Effects of cosolvents on the decaffeination of green tea by supercritical carbon dioxide

Due to the adverse effects of the caffeine in a variety of plant products, many methods have been explored for decaffeination, in efforts to remove or reduce the caffeine contained in plant materials. In this study, in order to remove caffeine from green tea (Camellia sinensis) leaves, we have employed supercritical carbon dioxide (SC–CO₂), which is known to be an ideal solvent, coupled with a cosolvent, such as ethanol or water. By varying the extraction conditions, changes not only in the amount of caffeine, but also in the quantities of the principal bioactive components of green tea, including catechins, such as epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG) and epicatechin (EC), were determined. The extraction conditions, including temperature, pressure and the cosolvent used, were determined to affect the efficacy of caffeine and catechin extraction. In particular, the type and concentration of a cosolvent used constituted critical factors for the caffeine removal, combined with minimal loss of catechins, especially EGCG. When the dry green tea leaves were extracted with SC–CO₂ modified with 95% (v/v) ethanol at 7.0 g per 100 g of CO₂ at 300 bar and 70 °C for 120 min, the caffeine content in the decaffeinated green tea leaves was reduced to 2.6% of the initial content. However, after the SC–CO₂ extraction, a substantial loss of EGCG, as much as 37.8% of original content, proved unavoidable.     

Extraction and fractionation of alkylresorcinols from triticale bran by two-step supercritical carbon dioxide

Bran from triticale cultivars Ultima and Pronghorn and Canadian Prairie Spring (CPS) wheat were fractionated using a two-step sequential supercritical carbon dioxide (SC-CO₂) extraction technique with and without ethanol to selectively extract and enrich high-value alkylresorcinols (ARs). Pure SC-CO₂ (70 °C; flow rate 25 g/min; pressure 35 MPa), was used for the first extraction step. The non-polar lipids yield ranged from 3.08 to 3.26 g/100 g and they were composed mainly of C18:2 (33-41 g/100 g), C18:1 (15-21 g/100 g), C16:0 (15-19 g/100 g), and C18:3 (4-5 g/100 g) fatty acids. The second extraction step, performed on the bran remaining after the first extraction, was carried out with SC-CO₂ plus 0.5 mL/min ethanol (70 °C; flow rate 5 g/min; pressure 35 MPa). The resulting total polar lipid yield of bran samples ranged between 0.62 and 0.86 g/100 g and the main compounds present in the polar lipids were ARs. The total AR contents of triticale cv Ultima, wheat, and triticale cv Pronghorn were 70, 68 and 36 mg/100 g, respectively. Saturated and unsaturated AR homologues of C15:0, C17:0, C19:0, C19:1, C21:0, C21:1, C23:0 and C25:0 were detected in all samples. Over 98% of the triticale ARs and over 95% of the wheat ARs were extracted under the SC-CO₂ conditions used.     

Proximate composition and extraction of carotenoids and lipids from Brazilian redspotted shrimp waste (Farfantepenaeus paulensis)

The chemical composition of redspotted shrimp (Penaeus paulensis) waste was investigated. The shrimp waste (freeze-dried heads, shells and tails) was found to have high protein (49% d.w.) and ash (27% d.w.) contents, but a low lipid content (4.9% d.w.) although the latter was higher than those found in other kinds of shrimp captured in Brazil. The fatty acid compositions showed that the lipids had a high content of unsaturated fatty acids, mainly EPA (C20:5; n-3) and DHA (C22:6; n-3). In order to establish an efficient and environmentally friendly recovery process for the astaxanthin (principal carotenoid and antioxidant present in the waste), the following processes were examined: traditional solvent extraction (TSE), super-critical fluid extraction (SC-CO₂) and super-critical fluid extraction with co-solvent (SC-CO₂ + ethanol). The temperature and pressure conditions for all the SC-CO₂ extractions were 50 °C and 30.0 MPa. The results showed that the mixture of 60% (v/v) n-hexane:isopropyl alcohol gave the highest (53 mg/kg waste) carotenoid extraction yield as compared to acetone, SC-CO₂ and SC-CO₂ + ethanol. The SC-CO₂ showed the lowest extraction yield of astaxanthin, but the addition of the entrainer (10% w/w) produced an important effect, increasing the astaxanthin extraction to values of 57.9%, similar to extraction with acetone (63.3%).     

