LWT - Food Science and Technology 164 (2022) 113643 Contents lists available at ScienceDirect LWT journal homepage: www.elsevier.com/locate/lwt Targeted analysis of sea buckthorn oil extracted by accelerated solvent extraction technique using green and conventional solvents Arnau Vilas-Franquesa a, Bibiana Juan a, *, Jordi Saldo a, b a Centre d’Innovació, Recerca i Transferència en Tecnologia dels Aliments (CIRTTA), XaRTA, TECNIO, MALTA-Consolider, Departament de Ciència Animal i dels Aliments, Facultat de Veterinària, Universitat Autònoma de Barcelona (Cerdanyola del Vallès), Barcelona, Spain b Instituto Politécnico Nacional (IPN), Centro de Desarrollo de Productos Bióticos (CEPROBI), Yautepec, Morelos, Mexico A R T I C L E I N F O A B S T R A C T Keywords: Sea buckthorn oil (SBO) was extracted using accelerated solvent extraction with two green solvents (ethanol and Sea buckthorn oil 2-methyltetrahydrofuran), and two petroleum-based solvents (hexane and diethyl ether) at four different tem­ Green solvents peratures (60, 90, 120 and 150 ◦ C), using one extraction cycle at 103.42 bar, 10 min static time, 5 min preheating Fatty acid profile and 30% flushing volume. β-carotene and α-tocopherol were quantified jointly using HPLC-UV/VIS and the fatty α-tocopherol β-carotene acid profile was analyzed using GC-MS. Extraction yield was also measured and the data was compared to a conventional extraction using Soxhlet. The extraction with ethanol achieved significantly higher concentrations of β-carotene in the final oil (1.67 mg/g oil) compared to the results of any other solvent (e.g. 1.23 mg/g oil for hexane ASE) or to the conventional extraction (1.27 mg/g oil). However, the use of green solvents led to the dragging of unwanted polar compounds (sugars), leading to lower oil yield values after purification. 1. Introduction Danh et al., 2013). In fact, green solvents are becoming nowadays more relevant, as the consumer is seeking for sustainable products and pro­ The oil from sea buckthorn (SB) can be extracted from two parts: (1) duction processes. Green solvents generally include but are not limited the seed or (2) the pulp and peel. The nutritional profile of the oil differs to ethanol or ethyl acetate, or the combination of water and a depending on the extracted part; the seed oil has greater amounts of water-soluble solvent (Castejón et al., 2018; Danh et al., 2013), and tocopherols and oleic acid, and the pulp oil contains greater amounts of recently terpenes (Kumar et al., 2017). Ethanol is used because of its low carotenoids – specially β-carotene – and palmitoleic acid, among other cost, and in cases in which the interest remains in obtaining somewhat bioactive lipophilic compounds. The intake of either one or both SBO polar compounds (e.g. extraction of slightly polar molecules such as has beneficial effects on human health, such as improvements in blood phospholipids). Another interesting green solvent that has been studied lipid profile (Guo, Yang, Cai, & Li, 2017) modulation of hypoxia, car­ recently is 2-methyltetrahydrofuran (2-MTHF). 2-MTHF is produced out dioprotective properties and other antioxidant properties (Olas, 2018). of carbohydrates from lignocellulose biomass, which represent the In addition, its nutritional profile makes it very valuable for the devel­ largest terrestrial biomass resource (Sicaire et al., 2014). Previous works opment of food products (Vilas-Franquesa, Saldo, & Juan, 2020). reporting extractions with 2-MTHF had yielded oils with (a) higher Several techniques have been exploited over the years for the re­ monounsaturated fatty acid levels when compared to hexane, obtaining covery of oils from vegetable matrices. The most exploited extraction a similar overall yield (such is the case of fennel and anise (Rebey et al., methodologies include cold pressing and solvent extraction (Danlami, 2019)), (b) a slightly higher polyunsaturated fatty acid concentration in Arsad, Zaini, and Sulaiman, 2014). Among the solvent extraction rapeseed oil (Sicaire et al., 2015), and (c) higher overall oil yield in methods, accelerated solvent extraction (ASE) is widely used as it allows caraway seeds when compared to hexane (Bourgou, Tounsi, Ksouri, for the application of high pressures, which translates into high tem­ Fauconnier, & Sellami, 2019; de Jesus, Ferreira, Fregolente, & Filho, peratures and shorter extraction times. In addition, ASE has shown 2018). promising results in the extraction of bioactive lipophilic compounds, The objective of this work was to comparatively evaluate oil yield, especially when using green solvents (Castejón, Luna, & Señoráns, 2018; the concentration of α-tocopherol and β-carotene (as the most important * Corresponding author. E-mail address: bibiana.juan@uab.cat (B. Juan). https://doi.org/10.1016/j.lwt.2022.113643 Received 22 February 2022; Received in revised form 31 May 2022; Accepted 5 June 2022 Available online 8 June 