Extraction of bioactive enriched fractions from Eruca sativa leaves by supercritical CO2 technology using different co-solvents

Supercritical fluid extraction from freeze-dried Eruca sativa leaves is assessed with the aim of studying the feasibility to obtain bioactive enriched fractions containing different classes of valuable compounds. Total extraction yields and compositions using pure CO₂ and CO₂ + selected co-solvents are compared. Overall extraction curves, fitted by the model of broken and intact cells developed by Sovová, are reported and the influence of the main parameters that affect the extraction process is analysed. The extract with the highest content in glucosinolates and phenols was collected at 30 MPa and 75 °C using 8% (w/w) of water with respect to the CO₂ flow rate, whereas the fraction richest in lipids was obtained using 8% (w/w) of ethanol as co-solvent at 45 °C and 30 MPa. A process including a first step with supercritical CO₂ extraction using water as co-solvent followed by a second step, where a fraction rich in lipids is extracted using ethanol as co-solvent, is proposed. SCCO₂ results are compared with Soxhlet and other methods that combine organic solvents with ultrasounds.     

Supercritical CO2 and low-pressure solvent extraction of mango (Mangifera indica) leaves: Global yield,extraction kinetics,chemical composition and cost of manufacturing

Low-pressure solvent extraction (LPSE) and supercritical fluid extraction (SFE) were used to obtain extracts from mango (Mangifera indica) leaves. Kinetics curves were determined for both methodologies. The extracts chemical compositions and manufacturing costs were determined for both processes. Global yield isotherms for SFE process were determined at 10–40 MPa and 313–323 K. The highest yield was 2.24% at 30 MPa and 323 K; the LPSE yield (9.3%) was almost three times higher than that of SFE (3.6%). Thin layer chromatography showed that mango leaves extracts have several classes of compounds as alkaloids, flavonoids and terpenoids, recovered by both methods. The cost of manufacturing (COM) mango leaves extracts were US$ 32/kg and US$ 92/kg for LPSE and SFE, respectively.     

Extraction of low-molar-mass phenolics and lipophilic compounds from Pinus pinaster wood with compressed CO2

Near-supercritical and supercritical CO₂ was used to extract low-molar-mass phenolics and lipophilic compounds from Pinus pinaster wood. Extraction of samples containing sapwood and knotwood was carried out at 10⿿25 MPa and 30⿿50 °C to assess the influence of the operational conditions on the yields of total extracts and phenolics, as well as on the radical scavenging capacity of extracts. The use of ethanol as a co-solvent increased both the extraction yields and the concentration of phenolics in extracts. Operating under selected conditions (25 MPa, 50 °C, 10% ethanol), the extraction yield accounted for 4.1 wt% of the oven-dry wood. The extracts contained up to 7.6 g of phenolic compounds (measured as gallic acid equivalents) per 100 g extract, and showed one third of the radical scavenging capacity of Trolox. Native resin acids accounted for about 24 g per 100 g extracts, whereas flavonoids, lignans, stilbenes and juvabiones were found at lower proportions.     

Enrichment of Nigella damascena extract with volatile compounds using supercritical fluid extraction

β-Elemene, germacrene A and damascenine were extracted from lady-in-a-mist (Nigella damascena L.) seeds with supercritical carbon dioxide at 10–30 MPa and 40–60 °C. The influence of supercritical fluid extraction (SFE) conditions on the yield and concentration of volatiles in the extract and the extraction kinetics were studied. The extraction yields and the apparent solubility of volatile compounds increased with increasing density of CO₂. The highest total yield was obtained at 30 MPa and 40 °C but the selectivity for volatiles was low under these conditions. With respect to both yield of volatiles and their concentration in extract, the best results were at 12 MPa and 40 °C, either with one separator or with additional separator maintained at 5 MPa and 25 °C. The yields of β-elemene, germacrene A and damascenine reached 0.72, 3.31 and 3.65 mg g⁻¹ and their concentration in the extract was 2.62, 12.04 and 13.28 wt.%, respectively. Though the yields of germacrene A and damascenine were by about 20% higher using Soxhlet extraction with hexane than using SFE, their concentration in the extract where fatty oil prevailed was only 1.19 and 1.20 wt.%, respectively. Under the conditions of hydrodistillation, partial conversion of germacrene A to β-elemene occurred and its yield was higher than using the other methods but the composition of volatiles in the SFE extracts better corresponds to the original raw material.     

