl esters; FID, flame ionization detection; GC, gas chromatography; HRT, hormone replacement therapy; MS, mass spectrometry; met_ara, methyl arachidate; met_beh, methyl behenate; met_E_11, methyl (E)-11-eicosenoate; met_ela, methyl elaidate; met_lind, methyl linolelaidate; met_lin, methyl linoleate; met_mar, Methyl margarate; met_oct1,2.3,4, Methyl octadecatrienoate isomer 1,2,3,4; met_ole, Methyl oleate; met_pal, methyl palmitate; met_pun, methyl punicate; met_ste, methyl stearate; met_Z_11, methyl (Z)-11-eicosenoate; MUFA, monounsaturated fatty acids; PSO, pomegranate seed oil; PUFA, polyunsaturated fatty acids; SFA, saturated fatty acids; scCO, 22supercritical carbon dioxide; SFE, supercritical fluid extraction ⁎ Corresponding author. E-mail address: [email protected] (D. Pljevljakušić). http://dx.doi.org/10.1016/j.indcrop.2017.04.024 Received 19 December 2016; Received in revised form 27 March 2017; Accepted 14 April 2017 0926-6690/ © 2017 Elsevier B.V. All rights reserved.
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2. Materials and methods
Considering all the beneficial properties of the oil rich in fatty acids, this study focuses on its extraction from pomegranate seed. Different techniques can be used for the extraction of oil from pomegranate seed. Techniques like normal stirring extraction, Soxhlet extraction, ultrasonic baths that employ organic solvents (e.g. n-hexane, petroleum, benzene, and acetone) and supercritical fluid extraction (SFE) have different extraction efficiency with the lowest value reported for SFE (Petrovic et al., 2012). Also, it is possible to obtain oil from seed by using cold pressing method and steam distillation. Cold pressing method provide high-quality oil but, in most cases, the process has a low extraction rate and consumes large amounts of energy. Steam distillation is a very simple process, but suffers of many disadvantages, such as thermal degradation, hydrolysis and solubilization of some compounds in water resulting in change of the flavour and fragrance profile of many oils extracted by this technique. Moreover, Goula (2013) obtained higher pomegranate seed oil yields by ultrasoundassisted extraction (302.3–446.3 g oil/kg seeds), compared to those by conventional extraction methods. SFE presents one of the clean and efficient techniques that have been developed in last decades. The most extensively used solvent in SFE process is supercritical carbon dioxide (scCO2) because of its low critical pressure (7.38 MPa) and temperature (31.1 °C), low cost and availability. It can be easily removed from final product without any residues making it suitable for use in medicine, food and pharmaceutical industry (Ivanovic et al., 2014). scCO2 is known for its high diffusion ability in organic matter and it is good solvent for many valuable compounds. It is predominantly used for extraction of non-polar components from plant material matrix such as fatty acids, sterols, terpenes without use of co-solvents (He et al., 2012). Many reports describe pretreatment of plant material with different techniques (microwave, rapid gas decompression, milling, etc.) in order to intensify extraction of valuable plant constitutes (Meyer et al., 2012; Sayyar et al., 2011). Microwave pretreatment has been reported as an effective method for increasing the oil yield from seeds (Ramesh et al., 1995). The use of microwave radiation offers reduced processing times and energy savings because the energy is delivered directly to materials through molecular interaction with the electromagnetic field resulting to heat generation throughout the material. Microwave radiation also allows rapid and uniform heating of relatively thick materials (Uquiche et al., 2008). Singh and Heldman (2001) reported that microwaves use radio waves to convey energy and convert it to heat at a frequency between 300 MHz and 300 GHz. In this frequency range, waves are mostly absorbed by water with a sufficiently polar oxygen group. Although, seeds are dried, they still contain traces of moisture that serves as the target for microwave heating. The moisture when heated up inside the materials due to microwave effect, evaporates and generates tremendous pressure on the seed cell membrane. The pressure pushes the cell membrane from inside, stretching and ultimately rupturing it, which facilitates leaching out of the active constituents from the pores (Wang and Weller, 2006). As a result, penetration of solvent into the seed cells as well as release of oil from inside of the seeds gets facilitated (Uquiche et al., 2008). To the best of our knowledge, there is no information in the available literature on the application of microwave radiation in a treatment of pomegranate seed prior to the oil extraction process. The objective of this work was to evaluate the effects of microwave pretreatment on the Soxhlet as well as supercritical fluid extraction efficiency of PSO from wild growing pomegranate and to compare qualitative and quantitative composition of fatty acids in obtained oil bearing in mind the potential use of the oil in the treatment of cardiovascular disease, certain types of cancers, and type II diabetes mellitus.
2.1. Plant material Wild growing pomegranate fruit was collected in Bosnia and Herzegovina in the village Do during November 2014 from a natural locality (GPS coordinates: 43.086°N 18.140°E, altitude 498 m). Pomegranate fruit skin and other impurities were previously separated from the seeds. Seeds were then washed with distilled water and air-dried at ambient temperature (4–6 days). Cleaned and dried seeds were ground with a high-speed mill (MMB 1000/05, Bosch). Ground seeds were fractionated by a set of sieves with mesh widths of 0.2, 0.5, and 1 mm to obtain the uniform particle size distribution. Fine powder ensure larger surface area, which provides better contact between the seed and the solvent, and which can enhance the extraction. Also, finer particles will allow improved or much deeper penetration of the microwave. Moisture content was determined by drying of the seed samples (8 g) at 105 ± 0.5 °C for 5 h. Determined moisture values were 2.58 and 5.01 wt.% for ground and whole (nonground) pomegranate seeds, respectively. 2.2. Microwave pretreatment Ground pomegranate seeds were placed in a single layer on a Pyrex petri dish (9 cm diameter), in the middle of the turntable plate of a microwave oven (NN-GD 469 M, Panasonic). Throughout microwave radiation, the sample rotates inside of the oven. This configuration allowed the samples to move through the equable electromagnetic field pattern formed inside the oven, allowing uniform energy absorption in the seeds. Samples were treated at a frequency of 2450 MHz for two times of radiation (2 and 6 min) and three levels of power (100, 250 and 600 W). The temperature of microwave heating varied from 63 to 136 °C. Microwave pretreated samples were subsequently placed in an apparatus for the extraction and extracted. Ground pomegranate seed sample without microwave radiation was used as a control. 2.3. Extraction procedures 2.3.1. Soxhlet extraction Approximately 20 g of ground pomegranate seeds with and without microwave radiation treatment were extracted with 250 mL n-hexane for 8 h by Soxhlet extraction. After extraction was completed, n-hexane was evaporated at 30 °C under reduced pressure using a rotary evaporator (Laboxact SEM842, KNF, UK). The quantity of obtained extracted oil was expressed in percentage, which is defined as mass of oil extracted over mass of the sample (pomegranate seeds) used for extraction. The obtained extracts were collected in vials and stored at −20 °C until further analysis. 2.3.2. Supercritical fluid extraction Supercritical fluid extraction (SFE) was performed in the high pressure unit (HPEA 500, Eurotechnica, Germany) previously described elsewhere (Ivanović et al., 2014) which was modified according to requirements of the process (Fig. 1). Sample of 10 g of ground pomegranate seed with and without microwave radiation treatment was filled into the stainless steel basket with a perforated bottom and top and then placed in stainless steel extraction vessel. Liquid CO2 supplied from CO2 cylinder (commercial CO2, 99% purity, Messer Tehnogas, Belgrade, Serbia) with a siphon tube was cooled in cryostat between the cylinder outlet and the pump to prevent CO2 vaporization. After the system was heated, the CO2 was pumped by a liquid metering pump (Milton Roy, France) until the required pressure is obtained. Than valve V-3 is opened and the extraction started. Depending on the specific flow mode, system pressure was maintained using a back-pressure valve (BPR). Applied operating pressure and temperature (47 °C and 37.9 MPa) were pre22
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Fig. 1. HPEA unit for supercritical extraction of pomegranate seed oil.
2.64.), compared with those from available literature (Adams, 2007), and used as additional tool to approve MS findings.
viously reported as optimum for obtaining high oil extraction yield (Liu et al., 2009). The extraction process lasted until the exhaustion of plant material (round 8.3 h). The flow rate of scCO2 (density of scCO2, 630 kg/m3) during the extraction process was 0.3 kg/h (calculated as the ratio of the mass of CO2 used for the extraction and extraction time). The obtained extracts were collected in vials and stored at −20 °C until further analysis.
2.5. Statistical analysis All analyses were performed at least three times with three independent samples and results in tables and figures are presented as mean ± standard deviation. Differences among treatments and control regarding the oil yield and PUFA content, as well as their fatty-acids content, were estimated through one-way ANOVA followed by post-hoc Duncan’s multiple range tests. Differences between mean values of individual oil yield and PUFA content for each extraction method were determined by Student’s t-test. All statistical analyses and output graphics were done using the software package STATISTICA v.7.0.
2.4. Analysis of fatty acids 2.4.1. Gas chromatography/flame ionization detection Prior to analysis, fatty acid methyl esters (FAME) were prepared according to the International Association of Official Analytical Communities (AOAC) (Official Surplus Method 965.4). Gas chromatography (GC/FID) analysis of FAME extracts isolated from pomegranate seeds was carried out on an gas chromatograph (Agilent Technologies, 7890A), equipped with split-less injector and automatic liquid sampler (ALS), attached to HP-5MS column (30 m · 0.25 mm, 0.25 μm film thickness) and fitted to flame ionization detector (FID). Carrier gas flow rate (H2) was 1 mL/min, injector temperature was 250 °C, detector temperature 300 °C, while column temperature was linearly programmed from 40 to 260 °C (at the rate of 4 °C/min), and held isothermally at 260 °C next 15 min. Solutions of tested samples in nhexane (15 μL/mL) were consecutively injected by ALS (2 μL, split mode 1:30). Area percent reports, obtained as result of standard processing of chromatograms, were used as base for the quantification purposes.