2022 0023-6438/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). A. Vilas-Franquesa et al. LWT 164 (2022) 113643 vitamers in SBO), and the fatty acid profile of SBO extracted from SB difference. The extracted oil was then stored in amber-tinted chroma­ dried berries with an accelerated solvent extractor using different sol­ tography vials at − 80 ◦ C for further analysis. vents (hexane, diethyl ether, ethanol and 2-MTHF) at different tem­ Ethanol and 2-MTHF dried extracts were further mixed with water peratures (60, 90, 120 and 150 ◦ C). Soxhlet extraction was used as a (at room temperature) since during the extraction some polar com­ reference method. pounds were dragged out because of the polarity of the solvents used (Table 1). The mixture of SBO extracted with ethanol and water resulted 2. Materials and methods in a clear solubilization and the solution was discarded. The oil was recovered from the walls of the lab flask by adding 20 ml of n-hexane. 2.1. Extraction solvents and material Dissolution was complete and SBO was obtained after solvent evapo­ ration at 45 ◦ C using a rotary evaporator as detailed above. In contrast, Hexane, ethanol, diethyl ether and 2-methyltetrahydrofuran were the mixture of SBO extracted with 2-MTHF and water resulted in a fuzzy used for the experiment. n-Hexane (with isomers) 99% purity (HPLC mixture. The content of the lab-flask was poured into a centrifugal vial grade) was purchased from Labbox Labware, S. L., Catalunya, Spain. 2- and centrifuged at 10,000 rpm for 10 min at room temperature. SBO was Methyltetrahydrofuran (2-MTHF) stabilized with 2,6-di-tert-butyl-4- recovered from the upper phase using a glass Pasteur pipette. methylphenol was purchased from Merck KGaA, Darmstadt, Germany. Diethyl ether, stabilized with 6 mg/L of BHT and ethanol absolute (99.8%) were purchased from Panreac Química S. A. U., Catalunya, 2.4. β-carotene and α-tocopherol analysis Spain. Polytetrafluoroethylene (PTFE) O-rings, PEEK seals, cell frites, 20 mm cellulose filters and PTFE-lined silicone septa for the ASE were A simultaneous quantification of β-carotene and α-tocopherol was purchased from Restek Corporation, Pennsylvania, U.S. adapted from Gimeno et al. (2000) with some modifications. Briefly, 400 mg of sea buckthorn oil (SBO) were mixed with 0.2 g of L-ascorbic acid (99%, Merck KGaA (Darmstadt, Germany), 15 mL of absolute 2.2. Sample preparation ethanol and 4 mL of a 76 g/100 ml KOH solution in that order in a centrifugation screw-capped tube. The tubes were then mixed gently Sun-dried sea buckthorn berries were purchased from a local and incubated at 70 ◦ C for 30 min with slow constant stirring. After harvester from the north-east region of Romania. The berries were cooling, 5 mL of sodium chloride at 25 g/L were added in each tube and categorized as the subspecies caucasica. Dried sea buckthorn berries the solution was vigorously mixed for 1 min and left undisturbed. Phase were grinded down with a Thermomix® TM 21 (Vorwerk, Wuppertal, separation occurred after 5 min. The resulting product was extracted Germany). The particle size distribution of the powder was measured by two times with 20 mL portions of n-hexane (≥95%, HPLC grade, Merck gravimetry using sieves of different mesh sizes. Particle size distribution KGaA, Darmstadt, Germany) and ethyl acetate (≥99.5% ACS reagent, was quantified twice. Merck KGaA, Darmstadt, Germany) at a ratio of 85:15 (v/v). The organic Approximately 8 g of dried and ground sea buckthorn berries were phase (supernatant) was recovered and brought to dryness at 40 ◦ C mixed thoroughly with diatomaceous earth at a ratio of 4:1. An accel­ under vacuum conditions. Finally, the residue was resuspended with 3 erated solvent extractor (ASE) extraction cell of 33 ml total volume was mL of methanol and passed through a 0.45 μm filter and directly injected filled with a cellulose membrane and the mix, in that order. The cell was to the HPLC. closed and inserted in the ASE cell rack. For Soxhlet extraction, the same The HPLC system consisted of a P680 HPLC Pump attached to an ASI- amount of sample was wrapped in a medium lab-working filter paper 100 Automated Sample Injector and a Thermostatted Column (Letslab delivering solutions S.L.U., Catalunya, Spain). The wrapped Compartment TCC-100 equipped with a reversed phase SunFire C18 sample was placed directly in the sample compartment of the Soxhlet column, 100 Å, 4.6 × 150 mm, 5 μm. The detection of the eluted com­ set-up. pounds was performed with a UVD170U detector. All the parts of the HPLC were from Dionex Corporation (California, U. S.). HPLC oven 2.3. Oil extraction and yield temperature was set at 45 ◦ C, sample volume was 50 μl and injected at 1.325 mL/min. Timespan was efficiently set at 20 min, and a gradient The oil was extracted from sea buckthorn dried berry powder by profile was 97:3 of solution A and solution B, respectively, for 6 min, using an