Selective Extraction of Phospholipid Mixtures by Supercritical Carbon Dioxide and Cosolvents

To optimize and enhance the value of a previously developed supercritical fluids (SF) process for removing oil from soybean flakes, we devised a two-step, sequential scheme for extraction of oil and phospholipid-containing fractions using SC-CO₂ alone or with ethanol. PLs were selectively removed from the flakes using the SC-CO₂/ethanol mixture. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were more readily solubilized in the SC-CO₂/cosolvent mixtures than were phosphatidylinositol and phosphatidic acid. The extent of recovery of PC and PE was a function of the molar fraction of cosolvent. Some fractionation of constituent phospholipids could be achieved by varying the molar fraction of ethanol. The extracts from the SF process were characterized by inductively coupled plasma spectroscopy (ICP) and HPLC with evaporative light scattering detection.     

Addition of ethanol to supercritical carbon dioxide enhances the inactivation of bacterial spores in the biofilm of Bacillus cereus

Supercritical carbon dioxide (SC-CO₂) was used to inactivate Bacillus cereus spores inside biofilms, which were grown on stainless steel. SC-CO₂ treatment was tested using various conditions, such as pressure treatment (10–30 MPa), temperature (35–60 °C), and time (10–120 min). B. cereus vegetative cells in the biofilm were completely inactivated by treatment with SC-CO₂ at 10 MPa and at 35 °C for 5 min. However, SC-CO₂ alone did not inactivate spores in biofilm even after the treatment time was extended to 120 min. When ethanol was used as a cosolvent with SC-CO₂ in the SC-CO₂ treatment using only 2–10 ml of ethanol in 100 ml of SC-CO₂ vessel for 60–90 min of treatment time at 10 MPa and 60 °C, B. cereus spores in the biofilm were found to be completely inactivated in the colony-forming test. We also assessed the viability of SC-CO₂-treated bacterial spores and vegetative cells in the biofilm by staining with SYTO 9 and propidium iodide. The membrane integrity of the vegetative cells was completely lost, while the integrity of the membrane was still maintained in most spores. However, when SC-CO₂ along with ethanol was used, both vegetative cells and spores lost their membrane integrity, indicating that the use of ethanol as a cosolvent with SC-CO₂ is efficient in inactivating the bacterial spores in the biofilm.     

Supercritical Carbon Dioxide Extraction of Squalene from <Emphasis Type="Italic">Amaranthus paniculatus</Emphasis>: Experiments and Process Characterization

Separation of squalene from Amaranthus paniculatus using supercritical carbon dioxide extraction (SC-CO₂) technology and optimization of its process parameters such as temperature, pressure, time of extraction, flow rate of carbon dioxide, and batch size have been conducted. The optimized conditions that provided the best yield of squalene were a sample size of 40 g of amaranth grains of particle diameter of 0.75 mm at a temperature of 100°C and pressure of 550 bar for 1.5 h extracting time at a flow rate of 0.2 L min⁻¹ of carbon dioxide. Solubility of squalene in SC-CO₂ under different operating conditions has also been evaluated using Chrastil equation which showed good agreement with the experimentally obtained yields. Various statistical analyses (regression equations, t test, and analysis of variance) conducted on the extraction parameters concluded that extraction pressure, time and sample batch size have significant effect on the yield of the squalene whereas extraction temperature and particle diameter do not. The results obtained are in accordance to the basic principle of supercritical fluid-phase equilibrium behavior and the solubility isobar and isotherm obtained showed similar trends with those reported for squalene. Using dimensionless numbers, an empirical correlation was also deduced for characterization of the extraction process of squalene in SC-CO₂.     

Extraction of boldo (Peumus boldus M.) leaves with supercritical CO2 and hot pressurized water