3. Results and discussion 3.1. Effect of microwave treatment on PSO yield Effect of microwave pretreatment on yield of PSO was determined through two different extraction techniques using n-hexane and scCO2. Results of extraction yields were compared with extraction yield of untreated pomegranate seed as a control sample. Yields of PSO obtained by extraction with n-hexane and supercritical fluid extraction with or without microwave pretreatment are presented in Table 1. The microwave pretreatment applied prior to extraction process influenced significantly PSO yield for both extraction techniques resulting in its increase. Microwave pretreatment prior to Soxhlet extraction contributed in increase in oil yield from 27.7% (for non-treated seeds) to 34.0–36.3% (Table 1.). These findings are in agreement with the previously published results for microwave pretreatment on extraction for some other plant material (Moreno et al., 2003; Li et al., 2004; Chemat et al., 2005; Duvernay et al., 2005; Cravotto et al., 2008; Uquiche et al., 2008; Azadmard-Damirchi et al., 2010). Moreno et al. (2003) used microwave pretreatment of 859 W during 11 min for the oil extraction from avocado pulp with n-hexane in Soxhlet apparatus and found that extraction efficiency increased from 54% to 97% when microwave pretreatment was used. Moreover, increase in microwave radiation power from 100 to 600 W in our study also contributed to slight
2.4.2. Gas chromatography/mass spectrometry The same chromatographic conditions as those mentioned for GC/ FID were employed for gas chromatography/mass spectrometry (GC/ MS analysis), using HP G 1800C Series II GCD system (Hewlett-Packard, Palo Alto, CA, USA). Instead of hydrogen, helium was used as carrier gas. Transfer line was heated at 260 °C. Mass spectra were acquired in EI mode (70 eV), in the range of 40–450 Da. Sample solutions were injected by ALS (2 μL, split mode 1:30). The constituents were identified by comparison of their mass spectra to those from Wiley275 and NIST/NBS libraries, using different search engines (PBM and NIST). In addition, the experimental values for retention indices were determined by the use of calibrated Automated Mass Spectral Deconvolution and Identification System software (AMDIS ver. 23
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Table 1 Estimated PSO yield for all observed treatments. #
1 2 3 4 5 6 7
Microwave treatment
n-hexane
SCCO2
Power [W]
Time [min]
Oil yield [%]*
Oil yield [%]*
Control 100 100 250 250 600 600
2 6 2 6 2 6
27.73 33.97 34.28 34.42 34.86 35.36 36.34
± ± ± ± ± ± ±
1.18 1.51 1.30 0.88 1.02 0.97 0.96
b a a a a a a
21.62 25.52 24.00 25.87 27.24 23.91 25.21
± ± ± ± ± ± ±
0.58 0.46 0.73 1.04 0.82 0.97 0.66
d b c b a c bc
a–d Means followed by different letters differ significantly, based on Duncan’s test at P < 0.05. * PSO yield is given as mean ± standard deviation (n = 3), expressed as% (w/w) on the dry-weight basis.
increase of extraction yield from 34.0 to 35.4% and from 34.3 to 36.3% for exposure time of 2 and 6 min, respectively. Although, pomegranate seed treated for 6 min at power of 600 W yielded more oil than that treated for 2 min at 100 W (36.3% compared with 34.0%, respectively), the oil obtained was black in color according to Pantone color scale. Similar results were obtained by Sayyar et al. (2011) for microwave treatment of jatropha seeds prior to extraction with n-hexane. They reported increase in extraction yield from 47.33% to 49.36% when microwave pretreatment time increased from 2 to 4 min. But with further increase in microwave pretreatment time to 6 min, they produced bunt/roasted seeds and reduction in extraction yield to 38.6%. Therefore it can be concluded that change in oil color from brown to black is probable the consequence of decomposition of the seed tissue during increased and prolonged microwave radiation which further results in extraction yield increase. Prolongation of microwave pretreatment increases cracking of the plant cells due to the built-up pressure caused by heating from inside. Cracked cells facilitate release of oil as well as easier penetration of extraction solvent into cells (Sayyar et al., 2011). Prolonged microwave radiation also leads to drying of plant material followed by plant material destruction when the heat generated is too high (Chow and Ma, 2007; Cheng et al., 2011). Microwave pretreatment prior to scCO2 extraction contributed to increase in PSO yield from 21.6% (for non-treated seeds) to 23.9–27.2% at tested conditions (Table 1) revealing positive influence of applied microwave pretreatment. The highest extraction PSO yield that Liu et al. (2009) obtained was 14.0% for scCO2 extraction at the pressure of 40 MPa and temperature of 60 °C. Unlike Soxhlet extraction, where increase in oil yield was perceived with increase in power of microwave treatment to 600 W and time to 6 min, in case of scCO2 extraction the highest PSO yield of 27.2%, was achieved for microwave pretreatment of 250 W during 6 min. Also, increased and prolonged microwave radiation did not affect color of the extracted oil (it remained yellow for all pretreatment conditions). Comparing PSO yield of untreated seeds with microwave pretreated seeds for Soxhelt and scCO2 extractions (Table 1), the positive effect of microwave pretreatment is evident (extraction yields increased up to 31% and up to 26% for Soxhlet and scCO2 extraction, respectively). Lower PSO yield of untreated seeds can be explained by the existence of undamaged cell which present resistance to oil extraction (Uquiche et al., 2008; Aguilera and Stanley, 1999). These cells can be raptured by microwave radiation due to vaporization of the water from the plant material microstructure and which increases pressure in its interior. Raptured cells enable for the oil to diffuse through the cell walls resulting in a higher mass transfer coefficients and extraction yields (Chemat et al., 2005; Uquiche et al., 2008). Also, drying of the plant material due to water vaporization during microwave pretreatment can lead to more brittle plant tissue. This dried tissue can be easily ruptured during extraction process leading to increase oil extraction yield (Uquiche et al., 2008). On the other hand, Wroniak et al. (2016)
Fig. 2. Average PSO yield obtained by scCO2 and n-hexane extraction after microwave pretreatment (n = 21), ** - denote statistically significant difference at P < 0.01 level.
reported prolonged exposition to microwave irradiation could decrease seed moisture content which directly influenced the amount of the extracted from the seeds. The extraction with n-hexane gave the higher average yield of PSO compared with scCO2 extraction for non-treated seeds (27.7% compared to 21.62%) and seeds treated with microwaves (36.3% compared to 27.2%), as it is shown in Table 1 and on Fig. 2. These results could be attributed to selectivity of used solvents. Namely, scCO2 is highly selective solvent while n-hexane is non-selective and causes the simultaneous extraction of non-volatile pigments and waxes. Solvent behavior of n-hexane increases extraction yield compared to scCO2 but also can lead to change in extract color. Also, according to Porto et al. (2016) the Hildebrand solubility parameter (δ) for n-hexane is 14.9 MPa½. Huang et al. (2013), for scCO2 under the experimental conditions 40 °C and 30 MPa, calculated the Hildebrand solubility parameter, whose value is 15.9 MPa ½, when the δ value is estimated by Marcus’s equation (Marcus, 2006; Huang et al., 2013).
3.2. Kinetic of PSO extraction with scCO2 To evaluate the effect of different microwave radiation power and exposure time on PSO extraction in more details, kinetic of SFE was analyzed. Changes in kinetic of pomegranate seeds oil extraction with change in microwave radiation are presented in Fig. 3 (A for 2 min; B for 6 min). Two extraction periods could be perceived: 1) constant increase in amount in extracted oil in the initial stage of the process, known as the washing or the fast extraction period and 2) slower increase in the amount of extracted oil as the extraction progresses, known as slow extract period (Petrovic et al., 2012). A constant increase in rate of oil extraction can be attributed to extraction of easy available oil from cells destroyed by milling and/or microwave pretreatment by simple washing of the plant material. Stage of the decreased rate can be attributed to extraction of oil due to diffusion of scCO2 through parts of plant material with oil fractions harder to access (Petrovic et al., 2012). It is interesting to notice that in first part of extraction the slope of the curve was the highest for non-treated seed, following by decrease in slope for seed treated with 250 W and 600 W, 24
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Fig. 3. Kinetic of oil extraction from pomegranate seeds with scCO2 after microwave pretreatment for A- 2 minutes and B - 6 minutes. Table 2 Content of fatty acids [m/m%] in pomegranate crude seed oil obtained from n-hexane extraction determined by GC/MS analysis. Factor levels solvent
n-hexane
n-hexane
n-hexane
n-hexane
n-hexane
n-hexane
n-hexane
power [W] time [min]
No treatment No treatment
100 2
100 6
250 2
250 6
600 2
600 6
Fatty acid components [%] met_pal SFAa,b met_mar SFA met_lin PUFA met_ole MUFA met_ela MUFA met_ste SFA met_lind PUFA met_pun PUFA met_oct1 PUFA met_oct2 PUFA met_oct3 PUFA met_oct4 SFA met_Z_11 MUFA met_E_11 MUFA met_ara SFA met_beh SFA
2.81 ± 0.09 0.06 ± 0.00 5.79 ± 0.24 6.32 ± 0.25 0.59 ± 0.02 2.57 ± 0.11 0.14 ± 0.01 59.52 ± 2.32 8.59 ± 0.18 1.90 ± 0.08 7.01 ± 0.31 2.42 ± 0.12 0.80 ± 0.02 0.80 ± 0.03 0.56 ± 0.02 0.12 ± 0.00
2.75 ± 0.06 0.05 ± 0.00 5.67 ± 0.22 6.38 ± 0.15 0.59 ± 0.03 2.62 ± 0.11 0.16 ± 0.01 60.28 ± 2.76 8.70 ± 0.28 1.75 ± 0.07 6.66 ± 0.30 2.27 ± 0.07 0.66 ± 0.02 0.78 ± 0.02 0.56 ± 0.01 0.12 ± 0.00
2.74 ± 0.10 0.05 ± 0.00 5.76 ± 0.24 6.50 ± 0.14 0.64 ± 0.01 2.66 ± 0.13 0.18 ± 0.02 54.62 ± 1.24 9.45 ± 0.34 2.99 ± 0.10 9.87 ± 0.46 2.37 ± 0.07 0.73 ± 0.02 0.79 ± 0.02 0.54 ± 0.01 0.12 ± 0.00
2.71 ± 0.08 0.06 ± 0.00 5.79 ± 0.29 6.39 ± 0.26 0.53 ± 0.02 2.59 ± 0.08 0.16 ± 0.01 54.40 ± 1.74 8.96 ± 0.38 3.44 ± 0.16 10.67 ± 0.32 2.27 ± 0.08 0.62 ± 0.03 0.78 ± 0.02 0.51 ± 0.02 0.12 ± 0.00
2.75 ± 0.08 0.05 ± 0.00 5.79 ± 0.22 6.51 ± 0.17 0.60 ± 0.03 2.64 ± 0.05 0.16 ± 0.01 52.92 ± 1.59 10.06 ± 0.36 3.32 ± 0.16 10.77 ± 0.22 2.33 ± 0.11 0.66 ± 0.02 0.81 ± 0.03 0.54 ± 0.01 0.09 ± 0.00
2.73 ± 0.11 0.06 ± 0.00 5.81 ± 0.13 6.36 ± 0.27 0.50 ± 0.02 2.60 ± 0.07 0.17 ± 0.01 54.19 ± 2.15 9.13 ± 0.41 3.40 ± 0.13 10.61 ± 0.52 2.32 ± 0.10 0.70 ± 0.02 0.75 ± 0.02 0.53 ± 0.02 0.14 ± 0.00
2.71 ± 0.06 0.06 ± 0.00 5.78 ± 0.20 6.43 ± 0.22 0.44 ± 0.02 2.58 ± 0.12 0.15 ± 0.01 53.88 ± 1.28 8.79 ± 0.40 3.71 ± 0.11 11.03 ± 0.48 2.32 ± 0.08 0.70 ± 0.03 0.77 ± 0.03 0.53 ± 0.03 0.11 ± 0.00
a b
Abbreviations: SFA – saturated fatty acid, MUFA – monounsaturated fatty acid, PUFA – polyunsaturated fatty acid. Standard deviations reported as value 0.00, were in fact lesser than 0.01.