Accelerated Solvent Extractor 200 (Dionex, Thermo Fisher then linear gradient to 100% A in 2 min, to an isocratic step of 100% Scientific, California, U.S). The pressure was set at 103.42 bar, static solution A for 10 min and finally a linear gradient to solution A and B time at 10 min, preheating at 5 min, flushing volume at 30% of the total (97:3) in 2 min, where solution A was methanol:butanol (92:8, respec­ cell volume, purging time at 30 s. Only one extraction cycle was used, as tively), and solution B was mili-Q water 100%. The wavelength for preliminary trials showed similar oil recoveries when using more cycles detection of α-tocopherol was set at 292 nm and the wavelength (results not shown). SBO was also extracted from the samples by using detection of β-carotene was set at 450 nm. DL-α-tocopherol acetate hexane in a Soxhlet experimental set-up for 5 h. After extraction, the (HPLC standard, Merck KGaA, Darmstadt, Germany) was used as an solvent was evaporated using a rotavapor equipment for 45 min at 45 ◦ C internal standard (1 mg/ml processed sample), giving steady recovery and 150 mbar of pressure. Yield was calculated based on weight values of 70%. α-tocopherol (synthetic, ≥96%, HPLC standard) and Table 1 Characteristics of the solvents used in the present study. Solvent Density (g/cm3) Molecular weight (g/mol) Purity (%) Boiling point (◦ C) Polaritya Molecule scheme Diethyl ether 0.71 74.12 ≥99.5 34.60 5.77 Hexane 0.66 86.18 ≥99 68.00 2.56 2-MTHF 0.85 86.13 ≥99 80.20 6.99b Ethanol 0.79 46.07 ≥99.5 78.37 8.05 a Polarity according to the Spectral Polarity Index developed by Freed, Biesecker, & Middleton (1990). b Polarity value from tetrahydrofuran. According to Aycock (2007), solvent polarity and Lewis base strength properties of MeTHF is somewhere between THF and diethyl ether. 2 A. Vilas-Franquesa et al. LWT 164 (2022) 113643 β-carotene (synthetic, ≥93%, analytical standard) from Merck KGaA 3. Results and discussion (Darmstadt, Germany) were used as standard (stock solutions were used at concentrations of 1 mg of α-tocopherol/ml of methanol (y = 0.0045x 3.1. Extraction yield of SBO extracted with different solvents + 0.0007, R2 = 0.99, concentrations from 0.6 to 0.08 mg/ml) and 0.5 mg β-carotene/ml of n-hexane (y = 0.0011x + 0.0046, R2 = 0.96, con­ The SBO yield using hexane, ethanol, diethyl ether and 2-MTHF were centrations from 0.15 to 0.02 mg/ml)). affected differently by temperature. Thus the most efficient temperature for extracting SBO was different depending on the solvent used, being 2.5. Fatty acid profile 120 ◦ C for hexane and diethyl ether (23.25% and 24.98% yield, respectively), 90 ◦ C for ethanol (21.75% yield), and 150 ◦ C for 2-MTHF The fatty acid composition of the extracted oils were determined by extraction (14.65% yield). The efficiency of extraction seemed to be derivatizing the fatty acids to fatty acid methyl esters (FAME) using the greater by increasing the extraction temperature when using 2-MTHF, previously reported method by Lamba, Modak, and Madras (2017) with which was partly true when using diethyl ether as an extraction sol­ minor changes. Briefly, 0.15 g of extracted SBO was poured into a 15 mL vent. However, the latter achieved the poorest extraction yield at screw-capped test tube and mixed with 2 mL of n-hexane (≥95%, HPLC 150 ◦ C, making evident the different behaviour of each solvent across grade, Merck KGaA, Germany) and 1 mL of 2 mol/L methanolic KOH different temperatures. Interestingly, the pattern clearly pointed out solution (methanol GC/HPLC GGR from Labbox Labware S. L. (Cata­ that the more polar the solvent was, the more was it affected by changes lunya, Spain); KOH pellets ACS reagent ≥85% from Merck KGaA in temperature. It is important to note that the solvent 2-MTHF was (Darmstadt, Germany)). The tubes were vigorously shaken for 30 s and strongly and significantly influenced by changes in temperature when incubated in a water bath previously heated at 70 ◦ C for 2 min. The tubes compared to the rest of the solvents. Results from the extraction yield were then taken out of the bath and cooled at room temperature for 2 reported by other authors indicated that the greater the temperature of min, and immediately after 1.2 mL of HCl 1 mol/L (Panreac Química S. the extraction, the greater the extraction yield (Castejón et al., 2018), L. U., Catalunya, Spain) were added to the tube. Gently stirring was differing from what was observed in the present experiment. Castejón applied for 10 min and then the mix was left undisturbed. Separation in et al. (2018) found that at temperatures of 150 ◦ C, the greatest yield was two phases occurred after 15 min. An aliquot of the upper phase was obtained when using ethanol or hexane in echium seed oil extraction directly injected into the gas chromatograph for analysis. when compared to lower temperatures. A capillary column VF-5 ms, 30 m × 0.25 mm with 0.25 μm film Interestingly, using