High-activity fractions in boldo leaves were extracted with supercritical CO₂ (SC-CO₂) and hot pressurized water (HPW). Total yield after 3 h of extraction (0.6–3.5%) in low-pressure SC-CO₂ experiments increased with process pressure (60–150 bar) and decreased with temperature (30–60 °C), as expected. The extract obtained with SC-CO₂ at 50 °C and 90 bar contained approximately 50% of essential oil components. In higher pressure experiments with solvent mixtures, the yield increased with pressure (300–450 bar) and modifier concentration (2–10% ethanol), ranging 0.14–1.95 ppm for the alkaloid boldine and 1.8–4.8% for total solids following 1.5 h treatment at 50 °C. Boldine recovery was solubility-controlled, reaching 7.4 ppm after 7-h extraction at 450 bar and 50 °C using an ethanol–SC-CO₂ mixture (5% co-solvent). Boldine solubility and yield decreased when using pure CO₂ at higher pressure (600 bar, 50 °C). The extract yield after 3 h batch-wise HPW extraction increased from 36.9% at 100 °C to 53.2% at 125 °C, and then decreased as temperature was increased to 175 °C. Boldine yield decreased from 26.8 ppm at 100 °C to 0.7 ppm at 125 °C, and was negligible at ⩾150 °C. The essential oil yield increased to a maximum at 110 °C and was negligible at ⩾150 °C also. The ranking of antioxidant potency of various extracts was as follows: HPW at 110 °C > methanol (soxhlet extraction) ≫ high-pressure SC-CO₂ with or without polar co-solvent > low-pressure SC-CO₂.     

Optimization of Supercritical Carbon Dioxide Extraction of Xanthones from Mangosteen Pericarp by Response Surface Methodology

Mangosteen fruit pericarp is one of the important sources of bioactive compound xanthone. Supercritical carbon dioxide (SC-CO₂) extraction was employed to extract xanthones from mangosteen fruit pericarp at three different levels of pressure (200–300 bar), temperature (40–60 °C) and solvent to material ratio (100–300 kg/kg). The optimal conditions for the total xanthone yield and the influence of parameters were determined by response surface methodology (RSM) using Box–Behnken design. In our study, the increase in total xanthone yield in SC-CO₂ fluid extraction depends more on the solute’s vapor effect. From response surface plots, pressure, temperature and solvent to material ratio exhibited independent and interactive effects on the extraction of xanthones. A regression equation for predicting the total xanthone yield was derived by statistical analysis, and a model with predictive ability of 0.99 was obtained. Maximum xanthone yield of 8.01% was predicted by RSM at 60 °C, 300 bar and a solvent to material ratio of 300 kg/kg while experimentally a yield of 7.56% was achieved. HPLC analysis was carried out for the optimum conditions for the identification and quantification of the xanthones. The antioxidant activities of the extracts were investigated by ferric reducing antioxidant power (FRAP) and the results showed that the extracts were enriched with antioxidant compound.     

Dynamic microwave-assisted extraction coupled on-line with clean-up for determination of caffeine in tea

A rapid method based on dynamic microwave-assisted extraction (DMAE) coupled on-line with clean-up was developed for the determination of caffeine in tea samples. A TM₀₁₀ microwave resonance cavity was applied to concentrate the microwave energy. An extraction vessel was placed in microwave irradiation zone of the TM₀₁₀ microwave resonance cavity. A silica gel column connected with the extraction vessel was used to remove chlorophyll in tea. The extraction and clean-up procedures were carried out simultaneously in a single step. The DMAE parameters were optimized by the Box-Behnken design. The maximum extraction efficiency was achieved using 70 W of microwave power, 3.5 mL of extraction solvent and 1.0 mL min⁻¹ of extraction solvent flow rate. The limit of detection obtained is 0.012 mg g⁻¹. The RSDs of intra- and inter-day of eight kinds of tea samples are 2.7%-5.4% and 5.1%-7.3%, respectively. The recovery of caffeine in the tea samples is in the range of 88.2%-99.3%.The proposed method has selectivity and sensitivity and considerably less labor and time for determination of caffeine in tea compared with conventional extraction methods, such assoxhlet extractionand liquid-liquid extraction.     

Response surface methodology applied to supercritical carbon dioxide extraction of Piper nigrum L. essential oil

This study was conducted to optimize the supercritical carbon dioxide (SC-CO₂) extraction of Piper nigrum L. essential oil using response surface methodology (RSM). In order to obtain the maximum yield of the essential oil, experiments were carried out using a three-factor central composite design (CCD) under following conditions: pressure of 15–30 MPa, temperature of 40–50 °C and dynamic extraction time of 40–80 min. A second-order polynomial regression model expressing the total extraction yield as a function of main SC-CO₂ variables was significantly (p < 0.05) fitted, with high coefficient of determination value (R² > 0.985). The results showed that the best extraction yield (2.16%) was obtained at 30 MPa, 50 °C and 80 min. Pressure showed the most significant (p < 0.05) effect on the yield variation. The chemical composition of the essential oil was determined using GC-flame ionization detection (FID) and gas chromatography–mass spectrometry (GC–MS) analysis. The main constituents (concentration > 3.0%, calculated as % peak area) in the P. nigrum L. essential oil obtained through SC-CO₂ extraction were determined to be β-caryophyllene (24.34%), limonene (15.84%), sabinene (15.04%), 3-carene (9.44%), β-pinene (9.27%) and copaene (4.52%).     