(SFA) were in range between 5.8 and 6.5% of total fatty acids, monounsaturated fatty acids (MUFA) were in the range from 13.9 to 16.1% and polyunsaturated fatty acids (PUFA) were the principal fatty acid class and they constitute between 84.3 and 86.1% of total fatty acids. Slightly larger average amount of PUFA was found in sample obtained by supercritical fluid extraction (85.6% of total fatty acids), compared with average amount of PUFA (85.5% of total fatty acid) obtained by extraction with n-hexane, but no statistically significant differences between them were observed (Fig. 4). The predominant fatty acid extracted by n-hexane and scCO2 extraction was punicic acid (approximately 60.0%). The applied microwave pretreatment influenced negligible increase of punicic acid from 59.5% (non-treated seeds) to 60.3% (100 W during 2 min) for nhexane extraction. Microwave pretreatment (250 W during 6 min) prior to scCO2 extraction contributed to a slight increase from 54.1% (for non-treated seeds) to 60.3%. Fadavi et al. (2006) studied fatty acid profile of seed oil from 25 different varieties of pomegranate obtained using Soxhlet extraction. They found that the punicic acid content ranged from 31.8 to 86.6%. In another study, super-heated n-hexane extraction of pomegranate seed oil resulted in 70.73% punicic acid (Eikani et al., 2012). Supercritical CO2 extraction used by Abbasi et al. (2008) resulted in punicic acid content from 69.8% to 79.0%. This amount is very important because punicic acid is the principal CLnA present in pomegranate seeds oil and it is connected with its potential
while seed treated with 100 W had the lowest slope in both cases (Fig. 3). Fiori (2007) reported that increase in the pressure for scCO2 extraction of grape seed oil leads to increase in slope of the curves for first part of the extraction. Increase in yield of first part of the extraction occurred because solubility of grape seed oil in scCO2 changed due to increase in pressure. Therefore, it can be assumed that change in PSO solubility occurred due to change in oil composition (Table 2). Extraction condition were constant but the slope of the curves for first part of the extraction changed. Variation in fatty acids composition of oil could be attributed to pretreatment conditions. Although, microwave pretreatment decreased yield of first part of extraction, it resulted in higher extraction yield for second period of extraction where diffusion of oil through plant material is limiting factor of extraction. 3.3. Effects of microwave pretreatment on fatty acids composition Quantitative and qualitative analysis of the fatty oil from the pomegranate seeds was carried out. Tables 2 and 3 present the fatty acid composition of the pomegranate seed oils obtained by extractions using n-hexane and scCO2. Sixteen fatty acids at different quantities were detected in PSO obtained by n-hexane and scCO2 extraction. Statistically, there were no significant differences between n-hexane and scCO2 obtained samples, suggesting both methods as satisfactory. Total saturated fatty acids 25
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Table 3 Content of fatty acids [m/m%] in pomegranate crude seed oil obtained from
SCCO2
extraction determined by GC/MS analysis.
Factor levels solvent
SCCO2
SCCO2
SCCO2
SCCO2
SCCO2
SCCO2
SCCO2
power [W] time [min]
No treatment No treatment
100 2
100 6
250 2
250 6
600 2
600 6
Fatty acid components [%] met_pal SFAa,b met_mar SFA met_lin PUFA met_ole MUFA met_ela MUFA met_ste SFA met_lind PUFA met_pun PUFA met_oct1 PUFA met_oct2 PUFA met_oct3 PUFA met_oct4 SFA met_Z_11 MUFA met_E_11 MUFA met_ara SFA met_beh SFA
3.20 ± 0.07 0.06 ± 0.00 6.25 ± 0.15 7.32 ± 0.17 0.43 ± 0.02 2.60 ± 0.11 0.15 ± 0.01 54.06 ± 1.12 8.82 ± 0.26 3.07 ± 0.14 9.72 ± 0.36 2.26 ± 0.08 0.74 ± 0.03 0.71 ± 0.02 0.53 ± 0.01 0.10 ± 0.00
2.77 ± 0.11 0.05 ± 0.00 5.79 ± 0.21 6.46 ± 0.23 0.41 ± 0.01 2.53 ± 0.07 0.14 ± 0.01 54.53 ± 1.69 8.85 ± 0.18 3.40 ± 0.07 10.59 ± 0.45 2.33 ± 0.10 0.77 ± 0.03 0.75 ± 0.03 0.50 ± 0.01 0.11 ± 0.00
2.75 ± 0.10 0.08 ± 0.00 5.69 ± 0.22 6.31 ± 0.24 0.38 ± 0.01 2.50 ± 0.07 0.12 ± 0.02 56.04 ± 2.71 8.55 ± 0.41 3.18 ± 0.10 9.95 ± 0.28 2.28 ± 0.05 0.72 ± 0.02 0.80 ± 0.02 0.52 ± 0.02 0.11 ± 0.00
2.83 ± 0.14 0.06 ± 0.00 5.81 ± 0.20 6.45 ± 0.19 0.41 ± 0.01 2.55 ± 0.12 0.13 ± 0.01 52.64 ± 1.33 9.37 ± 0.33 3.83 ± 0.08 11.45 ± 0.57 2.31 ± 0.06 0.69 ± 0.01 0.83 ± 0.04 0.52 ± 0.01 0.12 ± 0.00
2.74 ± 0.07 0.05 ± 0.00 5.65 ± 0.18 6.30 ± 0.24 0.34 ± 0.01 2.48 ± 0.10 0.11 ± 0.01 60.26 ± 2.48 8.50 ± 0.41 2.02 ± 0.05 7.05 ± 0.34 2.26 ± 0.06 0.73 ± 0.02 0.79 ± 0.03 0.52 ± 0.02 0.10 ± 0.00
2.76 ± 0.14 0.05 ± 0.00 5.70 ± 0.27 6.25 ± 0.30 0.33 ± 0.01 2.45 ± 0.10 0.12 ± 0.01 59.69 ± 1.75 8.13 ± 0.35 2.37 ± 0.10 7.84 ± 0.30 2.23 ± 0.10 0.73 ± 0.02 0.74 ± 0.03 0.52 ± 0.02 0.09 ± 0.00
2.79 ± 0.10 0.05 ± 0.00 5.79 ± 0.28 6.30 ± 0.17 0.33 ± 0.01 2.46 ± 0.06 0.11 ± 0.01 59.94 ± 2.46 8.25 ± 0.18 2.17 ± 0.09 7.44 ± 0.24 2.28 ± 0.06 0.65 ± 0.02 0.85 ± 0.04 0.50 ± 0.01 0.10 ± 0.00
a b
Abbreviations: SFA – saturated fatty acid, MUFA – monounsaturated fatty acid, PUFA – polyunsaturated fatty acid. Standard deviations reported as value 0.00, were in fact lesser than 0.01.
Hernandez et al., 2000; Tian et al., 2013). In our study, the levels of linoleic acid in pomegranate oil varied from 5.7 to 6.2%, which was lower than in some other species such as safflower (67.8–83.2%), soybean (48.0–59.0%), sunflower (48.3–74.0%), and flaxseed oils (15.2–15.9%) (Codex Alimentarius Commission, 1999; Choo et al., 2007). Although, there are observed differences between pretreated seeds and control group regarding levels of some fatty acids (i.e. palmitic, linoleic, oleic and stearic acids) statistical analysis has not shown any pattern, which could lead us to conclude any rule of prediction for future cases (Tables 2 and 3). Higher carbon content fatty acids including arachidic acid and behenic acid were also present in our oil samples at the level of trace amount. Arachidic acid content of pomegranate seed oils was determined to be in the range from 0 to 2.8% (Özgül-Yücel, 2005; Melgarejo and Artes, 2000). Nevertheless, these studies did not reported behenic acid in pomegranate seed oil. On the other hand, Fadavi et al. (2006) reported the presence of behenic acid in pomegranate seed oils ranging from 0 to 3.9%. Moreover, in our samples, lignoceric acid was present in trace amount in scCO2 extracted PSO using microwave pretreatment with power of 250 W during 6 min. Although our results correspond with some of the previously published papers, the difference in the content of fatty acids was also noticed. According to Kýralan et al. (2009) oil content could vary depending on genotype, location, harvest time, climatic conditions, etc.
4. Conclusion
Fig. 4. Average PUFA contents in oils obtained by scCO2 and n-hexane extraction (n = 21).
In this study, the effect of microwave pretreatment on the pomegranate seed oil (PSO) yield and its composition using Soxhlet and scCO2 extraction was perceived. Statistically significant difference was noticed in the PSO yield from non-treated and microwave treated seeds (extraction yields increased up to 31% and up to 26% for Soxhlet and scCO2 extraction, respectively after microwave pretreatment). The highest PSO yield by n-hexane extraction in Soxhlet apparatus was 36.3% after microwave pretreatment for 6 min with power of 600 W. The highest oil yield recorded using supercritical fluid extraction was 27.2% after microwave pretreatment at 250 W for 6 min. Oils obtained by scCO2 were yellow colored compared to oils obtained by Soxhlet extraction where color ranged from brown to black. This is probably due to greater extraction selectivity of scCO2. Qualitative and quantitative composition of fatty acids was similar in all samples. Punicic acid
biological and health beneficial effects. As reported by Özgül-Yücel (2005) pomegranate seed oil contained higher amount of CLnA than some well-known CLnA-rich seeds including pot marigold (29.5%), mahaleb (27.6%), and catalpa (27.5%). The second most abundant fatty acid in PSO was oleic (C18:1 C9) acid, which was in the range from 6.2 to 7.3%. According to Garima and Akoh (2009) and Jing et al. (2012), oleic acid was the most prevalent monounsaturated fatty acid in Georgians and Chinese PSO, respectively. On the other hand, in some studies of PSO obtained using different extraction methods (cold press, Soxhlet, superheated n-hexane and scCO2 extraction), linoleic and punicic acids were dominant polyunsaturated fatty acids (Fadavi et al., 2006; Kýralan et al., 2009; 26
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modeling. J. Food Eng. 117, 492–498. He, L., Zhang, X., Xu, H., Xu, C., Yuan, F., Knez, Z., Novak, Z., Gao, Y., 2012. Subcritical water extraction of phenolic compounds from pomegranate (Punica granatum L.) seed residues and investigation into their antioxidant activities with HPLC–ABTS•+ assay. Food Bioprod. Process 90, 215–223. Hernandez, F., Melgarejo, P., Olias, J.M., Artes, F., 2000. Symposium on Production, Processing and Marketing of Pomegranate in the Mediterranean Region: Advances in Research and Technology. CIHEAM‐IAMZ, Zaragoza (Spain). Huang, Z., Li, J.-H., Li, H.-S., Miao, H., Kawi, S., Goh, A., 2013. Effect of the polarmodifiers on supercritical extraction efficiency for template removal from hexagonal mesoporous silica materials: solubility parameter and polarity considerations. Sep. Purif. Technol. 118, 120–126. Ivanovic, J., Milovanovic, S., Stamenic, M., Fanovich, M.A., Jaeger, P., Zizovic, I., 2014. Application of an integrated supercritical extraction and impregnation process for incorporation of thyme extracts into different carriers. In: Jane Osborne (Ed.), Handbook on Supercritical Fluids, Fundaments, Properties and Applications. Nova Science Publishers, New York, pp. 257–280. Jing, P., Ye, T., Shi, H., Sheng, Y., Slavin, M., Gao, B., 2012. Antioxidant properties and phytochemical composition of China-grown pomegranate seeds. Food Chem. 132 (3), 1457–1464. Kýralan, M., Gölükcü, M., Tokgöz, H., 2009. Oil and conjugated linolenic acid contents of seeds from important pomegranate cultivars (Punica granatum L.) grown in Turkey. J. Am. Oil Chem. Soc. 86, 985–990. Lansky, E.P., Newman, R.A., 2007. Punica granatum (pomegranate) and its potential for prevention and treatment of inflammation and cancer. J. Ethnopharmacol. 