the Soxhlet technique (control) instead of ASE thickness containing 5% phenyl-methylpolysiloxane attached to a gas (using hexane as solvent) yielded significantly more oil. Nevertheless, chromatograph 6890 (Agilent Technologies, California, U. S.) was used this difference translated only into 0.65% more oil extracted by the for the chromatographic analysis. Helium was used as the carrier gas at former technique, which was in line with what previous authors found 1.7 mL/min, and oxygen and hydrogen served as fuel gases. The oven using hexane on flaxseeds (42.40% using Soxhlet and 41.90% using ASE temperature was raised from 75 to 240 ◦ C at 5 ◦ C/min and held at 240 ◦ C (Khattab & Zeitoun, 2013)) or echium seeds (31.3% using Soxhlet and for 20 min. The split value was 1:40 and isopropanol (≥99.5 ACS re­ 31.2% using ASE (Castejón et al., 2018)). One of the possible reasons agent, Merck KGaA, Darmstadt, Germany) was used as a rinsing agent. behind this could be the longer time the sample is left during Soxhlet Peak identification was performed using different techniques. First, a extraction, which may help to extract more oil when compared to the standard mix of alkanes (Standard Connecticut ETPH Calibration usually short static time employed in ASE extractions. Mixture (15 Components, C9 to C36 at concentration rate of 1000 μg/mL When comparing the same technique (ASE), ethyl ether was the dissolved in methylene chloride), Restek, Bellefonte, U.S.) was used to solvent achieving significantly greater extraction yield when compared selectively isolate and identify the hydrocarbons of the sample with the to ethanol, hexane or 2-MTHF (Fig. 1). The results indicated that SBO GC 6890 system. Secondly, the retention time of each fatty acid of in­ may contain mostly non-polar compounds that can be extracted by using terest appearing in the chromatogram was compared with the known a non-polar solvent such as hexane. However, there may also be other Kovats retention index. Finally, peak identification was performed with polar compounds found at lower concentrations that would benefit from a GC System 7890A attached to an MS triple-axis detector 5975C (Agi­ the extraction with diethyl ether. Similar results were reported using lent Technologies, California, U. S.) with the Wiley library and by Soxhlet extraction with diethyl ether on peach kernels, which achieved comparison of the retention time of bought standards of methylated significantly greater oil extraction yield when compared to all other fatty acids (Supelco 37 Component FAME Mix, Merck KGaA, Darmstadt, solvents, including hexane (Wu et al., 2011), or on potato peel oil Germany). Every sample analysis procedure was performed thrice, and mea­ surements by the appropriate equipment were performed in duplicate. 2.6. Statistical analysis All statistical analysis was performed with the software R-4.0. As­ sumptions were checked by first visually interpreting the Q-Q and boxplots from all analyses. Normality was double-checked by the Shapiro-Wilk test, which gave non-significant values for all the analysis, therefore proving the normality of the whole data. Subsequently, the statistical analysis of the data was performed. A two-way ANOVA was first run to understand the importance of the interaction between the temperature and the solvent on the final extraction yield, and concen­ tration of α-tocopherol and β-carotene as well as the fatty acid profile. Further analysis involved the use of Tukey’s post hoc tests to understand possible significant differences within variables. The statistical signifi­ cance of the results was set at p < .05. Fig. 1. Extraction yield of SBO by ASE using different solvents at high pressure compared to Soxhlet used as a conventional method (Control). Bars display the average value of the resulting extractions at different temperatures (60, 90, 120 and 150 ◦ C). Different letters mean significant differences at p < .05. 3 A. Vilas-Franquesa et al. LWT 164 (2022) 113643 extracted with different organic solvents, including ethanol, hexane and diethyl ether (Zia-ur-Rehman, Habib, & Shah, 2004). The extractions using 2-MTHF and ethanol yielded significantly lower oil yield when compared to the “non-green” solvents. Due to the relatively high polarity of both green solvents (Table 1), the extraction yield was affected greatly. Ethanol yielded less oil than hexane using pressurized liquid extractions at all temperatures (Fig. 1), similar to what was found in echium seeds at different temperatures (Castejón et al., 2018), probably due to the lipophilic nature of the extract. Ethanol can extract the part of lipids that have a slightly polar behaviour, such as phospholipids or waxes, or even proteins (Dunford & Zhang, 2003). In the case of SBO, proteins or phospholipids are not expected to interfere greatly. Nevertheless, ethanol could also extract other water-miscible components present in dried berries. An