Fractionated extraction of saponins from Brazilian ginseng by sequential process using supercritical CO2, ethanol and water

Saponins are surfactants that reduce the surface tension of aqueous solutions, besides having pharmacological actions. In order to extract and fractionate saponins from Pfaffia glomerata roots and Hebanthe eriantha roots using supercritical technology, fractionated extracts were obtained from a sequential process in fixed bed using supercritical CO₂ (scCO₂), ethanol, and water as solvents. All extractions were carried out in four sequential steps, at 50 °C and 300 bar. In the first step, pure scCO₂ was used as solvent, while (a) scCO₂/etanol (70:30, w/w); (b) ethanol, and (c) ethanol/water (70:30, v/v) were used as solvents in the three subsequent steps. The extracts were analyzed by thin layer chromatography (TLC) and surface tension. The extraction yields of the four steps were 0.16, 0.55, 1.00, and 6.90% for P. glomerata roots, and 0.17, 0.58, 0.89, and 28% for H. eriantha roots, showing a predominance of high polarity compounds in these species. TLC analysis showed that the extraction process was selective according to the polarity of the solvent, and provided extracts containing different saponins, except for scCO₂ extraction. The extracts from the extraction using ethanol + scCO₂ (Step 2) showed the greatest ability to reduce the surface tension of water from 72 mN m⁻¹ (pure water) to 25 mN m⁻¹, suggesting that this step was the best for extraction of less polar saponins in the extracts. The critical micelle concentration (CMC) values were approximately 2 and 8 g L⁻¹ for P. glomerata and H. Eriantha, respectively. These results confirmed the efficacy of the extraction process under study.     

Supercritical carbon dioxide extraction of Capsicum peppers: Global yield and capsaicinoid content

The objective of this study was to select a variety of pepper with high concentration of capsaicin and subject it to supercritical fluid extraction (SFE), in order to determine the best conditions of temperature (40, 50 and 60 °C) and pressure (15, 25 and 35 MPa) in terms of global yield (X₀) and capsaicinoids content of the extracts. The influence of drying process (freeze and oven drying) on X₀, capsaicin (C) and dihydrocapsaicin (DHC) contents and total phenolics was also analyzed. Capsicum frutescens showed the highest levels of capsaicinoids (1516 μg/g fresh fruit). For the responses C and DHC, the extraction conditions of 15 MPa and 40 °C provided the highest concentrations (C ⿿ 42 mg/g extract and DHC ⿿ 18.5 mg/g extract). The freeze drying process resulted in extracts with the highest concentration of capsaicinoids (61 mg/g extract), but in contrast, the phenolics were less susceptible to different drying processes, with a mean concentration of 35 mg GAE/g extract. The kinetics experiments indicated that the extraction rate of oleoresin was slightly slower than that of capsaicinoids at the operation conditions (40 °C and 15 MPa).     

Supercritical fluid extracts from tamarillo (Solanum betaceum Sendtn) epicarp and its application as protectors against lipid oxidation of cooked beef meat

The possibility of using the tamarillo (Solanum betaceum (Cav.) Sendtn (syn. Cyphomandra betacea)) epicarp as source of compounds with antioxidant activity in cooked beef meat (CBM) was explored. Extracts from tamarillo by supercritical fluid extraction (SFE) and Soxhlet extraction (SE) were obtained. The SFE was performed using pure CO₂ at different temperatures and pressures (40 and 50 °C; 10, 20 and 30 MPa) and CO₂ added with ethanol (CO₂/EtOH) as co-solvent (2, 5 and 8%, w/w). The SFE kinetics and mathematical modeling of the overall extraction curves (OEC) were also investigated. EtOH and hexane were used in the SE. The antioxidant activity (AA) of extracts was evaluated in CBM as well as the protection against lipid oxidation was determined by measuring lipid hydroperoxides (LHP) and thiobarbituric acid reactive species (TBARS). The extract obtained by SFE with CO₂/EtOH (50 °C/30 MPa and 2% of EtOH) showed the highest AA. In SFE, the co-solvent addition improved considerably the AA and the extraction yield. The extracts obtained by SFE with CO₂/EtOH showed a better AA compared with the synthetic antioxidant TBHQ. The highest yield values were achieved by SE with ethanol (7.7 ± 0.4%) and by SFE with 5% EtOH (1.9 ± 0.1%). The results indicate that extracts of tamarillo epicarp are a potential source of antioxidant compounds.     