Concentration of tocopherols by supercritical carbon dioxide with cosolvents

In order to optimize the conditions for enrichment of tocopherols, tocopherol extraction from chemically modified rapeseed deodorizer distillate (RDD) at three levels (2%, 4%, and 6%) of each cosolvent (methanol, ethanol, and mixed ethanol) was carried out by using supercritical carbon dioxide (SC-CO₂). After chemical modification, the fatty acid methyl esters (FAME) in RDD were produced and showed improved solubility in SC-CO₂. Since FAME were more volatile, they were extracted preferentially over tocopherols and other higher molecular weight compounds. Two steps were applied in the SC-CO₂ extraction. First, the FAME mixture was extracted under 120 bar until the weight of extracts did not increase anymore, and then, tocopherols were concentrated under 250 bar. Second, the concentrates from the first step under 250 bar were esterified and then extracted again under 120 bar until the weight of extracts did not increase anymore. Different cosolvents (methanol, ethanol, mixed ethanol) were used for tocopherol isolation under 250 bar in the second step. Tocopherol content, tocopherol recovery and the weight of target extract (those extracts with a concentration of tocopherols above 40%) increased while the extraction time decreased.     

Response surfaces of apricot kernel oil yield in supercritical carbon dioxide

Response surface methodology was used to determine the effects of solvent flow rate (2, 3 and 4 g/min), pressure (30, 37.5 and 45 MPa), temperature (40, 50 and 60 °C), and co-solvent concentration (0, 1.5 and 3 wt% ethanol) on oil yield of apricot (Prunus armeniaca L.) kernel oil in supercritical carbon dioxide (SC-CO₂). All the parameters had significant effects on oil yield as well as the interactions between solvent flow rate and pressure, and between pressure and temperature. Oil yield increased with increased parameters. The oil yield was represented by a second-degree polynomial equation. The maximum oil yield from the response surface equation was obtained as 0.26 g/g kernel for 15 min extraction of 5 g apricot kernel particles (particle diameter<0.850 mm) with 4 g/min solvent flow rate containing 3 wt% ethanol at 45 MPa and 60^oC. The response surface equation predicted the experimental oil yield with a 10% error. The fatty acid compositions of apricot kernel oils extracted with SC-CO₂ and hexane were similar.     

Extraction of oil from tiger nut (Cyperus esculentus L.) with supercritical carbon dioxide (SC-CO2)

Supercritical carbon dioxide (SC-CO₂) was used to extract oil from tiger nuts and the physicochemical properties and the impact of extraction conditions [i.e., temperature (40 °C–80 °C), pressure (20–40 MPa) and time (60–360 min)] on the oil yield were studied. The response surface analysis results revealed that the oil yield was significantly (P < 0.05) influenced by the main effect of the extraction pressure, extraction time and their quadratic effects respectively. However, the interaction between the extraction temperature and time had no significant (P > 0.05) effect on the oil yield. The highest oil yield was 26.28 g/100 g sample after 210 min of extraction time at 30.25 MPa and 60 °C respectively. The fatty acid composition of oils obtained by SC-CO₂ and Soxhlet showed marked variation. Also, the fatty acid composition varied depending on the operating conditions. The viscosity of the oil decreased with the increase in temperature.     