important group of hydrophil­ ic molecules in dried SB berries is carbohydrates, specially sugars. SB Fig. 2. Average concentration of α-tocopherol and β-carotene in SBO extracted sugars could be mostly extracted when using ethanol, yielding up to 75% with different solvents. Error bar show SD. Bars display the average value of the of the initial sugar content (Baümler, Carrín, & Carelli, 2016). The resulting extractions at different temperatures (60, 90, 120 and 150 ◦ C). applied further treatment allowed the confirmation of the theoretical Different letters mean significant differences at p < .05. Comparisons are not reasoning. Great values of extraction yield (around 50 g of oil/100 g of made across upper- and lower-case letters (i.e. across vitamers). dried berries) were achieved during the first exploration with ethanol (results not shown), but when water was added to rinse the outcoming The concentration of α-tocopherol in SBO was greater in the samples oil, the water drained most of the hydrophilic extracted compounds, extracted by ASE (hexane) when compared to the same solvent and including sugars, obtaining the final values represented in Fig. 1. Soxhlet technique (control). The longer extraction times in Soxhlet Finally, the lowest yield was recorded for the extraction with 2- extraction may trigger the destabilization of α-tocopherol to a relevant MTHF, roughly achieving an extraction yield of 13% (Fig. 1). A lower extent in which differences between these two groups become signifi­ extraction yield using 2-MTHF was recorded previously by other authors cant. In contrast, the concentration of α-tocopherol in SBO extracted on Tunisian date palm seeds when compared to other solvents with diethyl ether or 2-MTHF was significantly lower than the concen­ (Ben-Youssef, Fakhfakh, Breil, Abert-Vian, & Chemat, 2017), but higher tration in control samples (Fig. 2). No significant differences were extraction yields were recorded for anise seeds when using 2-MTHF observed in the concentration of α-tocopherol in oils extracted with (Rebey et al., 2019). Consonant to what resulted by using ethanol, diethyl ether or 2-MTHF. This was interesting since previous authors 2-MTHF effectively achieved a preliminary extraction of higher yield found greater saponification values from peach kernel oil extracted with from SB dried berries. The original extraction yield (around 32%) ethyl ether rather than oil extracted with hexane, translating into a dropped to 13% after applying the water rinsing process. Thus, it was greater extraction of unsaponifiable compounds, including tocopherols considered that most of the ‘extra’ yield was derived from the extraction and carotenoids (Wu et al., 2011). of sugars or other water-soluble compounds. The difference between the Significant differences were also observed in the concentration of final yield of 2-MTHF and ethanol could be because of two main reasons. α-tocopherol between the control group and the extraction at 120 and First, it could be a consequence from the polarity difference (Table 1); 150 ◦ C, being higher in the latter two. The greater the temperature, the ethanol then extracted a greater polar fraction than 2-MTHF, and part of greater the recovery of α-tocopherol, which achieved a plateau after the it could remain in the final oil yield (after rinsing it with water) due to its extraction temperature was set at 120 ◦ C (Fig. 3). This was in line with great amount. Second, it could be a consequence of distinct further what other authors observed, although using only one solvent (hexane) processing. While in ethanol a normal rinse would suffice, with 2-MTHF and observing the steady recovery of tocopherols above 100 ◦ C (Sanagi, a centrifugation was needed to allow phase separation, leading to See, Ibrahim, & Naim, 2005). Greater temperatures generally allow possible residues of oil left in the centrifuge vial. getting better mass transfer rates between the solvent and the sample matrices, which directly increase the capacity of the solvent to solubilize 3.2. Concentration of α-tocopherol in SBO analytes (Sanagi et al., 2005). Lower recoveries of α-tocopherol were There was a significant main effect of the solvent used in the extraction on the concentration of α-tocopherol in the extracted oil (Fig. 2). The concentration of α-tocopherol in extracted SBO was greater in the oil extracted with ethanol than in the oil extracted with hexane, and both were significantly greater than the oil extracted by Soxhlet (control). The lowest concentration of α-tocopherol was observed in SBO extracted with diethyl ether or 2-MTHF. A matrix of methanol/chloro­ form also extracted higher amounts of different tocopherols when extracting oil from almond, Brazil nuts, hazelnuts or pecan nuts when compared to other less polar compounds (Miraliakbari & Shahidi, 2008). In addition, other authors reported a higher extraction of to­ copherols – including