109, 177–206. Lansky, E.P., 1999. Phytoestrogen supplement prepared from pomegranate seed and a herbal mixture or coconut milk. US Patent 5 (891), 440. Li, H., Chen, B., Nie, N., Yao, S., 2004. Solvent effects on focused microwave assisted extraction of polyphenolic acids from Eucommia Ulmodies. Phytochem. Anal. 15, 306–312. Liu, G., Xu, X., Hao, Q., Gao, Y., 2009. Supercritical CO2 extraction optimization of pomegranate (Punica granatum L.) seed oil using response surface methodology. LWT Food Sci. Technol. 42, 1491–1495. Marcus, Y., 2006. Are solubility parameters relevant to supercritical fluids? J. Supercrit. Fluids 38, 7–12. Melgarejo, P., Artes, F., 2000. Total lipid content and fatty acid composition of oil seed from lesser known sweet pomegranate clones. J. Sci. Food Agric. 80, 1452–1454. Meyer, F., Jaeger, P., Eggers, R., Stamenic, M., Milovanovic, S., Zizovic, I., 2012. Effect of CO2 pre-treatment on scCO2 extraction of natural material. Chem. Eng. Process. Process Intensif. 56, 37–45. Moreno, A.O., Dorantes, L., Galindez, J., Guzman, R.I., 2003. Effect of different extraction methods on fatty acids volatile compounds, and physical and chemical properties of avocado (Persea americana mill.) oil. J. Agric. Food. Chem. 51, 2216–2221. Official Surplus Method 965.4 Fatty Acids in Oils and Fats, Preparation of Methyl Esters, Final Action 1984, Surplus 1965. Petrovic, S.S., Ivanovic, J., Milovanovic, S., Zizovic, I., 2012. Comparative analyses of the diffusion coefficients from thyme for different extraction processes. J. Serb. Chem. Soc. 77 (6), 799–813. Porto, C., Decorti, D., Natolino, A., 2016. Microwave pretreatment of Moringa oleifera seed: effect on oil obtained by pilot-scale supercritical carbon dioxide extraction and Soxhlet apparatus. J. Supercrit. Fluids 107, 38–43. Ramesh, M., Rao, P.H., Ramadoss, C.S., 1995. Microwave treatment of ground nut (Arachis hypogaea): extractability and quality of oil and its relation to lipase and lipoxygenase activity. LWT Food Sci. Technol. 28, 96–99. Sayyar, S., Abidin, Z.Z., Yunus, R., Muhammad, A., 2011. Solid liquid extraction of jatropha seeds by microwave pretreatment and ultrasound assisted methods. J. Appl. Sci. 11 (13), 2444–2447. Schubert, S.Y., Lansky, E.P., Neeman, I., 1999. Antioxidant and eicosanoid enzyme inhibition properties of Pomegranate seed oil and fermented juice flavonoids. J. Ethnopharmacol. 66 (1), 11–17. Singh, R.P., Heldman, D.R., 2001. Introduction to Food Process Engineering, third ed. Academic Presspp. 30–47. Tian, Y., Xu, Z., Zheng, B., Lo, Y.M., 2013. Optimization of ultrasonic-assisted extraction of pomegranate (Punica granatum L.) seed oil. Ultrason. Sonochem. 20, 202–208. Uquiche, E., Jerez, M., Ortiz, J., 2008. Effect of pretreatment with microwaves on mechanical extraction yield and quality of vegetable oil from Chilean hazelnuts (Gevuina avellana Mol.). Innov. Food Sci. Emerg. Technol. 9, 495–500. Wang, L., Weller, C.L., 2006. Recent advances in extraction of nutraceuticals from plants. Trends Food Sci. Technol. 17, 300–312. Wijendran, V., Hayes, K.C., 2004. Dietary n-6 and n-3 fatty acid balance and cardiovascular health. Annu. Rev. Nutr. 24, 597–615. Wroniak, M., Rekas, A., Siger, A., Janowicz, M., 2016. Microwave pretreatment effects on the changes in seeds microstructure, chemical composition and oxidative stability of rapeseed oil. LWT Food Sci. Technol. 68, 634–641.
was the major fatty acid found in all samples. This study showed that microwave treatment of pomegranate seeds prior to extraction increases oil yield without negative effect on fatty acid composition which is especially significant bearing in mind potential application of pomegranate seed oil in treatment of cardiovascular and skin diseases, type II diabetes mellitus, and cancer. Acknowledgments Financial support of this work from the Ministry of Education, Science and Technological Development of the Republic of Serbia (Project III 46013 and III 45017) is gratefully acknowledged. Authors also thanks Mr. Sreten Škrba who donated pomegranate fruits from his property. References Özgül-Yücel, S., 2005. Determination of conjugated linolenic acid content of selected oil seeds grown in Turkey. J. Am. Oil Chem. Soc. 82 (12), 893–897. Automated Mass Spectral Deconvolution and Identification System software (AMDIS ver. 2.64.), National Institute of Standards and Technology (NIST), Standard Reference Data Program, Gaithersburg, MD (USA). Abbasi, H., Rezaei, K., Rashidi, L., 2008. Extraction of essential oils form the seeds of pomegranate using organic solvents and supercritical CO2. J. Am. Oil Chem. Soc. 85, 83–89. Adams, R.P., 2007. Identification of Essential Oil Components by Gas Chromatography/ Mass Spectrometry, fourth ed. Allured Publishing Corporation, Carol Stream, Illinois, USA. Aguilera, J.M., Stanley, D.W., 1999. Microstructural Principles of Food Processing and Engineering, second ed. Aspen Publishers, Inc., Gaithersburg, MD, pp. 352–372. Al-Maiman, S.A., Ahmad, D., 2002. Changes in physical and chemical properties during pomegranate (Punica granatum L.) fruit maturation. Food Chem. 76, 437–441. Amarù, D., Field, C., 2009. Conjugated linoleic acid decreases MCF-7 human breast cancer cell growth and insulin-like growth factor-1 receptor levels. Lipids 44 (5), 449–458. Ashoori, F., Suzuki, S., Zhou, J.H., Isshiki, N., Miyachi, Y., 1994. Involvement of lipid peroxidation in necrosis of skin flaps and its suppression by ellagic acid. Plast. Reconstr. Surg. 94 (7), 1027–1037. Azadmard-Damirchi, S., Habibi-Nodeh, F., Hesari, J., Nemati, M., Fathi Achachlouei, B., 2010. Effect of pretreatment with microwaves on oxidative stability and nutraceuticals content of oil from rapeseed. Food Chem. 121, 1211–1215. Chemat, S., Ait-Amar, H., Lagha, A., Esveld, D.C., 2005. Microwave-assisted extraction kinetics of terpenes from caraway seeds. Chem. Eng. Process. 44, 1320–1326. Cheng, S.F., Mohd Nor, L., Chuah, C.H., 2011. Microwave pretreatment: a clean and dry method for palm oil production. Ind. Crops Prod. 34, 967–971. Choo, W.S., Birch, J., Dufour, J.P., 2007. Physicochemical and quality characteristics of cold-pressed flaxseed oils. J. Food Compos. Anal. 20, 202–211. Chow, M.C., Ma, A.N., 2007. Processing of fresh palm fruits using microwaves. J. Microw. Power Electromagn. Energy 40 (3), 165–173. Codex Alimentarius Commission, 1999. Codex stan 19. Edible Fats and Oils Not Covered by Individual Standards. Cravotto, G., Boffa, L., Mantegna, S., Perego, P., Avogadro, M., Cintas, P., 2008. Improved extraction of vegetable oils under high-intensity ultrasound and/or microwaves. Ultrason. Sonochem. 15, 898–902. Dubois, V., Breton, S., Linder, M., Fanni, J., Parmentier, M., 2007. Fatty acid profiles of 80 vegetable oils with regard to their nutritional potential. Eur. J. Lipid Sci. Technol. 109, 710–732. Duvernay, W.H., Assad, J.M., Sabliov, C.M., Lima, M., Xu, Z., 2005. Microwave extraction of antioxidant components from rice bran. Pharm. Eng. 25, 1–5. Eikani, M.H., Golmohammad, F., Homami, S.S., 2012. Extraction of pomegranate (Punica granatum L.) seed oil using superheated hexane. Food Bioprod. Process. 90, 32–36. FAO/WHO, 2010. Fats and Fatty Acids in Human Nutrition. Report of an Expert Consultation. FAO/WHO, Geneva, Switzerland. Fadavi, A., Barzegar, M., Azizi, M.H., 2006. Determination of fatty acids and total lipid content in oilseed of 25 pomegranates varieties grown in Iran. J. Food Compos. Anal. 19, 676–680. Fiori, L., 2007. Grape seed oil supercritical extraction kinetic and solubility data: critical approach and modeling. J. Supercrit. Fluids 43, 43–54. Garima, P., Akoh, C.C., 2009. Antioxidant capacity and lipid characterization of six Georgia-grown pomegranate cultivars. J. Agric. Food Chem. 57 (20), 9427–9436. Goula, A.M., 2013. Ultrasound-assisted extraction of pomegranate seed oil–Kinetic
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Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop
Improvement of supercritical CO2 and n-hexane extraction of wild growing pomegranate seed oil by microwave pretreatment
MARK
Sanja Đurđevića, Stoja Milovanovića, Katarina Šavikinb, Mihailo Ristićb, Nebojša Menkovićb, ⁎ Dejan Pljevljakušićb, , Slobodan Petrovića, Aleksandra Bogdanovića a b
University of Belgrade, Faculty of Technology and Metallurgy, Karnegijeva 4, 11000 Belgrade, Serbia Institute for Medicinal Plants Research “Dr Josif Pančić”, Tadeuša Koćuška 1, 11000 Belgrade, Serbia
A R T I C L E I N F O
A B S T R A C T
Keywords: Pomegranate seed oil Microwave pretreatment Supercritical fluid extraction Fatty acid composition Punicic acid
Microwave radiation was suggested as a pretreatment technique to increase the yield of pomegranate seed oil. Seeds were pretreated at 100, 250 and 600 W during 2 and 6 min and then extracted by supercritical carbon dioxide (scCO2) in high pressure unit as well as by n-hexane in Soxhlet apparatus. Even at the lowest microwave pretreatment parameters applied (100 W for 2 min) increased the yield of seed oil obtained by both extraction techniques compared with untreated seeds (from 27.7 to 34.0% and from 21.6 to 25.5% for Soxhlet and scCO2 extractions, respectively). Maximal oil yield in Soxhlet extraction (36.3%) was obtained with microwave radiation of 600 W for 6 min while for scCO2 extraction maximal oil yield (27.2%) was with 250 W for 6 min microwave radiation pretreatment. The qualitative and quantitative composition of fatty acids of the obtained oils was determined by gas chromatography/flame ionization detection and gas chromatography/mass spectrometry. Punicic acid was the most abundant fatty acid in pomegranate seed oil (> 60%). Microwave pretreatment of seeds showed negligible influence on profile and the amount of fatty acids in obtained extracts, compared with its significant influence on extraction yield. Our results recognize microwave pretreatment as a promising technique for intensification of oil extraction from pomegranate seeds.