Optimization of Supercritical Carbon Dioxide Extraction of Bioactive Flavonoid Compounds from Spearmint (<Emphasis Type="Italic">Mentha spicata</Emphasis> L.) Leaves by Using Response Surface Methodology

The bioactive flavonoid compounds of spearmint (Mentha spicata L.) leaves were obtained by using supercritical carbon dioxide (SC-CO₂) extraction. Extraction was carried out according to face-centred central composite design, and independent variables were pressure (100, 200 and 300 bar), temperature (40, 50 and 60 °C) and co-solvent amount (3, 6 and 9 g/min). The extraction process was optimized by using response surface methodology for the highest crude extraction yield of bioactive flavonoid compounds. The optimal conditions were identified as 209.39 bar pressure, 50.00 °C temperature and 7.39 g/min co-solvent amount. The obtained extract under optimum SC-CO₂ condition was analysed by high-performance liquid chromatography. Seven bioactive flavonoids including catechin, epicatechin, rutin, luteolin, myricetin, apigenin and naringenin were identified as major compounds. The results of quantification showed that spearmint leaves are potential source of antioxidant compounds.     

Decaffeination of fresh green tea leaf (Camellia sinensis) by hot water treatment

Hot water treatment was used to decaffeinate fresh tea leaf in the present study. Water temperature, extraction time and ratio of leaf to water had a statistically significant effect on the decaffeination. When fresh tea leaf was decaffeinated with a ratio of tea leaf to water of 1:20 (w/v) at 100 °C for 3 min, caffeine concentration was decreased from 23.7 to 4.0 mg g⁻¹, while total tea catechins decreased from 134.5 to 127.6 mg g⁻¹; 83% of caffeine was removed and 95% of total catechins was retained in the decaffeinated leaf. It is considered that the hot water treatment is a safe and inexpensive method for decaffeinating green tea. However, a large percentage of tea catechins was lost if rolled leaf and dry tea were decaffeinated by the hot water treatment and so the process is not suitable for processing black tea.     

Extraction of α-tocopherol and γ-oryzanol from rice bran

The aim of this research was to study the effect of operating mode (continuous versus batch+continuous), temperature, pressure and solvent on α-tocopherol and γ-oryzanol extraction from rice bran (Oryza sativa Linn.) and compare the efficiency of three extraction methods: SC-CO₂ extraction, solvent extraction and soxhlet extraction. Three sets of experiments were performed. First, extraction using SC-CO₂ was performed over a range of temperatures and pressures (45-65 °C and at 38 and 48 MPa), and at a CO₂ flow rate of 0.45 mL/min. The results showed that the best conditions for α-tocopherol extraction were 55 °C, 48 MPa in the batch+continuous mode. For γ-oryzanol, the best conditions were 65 °C, 48 MPa and in the continuous mode. In the second set of experiments, solvent extraction using hexane and ethanol at 32 and 55-60 °C was studied. The results showed that none of the solvents could extract α-tocopherol; however, ethanol at 55-60 °C was suitable for γ-oryzanol extraction. Finally, soxhlet extraction experiments using hexane for α-tocopherol and ethanol for γ-oryzanol were also performed. In summary, SC-CO₂ was found to be the best solvent for extracting both α-tocopherol and γ-oryzanol from rice bran, because of its higher yields and extraction rate.     

Effects of supercritical fluid extraction parameters on lycopene yield and antioxidant activity

The effects of various parameters of supercritical carbon dioxide (SC-CO₂) fluid extractions of tomato skins on the extraction yields and antioxidant activities of lycopene-rich extracts were investigated. A Box–Behnken design was applied to study the effects of three independent variables (temperature ranging from 40 to 100 °C, pressure ranging from 20 to 40 MPa, and flow rate ranging from 1.0 to 2.0 mL/min) on lycopene yield. The model showed good agreement with the experimental results, by the coefficient of determination (r² = 0.9834). Temperature, pressure, and the quadratic term for the temperature of SC-CO₂ extraction were large significantly positive factors affecting lycopene yield (P < 0.05). The maximum total lycopene content of 31.25 μg/g of raw tomato was extracted at the highest temperature of 100 °C, 40 MPa and 1.5 mL/min. TEAC assay was applied to assess the antioxidant activity of lycopene-rich extracts from SC-CO₂ fluid extraction. The effects of SC-CO₂ fluid extraction parameters on the antioxidant activities of the extracts differed with the yield. For each unit of lycopene extract, the antioxidant activity level was constant below 70 °C, but then gradually decreased above 70 °C due to isomerization occurring as a result of the higher temperature. The ratio of all-trans-lycopene to the cis-isomers changed from 1.70 to 1.32 when the operating temperature was adjusted from 40 to 100 °C, indicating an increased bioavailability due to the generation of the cis-isomers. No significant effects of pressure or flow rate of SC-CO₂ fluid extraction on the antioxidant activity were observed.     