Extraction of phenolic compounds and anthocyanins from blueberry (Vaccinium myrtillus L.) residues using supercritical CO2 and pressurized liquids

This work explored the potential of subcritical liquids and supercritical carbon dioxide (CO₂) in the recovery of extracts containing phenolic compounds, antioxidants and anthocyanins from residues of blueberry (Vaccinium myrtillus L.) processing. Supercritical CO₂ and pressurized liquids are alternatives to the use of toxic organic solvents or extraction methods that apply high temperatures. Blueberry is the fruit with the highest antioxidant and polyphenol content, which is present in both peel and pulp. In the extraction with pressurized liquids (PLE), water, ethanol and acetone were used at different proportions, with temperature, pressure and solvent flow rate kept constant at 40 °C, 20 MPa and 10 ml/min, respectively. The extracts were analyzed and the highest antioxidant activities and phenolic contents were found in the extracts obtained with pure ethanol and ethanol + water. The highest concentrations of anthocyanins were recovered with acidified water as solvent. In supercritical fluid extraction (SFE) with CO₂, water, acidified water, and ethanol were used as modifiers, and the best condition for all functional components evaluated was SFE with 90% CO₂, 5% water, and 5% ethanol. Sixteen anthocyanins were identified and quantified by ultra performance liquid chromatography (UPLC).     

Supercritical CO2 extraction of bioactive Tyrian purple precursors from the hypobranchial gland of a marine gastropod

Supercritical CO₂ provides considerable advantages over traditional solvents for the extraction of bioactive compounds from organic matter. Here we demonstrate the use of supercritical CO₂ as an efficient and safe alternative to traditional solvent extraction for the recovery of bioactive Tyrian purple precursors tyrindoleninone, 6-bromoisatin and tyriverdin from the marine mollusc Dicathais orbita. The effect of pressure on the selective extraction of brominated indoles was tested at 15, 30 and 50 MPa CO₂, and was compared to traditional chloroform extract composition and yields. Extracts obtained from 15 MPa selectively concentrated 6-bromoisatin, at 78% of the extract composition, whereas increased pressures of 30 and 50 MPa increased the solvating power of supercritical CO₂ to include the more lipophilic tyrindoleninone at 35 and 29% respectively, and tyriverdin at 23 and 40% respectively. This extraction method was also effective in separating the brominated indoles from toxic choline esters in the mollusc extracts. Extract yields from supercritical CO₂ were comparable to solvent extraction relative to whole whelk weight. This provides a viable alternative for nutraceutical development that does not rely on the use of toxic solvents.     

Extraction of anthocyanins and luteolin from Arrabidaea chica by sequential extraction in fixed bed using supercritical CO2, ethanol and water as solvents

Phenolic compounds from Arrabidaea chica Verlot leaves, besides conferring staining properties to their extracts, also have various biological activities including anti-inflammatory and wound-healing properties. To evaluate new possibilities for obtaining extracts with differentiated yield and composition, sequential extractions in fixed bed were performed at 40 and 50 °C, and 300 and 400 bar, using as extracting solvents pure supercritical carbon dioxide (scCO₂) in a first step, acidified ethanol in a second step and acidified water in a third extraction step. Four flavonoids of interest were investigated in the extracts, one of them being flavone (luteolin), and three anthocyanin compounds of type 3-desoxyanthocyanidins which were quantified by high performance liquid chromatography (HPLC). The extraction curves, the global yields and the concentration and yield of the compounds under study were evaluated. The results indicated that the cumulative total yields in the three steps ranged from 22% to 27% in all conditions of temperature and pressure, with the highest global yield at 50 °C and 300 bar. Although the lowest extraction yield was obtained using pure scCO₂, this step was highly selective, since only carajurin in its aglycone form was extracted among the compounds of interest and this was confirmed by analysis of MS/MS.     