1. Introduction Punica granatum L., known as pomegranate, is deep red colored fruit, with leathery skin, grenade-shaped and crowned by the pointed calyx. The fruit can be divided into three parts: the seeds (about 3% of the fruit weight), juice (about 30% of the fruit weight) and the peels, that also include the interior network of membranes (Lansky and Newman, 2007). Pomegranate seeds, usually considered as a waste of juice industry, comprise 12–20% of fatty oil (Lansky and Newman, 2007; Al-Maiman and Ahmad, 2002). According to previously published results, pomegranate seed oil (PSO) consists of 65–80% conjugated linolenic acids (CLnAs), among which the most important is punicic acid (Abbasi et al., 2008). Fatty acids are important components of human cell membranes and are known as precursors to many substances in the body. Preventive role of fatty acids in development of cardiovascular diseases and in alleviation of some other health
problem have been reported (Wijendran and Hayes, 2004). Also, they promote the reduction of both, total and HDL cholesterol (Dubois et al., 2007; FAO, 2010). According to their content of phytoestrogens, it is recommended that women in menopause could employ pomegranate seed oil as external and internal phytoestrogen medicaments, as a possible alternative or supplement to conventional hormone replacement therapy (HRT) (Lansky, 1999). Moreover, it has been reported that CLnA can up-regulate the tumor suppressor gene PTPRG, and may have anti-cancer properties (Amarù and Field, 2009). Also, because of low toxicity and confirmed effectiveness in lowering skin irritations, revitalizing dull or mature skin and wrinkles reduction, PSO are usually used in cosmetic industry products. Pomegranate oil inhibits two inflammatory enzymes, cyclooxygenase and lipoxygenase, which may help protect the skin against the age-accelerating threats of ultraviolet light and inflammation, which can help result in younger-looking skin (Ashoori et al., 1994; Schubert et al., 1999).
Abbreviations: ANOVA, analysis of variance; CLnAs, conjugated linolenic acids; FAME, fatty acid methyl esters; FID, flame ionization detection; GC, gas chromatography; HRT, hormone replacement therapy; MS, mass spectrometry; met_ara, methyl arachidate; met_beh, methyl behenate; met_E_11, methyl (E)-11-eicosenoate; met_ela, methyl elaidate; met_lind, methyl linolelaidate; met_lin, methyl linoleate; met_mar, Methyl margarate; met_oct1,2.3,4, Methyl octadecatrienoate isomer 1,2,3,4; met_ole, Methyl oleate; met_pal, methyl palmitate; met_pun, methyl punicate; met_ste, methyl stearate; met_Z_11, methyl (Z)-11-eicosenoate; MUFA, monounsaturated fatty acids; PSO, pomegranate seed oil; PUFA, polyunsaturated fatty acids; SFA, saturated fatty acids; scCO, 22supercritical carbon dioxide; SFE, supercritical fluid extraction ⁎ Corresponding author. E-mail address: [email protected] (D. Pljevljakušić). http://dx.doi.org/10.1016/j.indcrop.2017.04.024 Received 19 December 2016; Received in revised form 27 March 2017; Accepted 14 April 2017 0926-6690/ © 2017 Elsevier B.V. All rights reserved.
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2. Materials and methods
Considering all the beneficial properties of the oil rich in fatty acids, this study focuses on its extraction from pomegranate seed. Different techniques can be used for the extraction of oil from pomegranate seed. Techniques like normal stirring extraction, Soxhlet extraction, ultrasonic baths that employ organic solvents (e.g. n-hexane, petroleum, benzene, and acetone) and supercritical fluid extraction (SFE) have different extraction efficiency with the lowest value reported for SFE (Petrovic et al., 2012). Also, it is possible to obtain oil from seed by using cold pressing method and steam distillation. Cold pressing method provide high-quality oil but, in most cases, the process has a low extraction rate and consumes large amounts of energy. Steam distillation is a very simple process, but suffers of many disadvantages, such as thermal degradation, hydrolysis and solubilization of some compounds in water resulting in change of the flavour and fragrance profile of many oils extracted by this technique. Moreover, Goula (2013) obtained higher pomegranate seed oil yields by ultrasoundassisted extraction (302.3–446.3 g oil/kg seeds), compared to those by conventional extraction methods. SFE presents one of the clean and efficient techniques that have been developed in last decades. The most extensively used solvent in SFE process is supercritical carbon dioxide (scCO2) because of its low critical pressure (7.38 MPa) and temperature (31.1 °C), low cost and availability. It can be easily removed from final product without any residues making it suitable for use in medicine, food and pharmaceutical industry (Ivanovic et al., 2014). scCO2 is known for its high diffusion ability in organic matter and it is good solvent for many valuable compounds. It is predominantly used for extraction of non-polar components from plant material matrix such as fatty acids, sterols, terpenes without use of co-solvents (He et al., 2012). Many reports describe pretreatment of plant material with different techniques (microwave, rapid gas decompression, milling, etc.) in order to intensify extraction of valuable plant constitutes (Meyer et al., 2012; Sayyar et al., 2011). Microwave pretreatment has been reported as an effective method for increasing the oil yield from seeds (Ramesh et al., 1995). The use of microwave radiation offers reduced processing times and energy savings because the energy is delivered directly to materials through molecular interaction with the electromagnetic field resulting to heat generation throughout the material. Microwave radiation also allows rapid and uniform heating of relatively thick materials (Uquiche et al., 2008). Singh and Heldman (2001) reported that microwaves use radio waves to convey energy and convert it to heat at a frequency between 300 MHz and 300 GHz. In this frequency range, waves are mostly absorbed by water with a sufficiently polar oxygen group. Although, seeds are dried, they still contain traces of moisture that serves as the target for microwave heating. The moisture when heated up inside the materials due to microwave effect, evaporates and generates tremendous pressure on the seed cell membrane. The pressure pushes the cell membrane from inside, stretching and ultimately rupturing it, which facilitates leaching out of the active constituents from the pores (Wang and Weller, 2006). As a result, penetration of solvent into the seed cells as well as release of oil from inside of the seeds gets facilitated (Uquiche et al., 2008). To the best of our knowledge, there is no information in the available literature on the application of microwave radiation in a treatment of pomegranate seed prior to the oil extraction process. The objective of this work was to evaluate the effects of microwave pretreatment on the Soxhlet as well as supercritical fluid extraction efficiency of PSO from wild growing pomegranate and to compare qualitative and quantitative composition of fatty acids in obtained oil bearing in mind the potential use of the oil in the treatment of cardiovascular disease, certain types of cancers, and type II diabetes mellitus.
2.1. Plant material Wild growing pomegranate fruit was collected in Bosnia and Herzegovina in the village Do during November 2014 from a natural locality (GPS coordinates: 43.086°N 18.140°E, altitude 498 m). Pomegranate fruit skin and other impurities were previously separated from the seeds. Seeds were then washed with distilled water and air-dried at ambient temperature (4–6 days). Cleaned and dried seeds were ground with a high-speed mill (MMB 1000/05, Bosch). Ground seeds were fractionated by a set of sieves with mesh widths of 0.2, 0.5, and 1 mm to obtain the uniform particle size distribution. Fine powder ensure larger surface area, which provides better contact between the seed and the solvent, and which can enhance the extraction. Also, finer particles will allow improved or much deeper penetration of the microwave. Moisture content was determined by drying of the seed samples (8 g) at 105 ± 0.5 °C for 5 h. Determined moisture values were 2.58 and 5.01 wt.% for ground and whole (nonground) pomegranate seeds, respectively. 2.2. Microwave pretreatment Ground pomegranate seeds were placed in a single layer on a Pyrex petri dish (9 cm diameter), in the middle of the turntable plate of a microwave oven (NN-GD 469 M, Panasonic). Throughout microwave radiation, the sample rotates inside of the oven. This configuration allowed the samples to move through the equable electromagnetic field pattern formed inside the oven, allowing uniform energy absorption in the seeds. Samples were treated at a frequency of 2450 MHz for two times of radiation (2 and 6 min) and three levels of power (100, 250 and 600 W). The temperature of microwave heating varied from 63 to 136 °C. Microwave pretreated samples were subsequently placed in an apparatus for the extraction and extracted. Ground pomegranate seed sample without microwave radiation was used as a control. 2.3. Extraction procedures 2.3.1. Soxhlet extraction Approximately 20 g of ground pomegranate seeds with and without microwave radiation treatment were extracted with 250 mL n-hexane for 8 h by Soxhlet extraction. After extraction was completed, n-hexane was evaporated at 30 °C under reduced pressure using a rotary evaporator (Laboxact SEM842, KNF, UK). The quantity of obtained extracted oil was expressed in percentage, which is defined as mass of oil extracted over mass of the sample (pomegranate seeds) used for extraction. The obtained extracts were collected in vials and stored at −20 °C until further analysis. 2.3.2. Supercritical fluid extraction Supercritical fluid extraction (SFE) was performed in the high pressure unit (HPEA 500, Eurotechnica, Germany) previously described elsewhere (Ivanović et al., 2014) which was modified according to requirements of the process (Fig. 1). Sample of 10 g of ground pomegranate seed with and without microwave radiation treatment was filled into the stainless steel basket with a perforated bottom and top and then placed in stainless steel extraction vessel. Liquid CO2 supplied from CO2 cylinder (commercial CO2, 99% purity, Messer Tehnogas, Belgrade, Serbia) with a siphon tube was cooled in cryostat between the cylinder outlet and the pump to prevent CO2 vaporization. After the system was heated, the CO2 was pumped by a liquid metering pump (Milton Roy, France) until the required pressure is obtained. Than valve V-3 is opened and the extraction started. Depending on the specific flow mode, system pressure was maintained using a back-pressure valve (BPR). Applied operating pressure and temperature (47 °C and 37.9 MPa) were pre22
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Fig. 1. HPEA unit for supercritical extraction of pomegranate seed oil.
2.64.), compared with those from available literature (Adams, 2007), and used as additional tool to approve MS findings.
viously reported as optimum for obtaining high oil extraction yield (Liu et al., 2009). The extraction process lasted until the exhaustion of plant material (round 8.3 h). The flow rate of scCO2 (density of scCO2, 630 kg/m3) during the extraction process was 0.3 kg/h (calculated as the ratio of the mass of CO2 used for the extraction and extraction time). The obtained extracts were collected in vials and stored at −20 °C until further analysis.
2.5. Statistical analysis All analyses were performed at least three times with three independent samples and results in tables and figures are presented as mean ± standard deviation. Differences among treatments and control regarding the oil yield and PUFA content, as well as their fatty-acids content, were estimated through one-way ANOVA followed by post-hoc Duncan’s multiple range tests. Differences between mean values of individual oil yield and PUFA content for each extraction method were determined by Student’s t-test. All statistical analyses and output graphics were done using the software package STATISTICA v.7.0.