α-tocopherol – when using ethanol as compared to the extraction with hexane (Baümler et al., 2016). The results could be attributed to differences in solvent polarities (Table 1). Overall con­ centrations of extracted α-tocopherol ranged from 0.6 to 1.0 mg/g SBO, Fig. 3. Average concentration of α-tocopherol and β-carotene in SBO extracted values similar to what previous authors found in SB (Kallio, Yang, & at different temperatures. Error bars show SD. Bars display the average value of Peippo, 2002). It is important to mention that the values of the present the resulting extractions with different solvents (hexane, diethyl ether, ethanol research correspond to SB dried berries instead of different fractions and 2-MTHF). Different letters mean significant differences at p < .05. Com­ from the fruit (i.e. peel and seeds), differing from values found by other parisons are not made across upper- and lower-case letters (i.e. authors. across vitamers). 4 A. Vilas-Franquesa et al. LWT 164 (2022) 113643 observed in the control extraction, which may come from the longer temperatures up to 120 ◦ C, and significantly diminishing the concen­ extraction times and the subsequent oxidation of the analytes. tration of the analyte in the oil extracted at 150 ◦ C (Fig. 3). 3.3. Concentration of β-carotene in SBO 3.4. Concentration of monounsaturated (MUFA), polyunsaturated (PUFA) and saturated (SFA) fatty acids in SBO The recovery of β-carotene from sea buckthorn oil differed signifi­ cantly between all solvents, except the oil extracted with hexane using Considering the extraction with ASE – and excluding Soxhlet the technique ASE when compared to the same solvent using the Soxhlet extraction (Control) –, using less polar solvents yielded less SFA than methodology (control, Fig. 2). The use of different solvents was more using more polar solvents (Fig. 4). Due to the saturation degree of the relevant than the use of different techniques. However, more solvents SFA and the triglycerides in which they are packed, the less polar sol­ should be tried in the Soxhlet extraction in order to get more reliable vents should have been the solvents extracting more quantity (Tir, results to support this conclusion. Dutta, & Badjah-Hadj-Ahmed, 2012). Obtaining almost 10% more SFA The lowest concentration of β-carotene was found in oil extracted by Soxhlet extraction when compared to ASE extraction using hexane with diethyl ether and the greatest concentration was found in oil may serve as a proof that time could be an important factor when extracted with ethanol. Carotenoids had also been extracted in signifi­ considering the extraction of SFA from SB dried berries. At the extrac­ cantly greater amounts when using ethanol as the extraction solvent tion conditions used in the present research, it could be concluded that instead of other conventional solvents such as diethyl ether (Lich­ ethanol and 2-MTHF are suitable for the extraction of SFA from SBO, tenthaler & Wellburn, 1983) or acetone (Marsili & Callahan, 1993). yielding significantly greater concentrations than hexane or diethyl Ethanol was also found to be the most efficient solvent for the extraction ether, two solvents widely used for that purpose (Fig. 4). of β-carotene when compared to other green alternatives, such as ethyl The greatest concentration of MUFA in SBO was observed in the acetate and ethyl lactate (Ishida & Chapman, 2009). Thus, ethanol samples extracted with hexane – both extracted with ASE and Soxhlet –, comes out as a promising solvent for the extraction of β-carotene from followed by all other extractions (Fig. 4). Soxhlet extraction had been sea buckthorn dried berries. Like what was observed for α-tocopherol, also previously reported to extract greater amounts of MUFA than other the concentration of β-carotene in extracted SBO was significantly techniques, including supercritical fluid extraction and ASE, probably greater in the control samples when compared to the samples extracted due to the longer extraction time (Castejón et al., 2018; Rebey et al., with diethyl ether or 2-MTHF (Fig. 2). However, in this case, differences 2019; Reddy, Moodley, & Jonnalagadda, 2012). The higher extraction were not observed between the two extractions with hexane (control yield using hexane as compared with other solvents was in line with samples and ASE extraction), contrarily to what had been reported by what other authors observed (Bourgou et al., 2019). According to Saini and Keum (2018). Mezzomo, Mileo, Friedrich, Martínez, and Ferreira (2010), the reason Wu et al. (2011) found greater antioxidant capacity (in terms of for greater extraction may rely on the polarity index of the solvent, DPPH and Trolox equivalent antioxidant capacity assay (TEAC)) in the which is lower in apolar solvents when extracting long-chain fatty acids. peach kernel oil extracted with hexane when compared to the oil The fact that diethyl ether did not