Response surface optimization of supercritical CO2 extraction of α-tocopherol from gel and skin of Aloe vera and almond leaves

Supercritical fluid extraction (SFE) was studied as an alternative technology in the pharmaceutical industry for the separation of α-tocopherol from gel and skin of Aloe vera and almond leaves. The influence of operating conditions was investigated on the recovery of supercritical carbon dioxide (SC-CO₂) extraction of α-tocopherol from three-year old Aloe vera (Aloe barbadensis Miller) leaf gel. The obtained results were compared with the conventional Soxhlet extraction. Response surface methodology (RSM) was applied to optimize effective variables on the extracted recovery of α-tocopherol. The maximum α-tocopherol recovery of 53.41% from Aloe vera gel was obtained with employing RSM predicted optimal operating conditions of 32 MPa, 45.91 °C, 0.84 ml SC-CO₂/min and 140 min for extraction. The α-tocopherol extraction yield for gel and skin of Aloe vera and almond leaves at these optimal operating conditions were obtained 1.53, 16.29 and 2.61 mg/100 g dry sample, respectively.     

Obtaining phenolic compounds from jatoba (Hymenaea courbaril L.) bark by supercritical fluid extraction

The extraction of polyphenol compounds from jatoba (Hymenaea courbaril L. var stilbocarpa) bark using supercritical fluid extraction (SFE) with CO₂ and cosolvents has been investigated. Among the solvent systems studied, SFE using CO₂ and water (9:1, v/v), at 323 K and 35 MPa, presented the best results, with extract yield of 24%, and with high antioxidant activity (IC₅₀ of 0.2 mg/cm³). This solvent system was used to determine global yield isotherms, which were built at 323 and 333 K, and 15, 25, and 35 MPa, using a second lot of jatoba. The highest yield was 11.5% at 15 MPa and 323 K, with maximum total phenolic compounds (TPC) of 335.00 mg TAE/g extract (d.b.) and total tannins content of 1.8 g/100 g raw material. A kinetic experiment was performed using optimized conditions, yielding 18% extract, and the kinetic parameters were used to scale-up the process from laboratory to pilot scale. Chemical analyses showed high content of phenolic compounds in the extracts of jatoba bark mostly due to the presence of procyanidins.     

Extraction of phenolic compounds from pitanga (Eugenia uniflora L.) leaves by sequential extraction in fixed bed extractor using supercritical CO2, ethanol and water as solvents

With the goal of maximizing the extraction yield of phenolic compounds from pitanga leaves (Eugenia uniflora L.), a sequential extraction in fixed bed was carried out in three steps at 60 °C and 400 bar, using supercritical CO₂ (non-polar) as solvent in a first step, followed by ethanol (polarity: 5.2) and water (polarity: 9.0) in a second and third steps, respectively. All extracts were evaluated for global extraction yield, concentration and yield of both polyphenols and total flavonoids and antioxidant activity by DPPH method (in terms of EC₅₀). The nature of the solvent significantly influenced the process, since the extraction yield increased with solvent polarity. The aqueous extracts presented higher global extraction yield (22%), followed by ethanolic (16%) and supercritical extracts (5%). The study pointed out that the sequential extraction process is the most effective in terms of global extraction yield and yield of polyphenols and total flavonoids, because it produced the more concentrated extracts on phenolic compounds, since the supercritical ethanolic extract presented the highest phenolics content (240.5 mg GAE/g extract) and antioxidant capacity (EC₅₀ = 9.15 μg/mL). The most volatile fraction from the supercritical extract, which is similar to the essential oils obtained by steam distillation or hydrodistillation, presented as major compounds the germacrenos D and B + bicyclogermacrene (40.75%), selina-1,3,7(11)-trien-8-one + selina-1,3,7(11)-trien-8-one epoxide (27.7%) and trans-caryophyllene (14.18%).     