2.4. Analysis of fatty acids 2.4.1. Gas chromatography/flame ionization detection Prior to analysis, fatty acid methyl esters (FAME) were prepared according to the International Association of Official Analytical Communities (AOAC) (Official Surplus Method 965.4). Gas chromatography (GC/FID) analysis of FAME extracts isolated from pomegranate seeds was carried out on an gas chromatograph (Agilent Technologies, 7890A), equipped with split-less injector and automatic liquid sampler (ALS), attached to HP-5MS column (30 m · 0.25 mm, 0.25 μm film thickness) and fitted to flame ionization detector (FID). Carrier gas flow rate (H2) was 1 mL/min, injector temperature was 250 °C, detector temperature 300 °C, while column temperature was linearly programmed from 40 to 260 °C (at the rate of 4 °C/min), and held isothermally at 260 °C next 15 min. Solutions of tested samples in nhexane (15 μL/mL) were consecutively injected by ALS (2 μL, split mode 1:30). Area percent reports, obtained as result of standard processing of chromatograms, were used as base for the quantification purposes.
3. Results and discussion 3.1. Effect of microwave treatment on PSO yield Effect of microwave pretreatment on yield of PSO was determined through two different extraction techniques using n-hexane and scCO2. Results of extraction yields were compared with extraction yield of untreated pomegranate seed as a control sample. Yields of PSO obtained by extraction with n-hexane and supercritical fluid extraction with or without microwave pretreatment are presented in Table 1. The microwave pretreatment applied prior to extraction process influenced significantly PSO yield for both extraction techniques resulting in its increase. Microwave pretreatment prior to Soxhlet extraction contributed in increase in oil yield from 27.7% (for non-treated seeds) to 34.0–36.3% (Table 1.). These findings are in agreement with the previously published results for microwave pretreatment on extraction for some other plant material (Moreno et al., 2003; Li et al., 2004; Chemat et al., 2005; Duvernay et al., 2005; Cravotto et al., 2008; Uquiche et al., 2008; Azadmard-Damirchi et al., 2010). Moreno et al. (2003) used microwave pretreatment of 859 W during 11 min for the oil extraction from avocado pulp with n-hexane in Soxhlet apparatus and found that extraction efficiency increased from 54% to 97% when microwave pretreatment was used. Moreover, increase in microwave radiation power from 100 to 600 W in our study also contributed to slight
2.4.2. Gas chromatography/mass spectrometry The same chromatographic conditions as those mentioned for GC/ FID were employed for gas chromatography/mass spectrometry (GC/ MS analysis), using HP G 1800C Series II GCD system (Hewlett-Packard, Palo Alto, CA, USA). Instead of hydrogen, helium was used as carrier gas. Transfer line was heated at 260 °C. Mass spectra were acquired in EI mode (70 eV), in the range of 40–450 Da. Sample solutions were injected by ALS (2 μL, split mode 1:30). The constituents were identified by comparison of their mass spectra to those from Wiley275 and NIST/NBS libraries, using different search engines (PBM and NIST). In addition, the experimental values for retention indices were determined by the use of calibrated Automated Mass Spectral Deconvolution and Identification System software (AMDIS ver. 23
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Table 1 Estimated PSO yield for all observed treatments. #
1 2 3 4 5 6 7
Microwave treatment
n-hexane
SCCO2
Power [W]
Time [min]
Oil yield [%]*
Oil yield [%]*
Control 100 100 250 250 600 600
2 6 2 6 2 6
27.73 33.97 34.28 34.42 34.86 35.36 36.34
± ± ± ± ± ± ±
1.18 1.51 1.30 0.88 1.02 0.97 0.96
b a a a a a a
21.62 25.52 24.00 25.87 27.24 23.91 25.21
± ± ± ± ± ± ±
0.58 0.46 0.73 1.04 0.82 0.97 0.66
d b c b a c bc
a–d Means followed by different letters differ significantly, based on Duncan’s test at P < 0.05. * PSO yield is given as mean ± standard deviation (n = 3), expressed as% (w/w) on the dry-weight basis.
increase of extraction yield from 34.0 to 35.4% and from 34.3 to 36.3% for exposure time of 2 and 6 min, respectively. Although, pomegranate seed treated for 6 min at power of 600 W yielded more oil than that treated for 2 min at 100 W (36.3% compared with 34.0%, respectively), the oil obtained was black in color according to Pantone color scale. Similar results were obtained by Sayyar et al. (2011) for microwave treatment of jatropha seeds prior to extraction with n-hexane. They reported increase in extraction yield from 47.33% to 49.36% when microwave pretreatment time increased from 2 to 4 min. But with further increase in microwave pretreatment time to 6 min, they produced bunt/roasted seeds and reduction in extraction yield to 38.6%. Therefore it can be concluded that change in oil color from brown to black is probable the consequence of decomposition of the seed tissue during increased and prolonged microwave radiation which further results in extraction yield increase. Prolongation of microwave pretreatment increases cracking of the plant cells due to the built-up pressure caused by heating from inside. Cracked cells facilitate release of oil as well as easier penetration of extraction solvent into cells (Sayyar et al., 2011). Prolonged microwave radiation also leads to drying of plant material followed by plant material destruction when the heat generated is too high (Chow and Ma, 2007; Cheng et al., 2011). Microwave pretreatment prior to scCO2 extraction contributed to increase in PSO yield from 21.6% (for non-treated seeds) to 23.9–27.2% at tested conditions (Table 1) revealing positive influence of applied microwave pretreatment. The highest extraction PSO yield that Liu et al. (2009) obtained was 14.0% for scCO2 extraction at the pressure of 40 MPa and temperature of 60 °C. Unlike Soxhlet extraction, where increase in oil yield was perceived with increase in power of microwave treatment to 600 W and time to 6 min, in case of scCO2 extraction the highest PSO yield of 27.2%, was achieved for microwave pretreatment of 250 W during 6 min. Also, increased and prolonged microwave radiation did not affect color of the extracted oil (it remained yellow for all pretreatment conditions). Comparing PSO yield of untreated seeds with microwave pretreated seeds for Soxhelt and scCO2 extractions (Table 1), the positive effect of microwave pretreatment is evident (extraction yields increased up to 31% and up to 26% for Soxhlet and scCO2 extraction, respectively). Lower PSO yield of untreated seeds can be explained by the existence of undamaged cell which present resistance to oil extraction (Uquiche et al., 2008; Aguilera and Stanley, 1999). These cells can be raptured by microwave radiation due to vaporization of the water from the plant material microstructure and which increases pressure in its interior. Raptured cells enable for the oil to diffuse through the cell walls resulting in a higher mass transfer coefficients and extraction yields (Chemat et al., 2005; Uquiche et al., 2008). Also, drying of the plant material due to water vaporization during microwave pretreatment can lead to more brittle plant tissue. This dried tissue can be easily ruptured during extraction process leading to increase oil extraction yield (Uquiche et al., 2008). On the other hand, Wroniak et al. (2016)
Fig. 2. Average PSO yield obtained by scCO2 and n-hexane extraction after microwave pretreatment (n = 21), ** - denote statistically significant difference at P < 0.01 level.
reported prolonged exposition to microwave irradiation could decrease seed moisture content which directly influenced the amount of the extracted from the seeds. The extraction with n-hexane gave the higher average yield of PSO compared with scCO2 extraction for non-treated seeds (27.7% compared to 21.62%) and seeds treated with microwaves (36.3% compared to 27.2%), as it is shown in Table 1 and on Fig. 2. These results could be attributed to selectivity of used solvents. Namely, scCO2 is highly selective solvent while n-hexane is non-selective and causes the simultaneous extraction of non-volatile pigments and waxes. Solvent behavior of n-hexane increases extraction yield compared to scCO2 but also can lead to change in extract color. Also, according to Porto et al. (2016) the Hildebrand solubility parameter (δ) for n-hexane is 14.9 MPa½. Huang et al. (2013), for scCO2 under the experimental conditions 40 °C and 30 MPa, calculated the Hildebrand solubility parameter, whose value is 15.9 MPa ½, when the δ value is estimated by Marcus’s equation (Marcus, 2006; Huang et al., 2013).
3.2. Kinetic of PSO extraction with scCO2 To evaluate the effect of different microwave radiation power and exposure time on PSO extraction in more details, kinetic of SFE was analyzed. Changes in kinetic of pomegranate seeds oil extraction with change in microwave radiation are presented in Fig. 3 (A for 2 min; B for 6 min). Two extraction periods could be perceived: 1) constant increase in amount in extracted oil in the initial stage of the process, known as the washing or the fast extraction period and 2) slower increase in the amount of extracted oil as the extraction progresses, known as slow extract period (Petrovic et al., 2012). A constant increase in rate of oil extraction can be attributed to extraction of easy available oil from cells destroyed by milling and/or microwave pretreatment by simple washing of the plant material. Stage of the decreased rate can be attributed to extraction of oil due to diffusion of scCO2 through parts of plant material with oil fractions harder to access (Petrovic et al., 2012). It is interesting to notice that in first part of extraction the slope of the curve was the highest for non-treated seed, following by decrease in slope for seed treated with 250 W and 600 W, 24
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Fig. 3. Kinetic of oil extraction from pomegranate seeds with scCO2 after microwave pretreatment for A- 2 minutes and B - 6 minutes. Table 2 Content of fatty acids [m/m%] in pomegranate crude seed oil obtained from n-hexane extraction determined by GC/MS analysis. Factor levels solvent
n-hexane
n-hexane
n-hexane
n-hexane
n-hexane
n-hexane
n-hexane
power [W] time [min]
No treatment No treatment
100 2
100 6
250 2
250 6
600 2
600 6
Fatty acid components [%] met_pal SFAa,b met_mar SFA met_lin PUFA met_ole MUFA met_ela MUFA met_ste SFA met_lind PUFA met_pun PUFA met_oct1 PUFA met_oct2 PUFA met_oct3 PUFA met_oct4 SFA met_Z_11 MUFA met_E_11 MUFA met_ara SFA met_beh SFA
2.81 ± 0.09 0.06 ± 0.00 5.79 ± 0.24 6.32 ± 0.25 0.59 ± 0.02 2.57 ± 0.11 0.14 ± 0.01 59.52 ± 2.32 8.59 ± 0.18 1.90 ± 0.08 7.01 ± 0.31 2.42 ± 0.12 0.80 ± 0.02 0.80 ± 0.03 0.56 ± 0.02 0.12 ± 0.00
2.75 ± 0.06 0.05 ± 0.00 5.67 ± 0.22 6.38 ± 0.15 0.59 ± 0.03 2.62 ± 0.11 0.16 ± 0.01 60.28 ± 2.76 8.70 ± 0.28 1.75 ± 0.07 6.66 ± 0.30 2.27 ± 0.07 0.66 ± 0.02 0.78 ± 0.02 0.56 ± 0.01 0.12 ± 0.00
2.74 ± 0.10 0.05 ± 0.00 5.76 ± 0.24 6.50 ± 0.14 0.64 ± 0.01 2.66 ± 0.13 0.18 ± 0.02 54.62 ± 1.24 9.45 ± 0.34 2.99 ± 0.10 9.87 ± 0.46 2.37 ± 0.07 0.73 ± 0.02 0.79 ± 0.02 0.54 ± 0.01 0.12 ± 0.00
2.71 ± 0.08 0.06 ± 0.00 5.79 ± 0.29 6.39 ± 0.26 0.53 ± 0.02 2.59 ± 0.08 0.16 ± 0.01 54.40 ± 1.74 8.96 ± 0.38 3.44 ± 0.16 10.67 ± 0.32 2.27 ± 0.08 0.62 ± 0.03 0.78 ± 0.02 0.51 ± 0.02 0.12 ± 0.00
2.75 ± 0.08 0.05 ± 0.00 5.79 ± 0.22 6.51 ± 0.17 0.60 ± 0.03 2.64 ± 0.05 0.16 ± 0.01 52.92 ± 1.59 10.06 ± 0.36 3.32 ± 0.16 10.77 ± 0.22 2.33 ± 0.11 0.66 ± 0.02 0.81 ± 0.03 0.54 ± 0.01 0.09 ± 0.00
2.73 ± 0.11 0.06 ± 0.00 5.81 ± 0.13 6.36 ± 0.27 0.50 ± 0.02 2.60 ± 0.07 0.17 ± 0.01 54.19 ± 2.15 9.13 ± 0.41 3.40 ± 0.13 10.61 ± 0.52 2.32 ± 0.10 0.70 ± 0.02 0.75 ± 0.02 0.53 ± 0.02 0.14 ± 0.00
2.71 ± 0.06 0.06 ± 0.00 5.78 ± 0.20 6.43 ± 0.22 0.44 ± 0.02 2.58 ± 0.12 0.15 ± 0.01 53.88 ± 1.28 8.79 ± 0.40 3.71 ± 0.11 11.03 ± 0.48 2.32 ± 0.08 0.70 ± 0.03 0.77 ± 0.03 0.53 ± 0.03 0.11 ± 0.00
a b
Abbreviations: SFA – saturated fatty acid, MUFA – monounsaturated fatty acid, PUFA – polyunsaturated fatty acid. Standard deviations reported as value 0.00, were in fact lesser than 0.01.