achieve similar extraction results for extracted with ethyl ether. Greater antioxidant capacity does not imply MUFA when compared to hexane extraction may be due to its higher greater vitamin concentration, yet vitamins could contribute signifi­ polarity index (4.4, compared to hexane (0) and ethanol (5.2) (Mezzomo cantly to this attribute. Hexane could then extract oil with greater et al., 2010)). antioxidant activity, and thus probably with greater vitamin concen­ All solvents achieved significantly different concentrations of PUFA tration, as observed in the present experiment. Nevertheless, different in the resulting SBO (Fig. 4). Control samples (Soxhlet) extracted vitamins could be present in the matrix, as it was observed in peach significantly lower amounts of PUFA when compared to any ASE kernel (tocopherols, carotenoids (Wu et al., 2011)), and it is thereupon extraction. Other authors also reported lower amounts of PUFA in oil difficult to assume whether the greater antioxidant capacity could come from caraway (Carum carvi) seeds extracted by using the Soxhlet tech­ from one vitamin or another. nique (and hexane as solvent) when compared to other techniques and The concentration of β-carotene extracted with diethyl ether was greener solvents (Bourgou et al., 2019). The longer extraction time when significantly lower compared to the values obtained by 2-MTHF using Soxhlet is a great barrier to the efficient recovery of PUFA in SBO. extraction. Diethyl ether is a less polar solvent when compared to 2- The long extraction time implies prolonged contact with air and pro­ MTHF, closer to the polarity of hexane (Table 1). Rebey et al. (2019) longed temperature exposure of the sample, two critical factors when reported higher antioxidant values of fennel and anise oils extracted extracting polyunsaturated molecules, as they may lead to degradation with hexane compared to oils extracted with 2-MTHF. β-carotene, together with lycopene, are highly lipophilic non-polar carotenoids mainly because of the lack of a functional polar groups in their structure (Saini & Keum, 2018), therefore making them theoretically chemically more prone to their extraction with non-polar solvents. Significant differences were also observed in the concentration of β-carotene between the control group and the extraction at any other temperature, being higher in the former (Fig. 3). It seemed that the degradation of β-carotene during extraction was slightly more influ­ enced by the temperature rather than by the exposure of the compound to the light and oxygen during the extraction (control extraction). This was clearly in contrast with what was observed during the extraction of α-tocopherol (Fig. 3). The concentration of β-carotene was significantly lower in the oil extracted at 150 ◦ C when compared to all other tem­ peratures. Other authors already reported losses of antioxidant capacity of carotenoids extracts – from Haematococcus pluvialis microalga – when Fig. 4. Average concentration of SFA, MUFA and PUFA in SBO extracted using increasing the extraction temperature (Jaime et al., 2010). The loss of different solvents. Error bars show SD. Bars display the average value of the antioxidant capacity was attributed to the loss of important carotenoid resulting extractions at different temperatures (60, 90, 120 and 150 ◦ C). fractions, being severe above 100 ◦ C. The present work adds evidence to Different letters - upper-, lower-case and Greek letters - mean significant dif­ this fact in a new matrix, with a stable extraction of β-carotene over ferences at p < .05. 5 A. Vilas-Franquesa et al. LWT 164 (2022) 113643 of the most unsaturated fatty acids. The results from the ASE extraction indicated that the less polar solvents achieved greater amounts of PUFA in the extracted SBO when compared to greener solvents (i.e. ethanol, 2-MTHF). The greater extraction of more polar lipids with apolar sol­ vents may be due to the form in which these are found in the original matrix; they may be bond to a triglyceride molecule (more apolar than the fatty acid itself) and thus more difficult for them to be extracted using polar solvents. The extract using diethyl ether yielded more quantity of PUFA when compared to its non-polar counterpart hexane, probably due to its relatively higher polarity (Mezzomo et al., 2010; Tir et al., 2012). Nevertheless, other authors have found hexane to yield higher amounts of PUFA in extracted oils when compared to other non-polar solvents (Wu et al., 2011), although the extraction was per­ formed using a Soxhlet apparatus, differing from what had been used in the present experiment. Extracting SBO with 2-MTHF yielded signifi­ cantly higher PUFA when compared to ethanol. Ethanol is stated to be Fig. 5. Average concentration of SFA, MUFA and PUFA in SBO extracted using slightly more polar than 2-MTHF, which in turn could influence the different temperatures. Error bars show