SUPERCRITICAL CO2/ETHANOL EXTRACTION OF ASTAXANTHIN FROM BLUE CRAB (CALLINECTES SAPIDUS) SHELL WASTE

Astaxanthin (AX) is the major naturally occurring carotenoid pigment in marine crustaceans and the flesh of salmonids. These organisms are unable to synthesize AX de novo and when farmed commercially, require it in their feed. The high cost of synthetic AX has promoted research into new natural sources of ihe pigment, such as crustacean wastes. In this work, AX from demineralized crab (Callinectes sapidusj shell waste was extracted with a mixture of supercritical C₂ and ethanol as a cosolvent. The effect of total solids load, pressure and temperature was assessed by response surface methodology (RSM). Extracted AX was determined by HPLC. The experimental data were fined to a second order model whereby the conditions for maximum extraction yield were defined (≥ 34 MPa, 45C and solids load of 25 g). Pressure and solids load were the most important factors affecting AX extraction yields.     

Preparation of anthocyanin-loaded liposomes using an improved supercritical carbon dioxide method

Anthocyanin-loaded liposomes were prepared via a single step supercritical carbon dioxide (SC-CO₂) process. Phospholipid/anthocyanin suspension equilibrated with CO₂ was depressurized at a constant pressure and rate. The effects of pressure, depressurization rate and temperature on the characteristics of liposomes were investigated. Liposomes obtained had a mean diameter of 160 ± 2 nm, polydispersity index of 0.26 ± 0.01, encapsulation efficiency of 52.2 ± 2.1% and zeta potential of − 44.3 ± 2.9 mV. Elevated pressure and depressurization rate generated smaller particles with higher uniformity while high temperature led to reduced sphericity. The SC-CO₂ method produced liposomes with enhanced intactness, sphericity and uniformity compared to the thin film hydration method. This method offers the possibility to utilize dense phase CO₂ to process the phospholipid aggregates into nano/micro particles and control their characteristics via tuning of processing parameters. SC-CO₂ method shows promise in scalable production of liposomes loaded with a variety of bioactives, targeting food applications.Industrial relevanceHigh quality anthocyanin-loaded liposomes were prepared using SC-CO₂. In this process, heterogenous phospholipid aggregates suspended in an aqueous medium are transformed into unilamellar and spherical liposomes with a narrow size distribution and other characteristics that can be regulated via tuning of the processing parameters (pressure, depressurization rate and temperature). The SC-CO₂ method resulted in superior particle characteristics over those prepared via the traditional thin film hydration method and also overcame some drawbacks like organic solvent residue and vesicle leakage associated with the traditional method. Anthocyanin encapsulated into liposomes can be protected from adverse external conditions with potential benefits in food and nutraceutical formulations for improved efficacy and health benefits. This improved SC–CO₂ process shows great promise for potential scale up of liposome production in the industry to encapsulate a variety of bioactives, targeting food and nutraceutical applications.     

Optimization of supercritical CO2 extraction of phytosterol-enriched oil from Kalahari melon seeds

Supercritical carbon dioxide (SC-CO₂) extraction of oil from Kalahari melon seeds was investigated in this study. Response surface methodology was applied to model and optimize the extraction, namely pressure (200–400 bar), temperature (40–80 °C), and supercritical fluid flow rate (10–20 mL/min). Well-fitting models were successfully established for oil recovery (R ² = 0.9672) and phytosterol concentration (milligrams per 100 g; R ² = 0.8150) through multiple linear regressions with backward elimination. The effect of supercritical fluid flow rate was the most significant (P < 0.05) factor that affected oil recovery but this factor had no significant (P > 0.05) effect on phytosterol concentration. The optimal processing conditions for oil recovery and phytosterol concentration were pressure of 300 bar, temperature at 40 °C, and supercritical fluid flow rate of 12 mL/min. These optimal conditions yielded a 76.3% oil recovery and 836.5 mg/100 g of phytosterol concentration. The oil content in the Kalahari melon seeds as estimated by Soxhlet extraction was around 30.5/100 g. The phytosterol concentration in the oil extracted with SC-CO₂ extraction was 94% higher than that obtained with solvent extraction.