(SFA) were in range between 5.8 and 6.5% of total fatty acids, monounsaturated fatty acids (MUFA) were in the range from 13.9 to 16.1% and polyunsaturated fatty acids (PUFA) were the principal fatty acid class and they constitute between 84.3 and 86.1% of total fatty acids. Slightly larger average amount of PUFA was found in sample obtained by supercritical fluid extraction (85.6% of total fatty acids), compared with average amount of PUFA (85.5% of total fatty acid) obtained by extraction with n-hexane, but no statistically significant differences between them were observed (Fig. 4). The predominant fatty acid extracted by n-hexane and scCO2 extraction was punicic acid (approximately 60.0%). The applied microwave pretreatment influenced negligible increase of punicic acid from 59.5% (non-treated seeds) to 60.3% (100 W during 2 min) for nhexane extraction. Microwave pretreatment (250 W during 6 min) prior to scCO2 extraction contributed to a slight increase from 54.1% (for non-treated seeds) to 60.3%. Fadavi et al. (2006) studied fatty acid profile of seed oil from 25 different varieties of pomegranate obtained using Soxhlet extraction. They found that the punicic acid content ranged from 31.8 to 86.6%. In another study, super-heated n-hexane extraction of pomegranate seed oil resulted in 70.73% punicic acid (Eikani et al., 2012). Supercritical CO2 extraction used by Abbasi et al. (2008) resulted in punicic acid content from 69.8% to 79.0%. This amount is very important because punicic acid is the principal CLnA present in pomegranate seeds oil and it is connected with its potential
while seed treated with 100 W had the lowest slope in both cases (Fig. 3). Fiori (2007) reported that increase in the pressure for scCO2 extraction of grape seed oil leads to increase in slope of the curves for first part of the extraction. Increase in yield of first part of the extraction occurred because solubility of grape seed oil in scCO2 changed due to increase in pressure. Therefore, it can be assumed that change in PSO solubility occurred due to change in oil composition (Table 2). Extraction condition were constant but the slope of the curves for first part of the extraction changed. Variation in fatty acids composition of oil could be attributed to pretreatment conditions. Although, microwave pretreatment decreased yield of first part of extraction, it resulted in higher extraction yield for second period of extraction where diffusion of oil through plant material is limiting factor of extraction. 3.3. Effects of microwave pretreatment on fatty acids composition Quantitative and qualitative analysis of the fatty oil from the pomegranate seeds was carried out. Tables 2 and 3 present the fatty acid composition of the pomegranate seed oils obtained by extractions using n-hexane and scCO2. Sixteen fatty acids at different quantities were detected in PSO obtained by n-hexane and scCO2 extraction. Statistically, there were no significant differences between n-hexane and scCO2 obtained samples, suggesting both methods as satisfactory. Total saturated fatty acids 25
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Table 3 Content of fatty acids [m/m%] in pomegranate crude seed oil obtained from
SCCO2
extraction determined by GC/MS analysis.
Factor levels solvent
SCCO2
SCCO2
SCCO2
SCCO2
SCCO2
SCCO2
SCCO2
power [W] time [min]
No treatment No treatment
100 2
100 6
250 2
250 6
600 2
600 6
Fatty acid components [%] met_pal SFAa,b met_mar SFA met_lin PUFA met_ole MUFA met_ela MUFA met_ste SFA met_lind PUFA met_pun PUFA met_oct1 PUFA met_oct2 PUFA met_oct3 PUFA met_oct4 SFA met_Z_11 MUFA met_E_11 MUFA met_ara SFA met_beh SFA
3.20 ± 0.07 0.06 ± 0.00 6.25 ± 0.15 7.32 ± 0.17 0.43 ± 0.02 2.60 ± 0.11 0.15 ± 0.01 54.06 ± 1.12 8.82 ± 0.26 3.07 ± 0.14 9.72 ± 0.36 2.26 ± 0.08 0.74 ± 0.03 0.71 ± 0.02 0.53 ± 0.01 0.10 ± 0.00
2.77 ± 0.11 0.05 ± 0.00 5.79 ± 0.21 6.46 ± 0.23 0.41 ± 0.01 2.53 ± 0.07 0.14 ± 0.01 54.53 ± 1.69 8.85 ± 0.18 3.40 ± 0.07 10.59 ± 0.45 2.33 ± 0.10 0.77 ± 0.03 0.75 ± 0.03 0.50 ± 0.01 0.11 ± 0.00
2.75 ± 0.10 0.08 ± 0.00 5.69 ± 0.22 6.31 ± 0.24 0.38 ± 0.01 2.50 ± 0.07 0.12 ± 0.02 56.04 ± 2.71 8.55 ± 0.41 3.18 ± 0.10 9.95 ± 0.28 2.28 ± 0.05 0.72 ± 0.02 0.80 ± 0.02 0.52 ± 0.02 0.11 ± 0.00
2.83 ± 0.14 0.06 ± 0.00 5.81 ± 0.20 6.45 ± 0.19 0.41 ± 0.01 2.55 ± 0.12 0.13 ± 0.01 52.64 ± 1.33 9.37 ± 0.33 3.83 ± 0.08 11.45 ± 0.57 2.31 ± 0.06 0.69 ± 0.01 0.83 ± 0.04 0.52 ± 0.01 0.12 ± 0.00
2.74 ± 0.07 0.05 ± 0.00 5.65 ± 0.18 6.30 ± 0.24 0.34 ± 0.01 2.48 ± 0.10 0.11 ± 0.01 60.26 ± 2.48 8.50 ± 0.41 2.02 ± 0.05 7.05 ± 0.34 2.26 ± 0.06 0.73 ± 0.02 0.79 ± 0.03 0.52 ± 0.02 0.10 ± 0.00
2.76 ± 0.14 0.05 ± 0.00 5.70 ± 0.27 6.25 ± 0.30 0.33 ± 0.01 2.45 ± 0.10 0.12 ± 0.01 59.69 ± 1.75 8.13 ± 0.35 2.37 ± 0.10 7.84 ± 0.30 2.23 ± 0.10 0.73 ± 0.02 0.74 ± 0.03 0.52 ± 0.02 0.09 ± 0.00
2.79 ± 0.10 0.05 ± 0.00 5.79 ± 0.28 6.30 ± 0.17 0.33 ± 0.01 2.46 ± 0.06 0.11 ± 0.01 59.94 ± 2.46 8.25 ± 0.18 2.17 ± 0.09 7.44 ± 0.24 2.28 ± 0.06 0.65 ± 0.02 0.85 ± 0.04 0.50 ± 0.01 0.10 ± 0.00
a b
Abbreviations: SFA – saturated fatty acid, MUFA – monounsaturated fatty acid, PUFA – polyunsaturated fatty acid. Standard deviations reported as value 0.00, were in fact lesser than 0.01.
Hernandez et al., 2000; Tian et al., 2013). In our study, the levels of linoleic acid in pomegranate oil varied from 5.7 to 6.2%, which was lower than in some other species such as safflower (67.8–83.2%), soybean (48.0–59.0%), sunflower (48.3–74.0%), and flaxseed oils (15.2–15.9%) (Codex Alimentarius Commission, 1999; Choo et al., 2007). Although, there are observed differences between pretreated seeds and control group regarding levels of some fatty acids (i.e. palmitic, linoleic, oleic and stearic acids) statistical analysis has not shown any pattern, which could lead us to conclude any rule of prediction for future cases (Tables 2 and 3). Higher carbon content fatty acids including arachidic acid and behenic acid were also present in our oil samples at the level of trace amount. Arachidic acid content of pomegranate seed oils was determined to be in the range from 0 to 2.8% (Özgül-Yücel, 2005; Melgarejo and Artes, 2000). Nevertheless, these studies did not reported behenic acid in pomegranate seed oil. On the other hand, Fadavi et al. (2006) reported the presence of behenic acid in pomegranate seed oils ranging from 0 to 3.9%. Moreover, in our samples, lignoceric acid was present in trace amount in scCO2 extracted PSO using microwave pretreatment with power of 250 W during 6 min. Although our results correspond with some of the previously published papers, the difference in the content of fatty acids was also noticed. According to Kýralan et al. (2009) oil content could vary depending on genotype, location, harvest time, climatic conditions, etc.
4. Conclusion
Fig. 4. Average PUFA contents in oils obtained by scCO2 and n-hexane extraction (n = 21).