SD. Bars display the average value of efficiency in recovery of fatty acids, especially PUFA (Fig. 4). the resulting extractions with different solvents (hexane, diethyl ether, ethanol and 2-MTHF). Different letters mean significant differences at p < .05. Com­ The results showed significant differences between the tested tem­ parisons are not made across upper-, lower-case and Greek letters (i.e. peratures on the recovery of SFA, MUFA and PUFA from SBO. SFA across groups). concentration in control samples was greater when compared to all other samples (Fig. 5). When it comes to ASE extractions, temperatures time when using Soxhlet (5h) when compared to the extraction time of of 120 and 150 ◦ C achieved statistically significantly lower concentra­ ASE (10 min). tions of SFA when compared to temperatures of 60 and 90 ◦ C (Fig. 5). In The extraction using green solvents achieves nutritionally good the case of polar solvents, such as ethanol and 2-MTHF, polarizability values when compared to the petroleum-based solvents, especially when may be negatively affected – so the polarity of the solvent is reduced – at using ethanol. The values of β-carotene concentration are far better in higher temperatures (Lu, Boughner, Liotta, & Eckert, 2002), which SBO after ethanol extraction when compared to all other solvents. For could then trigger a greater extraction of SFA at higher temperatures. that reason, ethanol should be considered positively when evaluating Results herein show otherwise, indicating a drop in the concentration of the extraction and isolation of β-carotene from sea buckthorn pulp, even SFA at higher temperatures (Fig. 5). There may be the need of rising when considering that the extraction with ethanol intrinsically carries more the temperature in order to see improvements in the extraction of the application of an extra processing step to wash the dragged polar SFA from SBO. Even though it may have an influence, it is also note­ compounds that influence the yield. In contrast, the fatty acid profile of worthy that the results show the effect on the overall extraction of SFA, SBO oil extracted with green solvents is worse than petroleum-based not only the effect of the extraction using the most polar solvents. solvents, with the noteworthy greater extraction of SFA. In contrast, extractions at higher temperatures (120 and 150 ◦ C) yielded a greater concentration of MUFA when compared to extractions at lower temperatures (60 and 90 ◦ C; Fig. 5). The fact that higher tem­ CRediT authorship contribution statement peratures may decrease the polarity of polar solvents may be the reason that could explain why at higher temperatures the extraction of MUFA Arnau Vilas-Franquesa: Investigation, Conceptualization, Meth­ was higher when compared to lower temperatures. However, all sol­ odology, Data curation, Writting and Editing. Bibiana Juan: Validation, vents may influence the results in the temperature, making it closer to a Reviewing and Editing. Jordi Saldo: Validation, Reviewing and Editing. conjecture than to a conclusion. At last, the concentration of PUFA in the SBO from control samples Declaration of competing interest was significantly lower than any other temperature (Fig. 5). PUFAs are the most oxidizable fatty acid molecules herein explored and are espe­ The authors declare that there is no conflict of interest. cially vulnerable at high temperatures, which makes them prone to degradation. The use of longer processing times in more cycles when using the Soxhlet extraction technique and the high extraction temper­ Acknowledgements atures employed in this technique could explain the lower concentration obtained in the resulting oil. In fact, other authors already observed Authors would like to express their gratitude to “Agència de Gestió lower recoveries of PUFA from Soxhlet when compared to ASE extrac­ d’Ajuts Universitaris i de Recerca (AGAUR)”, a public entity attached to tions (Castejón et al., 2018). From all other temperatures, the recovery the “Departament d’Empresa i Coneixement” of the “Generalitat de of PUFA seemed steady, except for 90 ◦ C, in which the recovery of PUFA Catalunya”, from which the first author received a predoctoral stu­ was significantly greater compared to 60 or 120 ◦ C. Interestingly, the dentship grant coded ‘2017 DI 079’. concentration of PUFA in SBO extracted at 150 ◦ C was non-significantly different from that obtained at 90 ◦ C (Fig. 5). This seemed to indicate References that there could exist an ideal temperature value for the extraction of Aycock, D. F. (2007). Solvent applications of 2-methyltetrahydrofuran in organometallic PUFA from SBO. and biphasic reactions. Organic Process Research & Development, 11(1), 156–159. https://doi.org/10.1021/op060155c 4. Conclusions Baümler, E. R., Carrín, M. E., & Carelli, A. A. (2016). Extraction of sunflower oil using ethanol as solvent. 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