In this study, the effect of microwave pretreatment on the pomegranate seed oil (PSO) yield and its composition using Soxhlet and scCO2 extraction was perceived. Statistically significant difference was noticed in the PSO yield from non-treated and microwave treated seeds (extraction yields increased up to 31% and up to 26% for Soxhlet and scCO2 extraction, respectively after microwave pretreatment). The highest PSO yield by n-hexane extraction in Soxhlet apparatus was 36.3% after microwave pretreatment for 6 min with power of 600 W. The highest oil yield recorded using supercritical fluid extraction was 27.2% after microwave pretreatment at 250 W for 6 min. Oils obtained by scCO2 were yellow colored compared to oils obtained by Soxhlet extraction where color ranged from brown to black. This is probably due to greater extraction selectivity of scCO2. Qualitative and quantitative composition of fatty acids was similar in all samples. Punicic acid
biological and health beneficial effects. As reported by Özgül-Yücel (2005) pomegranate seed oil contained higher amount of CLnA than some well-known CLnA-rich seeds including pot marigold (29.5%), mahaleb (27.6%), and catalpa (27.5%). The second most abundant fatty acid in PSO was oleic (C18:1 C9) acid, which was in the range from 6.2 to 7.3%. According to Garima and Akoh (2009) and Jing et al. (2012), oleic acid was the most prevalent monounsaturated fatty acid in Georgians and Chinese PSO, respectively. On the other hand, in some studies of PSO obtained using different extraction methods (cold press, Soxhlet, superheated n-hexane and scCO2 extraction), linoleic and punicic acids were dominant polyunsaturated fatty acids (Fadavi et al., 2006; Kýralan et al., 2009; 26
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modeling. J. Food Eng. 117, 492–498. He, L., Zhang, X., Xu, H., Xu, C., Yuan, F., Knez, Z., Novak, Z., Gao, Y., 2012. Subcritical water extraction of phenolic compounds from pomegranate (Punica granatum L.) seed residues and investigation into their antioxidant activities with HPLC–ABTS•+ assay. Food Bioprod. Process 90, 215–223. Hernandez, F., Melgarejo, P., Olias, J.M., Artes, F., 2000. Symposium on Production, Processing and Marketing of Pomegranate in the Mediterranean Region: Advances in Research and Technology. CIHEAM‐IAMZ, Zaragoza (Spain). Huang, Z., Li, J.-H., Li, H.-S., Miao, H., Kawi, S., Goh, A., 2013. Effect of the polarmodifiers on supercritical extraction efficiency for template removal from hexagonal mesoporous silica materials: solubility parameter and polarity considerations. Sep. Purif. Technol. 118, 120–126. Ivanovic, J., Milovanovic, S., Stamenic, M., Fanovich, M.A., Jaeger, P., Zizovic, I., 2014. Application of an integrated supercritical extraction and impregnation process for incorporation of thyme extracts into different carriers. In: Jane Osborne (Ed.), Handbook on Supercritical Fluids, Fundaments, Properties and Applications. Nova Science Publishers, New York, pp. 257–280. Jing, P., Ye, T., Shi, H., Sheng, Y., Slavin, M., Gao, B., 2012. Antioxidant properties and phytochemical composition of China-grown pomegranate seeds. Food Chem. 132 (3), 1457–1464. Kýralan, M., Gölükcü, M., Tokgöz, H., 2009. Oil and conjugated linolenic acid contents of seeds from important pomegranate cultivars (Punica granatum L.) grown in Turkey. J. Am. Oil Chem. Soc. 86, 985–990. Lansky, E.P., Newman, R.A., 2007. Punica granatum (pomegranate) and its potential for prevention and treatment of inflammation and cancer. J. Ethnopharmacol. 109, 177–206. Lansky, E.P., 1999. Phytoestrogen supplement prepared from pomegranate seed and a herbal mixture or coconut milk. US Patent 5 (891), 440. Li, H., Chen, B., Nie, N., Yao, S., 2004. Solvent effects on focused microwave assisted extraction of polyphenolic acids from Eucommia Ulmodies. Phytochem. Anal. 15, 306–312. Liu, G., Xu, X., Hao, Q., Gao, Y., 2009. Supercritical CO2 extraction optimization of pomegranate (Punica granatum L.) seed oil using response surface methodology. LWT Food Sci. Technol. 42, 1491–1495. Marcus, Y., 2006. Are solubility parameters relevant to supercritical fluids? J. Supercrit. Fluids 38, 7–12. Melgarejo, P., Artes, F., 2000. Total lipid content and fatty acid composition of oil seed from lesser known sweet pomegranate clones. J. Sci. Food Agric. 80, 1452–1454. Meyer, F., Jaeger, P., Eggers, R., Stamenic, M., Milovanovic, S., Zizovic, I., 2012. Effect of CO2 pre-treatment on scCO2 extraction of natural material. Chem. Eng. Process. Process Intensif. 56, 37–45. Moreno, A.O., Dorantes, L., Galindez, J., Guzman, R.I., 2003. Effect of different extraction methods on fatty acids volatile compounds, and physical and chemical properties of avocado (Persea americana mill.) oil. J. Agric. Food. Chem. 51, 2216–2221. Official Surplus Method 965.4 Fatty Acids in Oils and Fats, Preparation of Methyl Esters, Final Action 1984, Surplus 1965. Petrovic, S.S., Ivanovic, J., Milovanovic, S., Zizovic, I., 2012. Comparative analyses of the diffusion coefficients from thyme for different extraction processes. J. Serb. Chem. Soc. 77 (6), 799–813. Porto, C., Decorti, D., Natolino, A., 2016. Microwave pretreatment of Moringa oleifera seed: effect on oil obtained by pilot-scale supercritical carbon dioxide extraction and Soxhlet apparatus. J. Supercrit. Fluids 107, 38–43. Ramesh, M., Rao, P.H., Ramadoss, C.S., 1995. Microwave treatment of ground nut (Arachis hypogaea): extractability and quality of oil and its relation to lipase and lipoxygenase activity. LWT Food Sci. Technol. 28, 96–99. Sayyar, S., Abidin, Z.Z., Yunus, R., Muhammad, A., 2011. Solid liquid extraction of jatropha seeds by microwave pretreatment and ultrasound assisted methods. J. Appl. Sci. 11 (13), 2444–2447. Schubert, S.Y., Lansky, E.P., Neeman, I., 1999. Antioxidant and eicosanoid enzyme inhibition properties of Pomegranate seed oil and fermented juice flavonoids. J. Ethnopharmacol. 66 (1), 11–17. Singh, R.P., Heldman, D.R., 2001. Introduction to Food Process Engineering, third ed. Academic Presspp. 30–47. Tian, Y., Xu, Z., Zheng, B., Lo, Y.M., 2013. Optimization of ultrasonic-assisted extraction of pomegranate (Punica granatum L.) seed oil. Ultrason. Sonochem. 20, 202–208. Uquiche, E., Jerez, M., Ortiz, J., 2008. Effect of pretreatment with microwaves on mechanical extraction yield and quality of vegetable oil from Chilean hazelnuts (Gevuina avellana Mol.). Innov. Food Sci. Emerg. Technol. 9, 495–500. Wang, L., Weller, C.L., 2006. Recent advances in extraction of nutraceuticals from plants. Trends Food Sci. Technol. 17, 300–312. Wijendran, V., Hayes, K.C., 2004. Dietary n-6 and n-3 fatty acid balance and cardiovascular health. Annu. Rev. Nutr. 24, 597–615. Wroniak, M., Rekas, A., Siger, A., Janowicz, M., 2016. Microwave pretreatment effects on the changes in seeds microstructure, chemical composition and oxidative stability of rapeseed oil. LWT Food Sci. Technol. 68, 634–641.
was the major fatty acid found in all samples. This study showed that microwave treatment of pomegranate seeds prior to extraction increases oil yield without negative effect on fatty acid composition which is especially significant bearing in mind potential application of pomegranate seed oil in treatment of cardiovascular and skin diseases, type II diabetes mellitus, and cancer. Acknowledgments Financial support of this work from the Ministry of Education, Science and Technological Development of the Republic of Serbia (Project III 46013 and III 45017) is gratefully acknowledged. Authors also thanks Mr. Sreten Škrba who donated pomegranate fruits from his property. References Özgül-Yücel, S., 2005. Determination of conjugated linolenic acid content of selected oil seeds grown in Turkey. J. Am. Oil Chem. Soc. 82 (12), 893–897. Automated Mass Spectral Deconvolution and Identification System software (AMDIS ver. 2.64.), National Institute of Standards and Technology (NIST), Standard Reference Data Program, Gaithersburg, MD (USA). Abbasi, H., Rezaei, K., Rashidi, L., 2008. Extraction of essential oils form the seeds of pomegranate using organic solvents and supercritical CO2. J. Am. Oil Chem. Soc. 85, 83–89. Adams, R.P., 2007. Identification of Essential Oil Components by Gas Chromatography/ Mass Spectrometry, fourth ed. Allured Publishing Corporation, Carol Stream, Illinois, USA. Aguilera, J.M., Stanley, D.W., 1999. Microstructural Principles of Food Processing and Engineering, second ed. Aspen Publishers, Inc., Gaithersburg, MD, pp. 352–372. Al-Maiman, S.A., Ahmad, D., 2002. Changes in physical and chemical properties during pomegranate (Punica granatum L.) fruit maturation. Food Chem. 76, 437–441. Amarù, D., Field, C., 2009. Conjugated linoleic acid decreases MCF-7 human breast cancer cell growth and insulin-like growth factor-1 receptor levels. Lipids 44 (5), 449–458. Ashoori, F., Suzuki, S., Zhou, J.H., Isshiki, N., Miyachi, Y., 1994. Involvement of lipid peroxidation in necrosis of skin flaps and its suppression by ellagic acid. Plast. Reconstr. Surg. 94 (7), 1027–1037. Azadmard-Damirchi, S., Habibi-Nodeh, F., Hesari, J., Nemati, M., Fathi Achachlouei, B., 2010. Effect of pretreatment with microwaves on oxidative stability and nutraceuticals content of oil from rapeseed. Food Chem. 121, 1211–1215. Chemat, S., Ait-Amar, H., Lagha, A., Esveld, D.C., 2005. Microwave-assisted extraction kinetics of terpenes from caraway seeds. Chem. Eng. Process. 44, 1320–1326. Cheng, S.F., Mohd Nor, L., Chuah, C.H., 2011. Microwave pretreatment: a clean and dry method for palm oil production. Ind. Crops Prod. 34, 967–971. Choo, W.S., Birch, J., Dufour, J.P., 2007. Physicochemical and quality characteristics of cold-pressed flaxseed oils. J. Food Compos. Anal. 20, 202–211. Chow, M.C., Ma, A.N., 2007. Processing of fresh palm fruits using microwaves. J. Microw. Power Electromagn. Energy 40 (3), 165–173. Codex Alimentarius Commission, 1999. Codex stan 19. Edible Fats and Oils Not Covered by Individual Standards. Cravotto, G., Boffa, L., Mantegna, S., Perego, P., Avogadro, M., Cintas, P., 2008. Improved extraction of vegetable oils under high-intensity ultrasound and/or microwaves. Ultrason. Sonochem. 15, 898–902. Dubois, V., Breton, S., Linder, M., Fanni, J., Parmentier, M., 2007. Fatty acid profiles of 80 vegetable oils with regard to their nutritional potential. Eur. J. Lipid Sci. Technol. 109, 710–732. Duvernay, W.H., Assad, J.M., Sabliov, C.M., Lima, M., Xu, Z., 2005. Microwave extraction of antioxidant components from rice bran. Pharm. Eng. 25, 1–5. Eikani, M.H., Golmohammad, F., Homami, S.S., 2012. Extraction of pomegranate (Punica granatum L.) seed oil using superheated hexane. Food Bioprod. Process. 90, 32–36. FAO/WHO, 2010. Fats and Fatty Acids in Human Nutrition. Report of an Expert Consultation. FAO/WHO, Geneva, Switzerland. Fadavi, A., Barzegar, M., Azizi, M.H., 2006. Determination of fatty acids and total lipid content in oilseed of 25 pomegranates varieties grown in Iran. J. Food Compos. Anal. 19, 676–680. Fiori, L., 2007. Grape seed oil supercritical extraction kinetic and solubility data: critical approach and modeling. J. Supercrit. Fluids 43, 43–54. Garima, P., Akoh, C.C., 2009. Antioxidant capacity and lipid characterization of six Georgia-grown pomegranate cultivars. J. Agric. Food Chem. 57 (20), 9427–9436. Goula, A.M., 2013. Ultrasound-assisted extraction of pomegranate seed oil–Kinetic
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