Water and ethanol as co-solvent in supercritical fluid extraction of proanthocyanidins from grape marc: A comparison and a proposal

Supercritical carbon dioxide (SC-CO₂) extraction of grape marc was studied using water (W) and ethanol (EtOH) as co-solvent at 15% (w/w), 100 and 200 MPa, and 313.15, 323.15 and 333.15 K to analyze their influence upon total phenols of the extracts. The overall extraction curves were determined and suggested 10 MPa and 313.15 K as the best operating conditions for SC-CO₂ + 15%W extraction, and 10 MPa and 333.15 K for SC-CO₂ + 15% EtOH. The phenolic yields obtained were 63.4 g/kg of extract for SC-CO₂ + 15% W and 38.8 g/kg of extract for SC-CO₂ + 15% EtOH. An alternative method combining Sc-CO₂ + 15% W extraction, followed by SC-CO₂ + 15% EtOH was tested. This procedure provided the best results allowing to obtain the highest phenolic yield (68.0 g/kg of extract), phenol content (733.6 mg GAE/100 g DM), proanthocyanidins concentration (572.8 mg catechin/100 g DM) and antioxidant activity (2649.6 mg α-tocopherol/100 g DM). SC-CO₂ methods were compared with methanol extraction.     

Screening of twelve Clostridium botulinum (group I) spores for high-pressure resistance at elevated-temperatures

Twelve strains of Clostridium botulinum group I spores, suspended in phosphate buffer (0.1 M) at approximately 10⁷ CFU/ml concentration, were subjected to high pressure treatments (800 and 900 MPa; 0.5–15 min) at elevated temperatures (90 and 100 °C). The treatments were chosen to have a range of pressure/temperature severity to be able to discriminate the spore strains for their pressure resistance. An insulated test chamber was used to achieve temperature stability during treatment. Preliminary tests showed the need for an 8 day anaerobic incubation for enumeration. Results showed that strains PA9508B, HO9504A and CK2-A had higher pressure resistance than others among the 12 strains studied. Strain 62A was least resistant and completely inactivated by the treatment. Estimated D values of the more resistant strains were in the 0.66–1.8 min range at 900 MPa at 100 °C treatment. The temperature sensitivity parameter (Z_P value) in the 800–900 MPa pressure range varied between 10 and 16 °C, and pressure sensitivity parameter (Z_T value) in the 90–100 °C temperature range varied between 340 and 760 MPa. The most pressure resistant strain was PA9508B with an estimated Z_P value of 16.0 °C and Z_T value of 470 MPa. Since the pathogenic strains of C. botulinum have different pressure resistance, the most resistance strain should be selected as the basis for process establishment.     

Supercritical carbon dioxide extraction of squalene and tocopherols from amaranth and assessment of extracts antioxidant activity

Squalene and tocopherols are the most important bioactive constituents in lipophilic amaranth fraction. Therefore, developments of processes of isolation of amaranth extracts enriched with these compounds are of interest. In this study the lipophilic fraction of amaranth seeds was extracted by supercritical fluid extraction with carbon dioxide (SCE-CO₂) under different pressure conditions and by adding 2 and 5% of cosolvent ethanol. The yield of extract varied from 1.37 (15 MPa without cosolvent) to 5.12% (55 MPa and 5% of cosolvent). The highest content of unsaponifiables (21.1%) in the extract was at 55 MPa and 5% of cosolvent; at these conditions the yields of tocopherols and squalene from amaranth seeds were 317.3 mg/kg and 0.289 g/100 g, respectively. Tocopherol isomers in amaranth oil were distributed at the approximate ratio of 1(α-T):27(β-T):6.5(γ-T):5(δ-T). The extract was fractionated in the two separators by gradual decrease of the pressure and it was found that the fraction obtained at ambient conditions contained the highest concentration of tocopherols (up to 7.6 mg/g) and squalene (up to 17.9 g/100 g oil). The highest antioxidant activity measured by the L-ORAC assay possessed the fractions with the highest concentrations of squalene and tocopherols and obtained at 15 MPa with pure CO₂ (235.1 μmol TE/g) and 2% of cosolvent (257.6 μmol TE/g).     

Allelopathic properties of the fractions obtained from sunflower leaves using supercritical carbon dioxide: The effect of co-solvent addition

The work described here is a continuation of a previous study centered on the extraction, using supercritical carbon dioxide, of bioactive substances from sunflower leaves of the Helianthus annuus L. variety Arianna. In this study the addition of 9% of ethanol as co-solvent was analyzed. The extraction was carried out (P = 100/400 bar, T = 35/55 °C, ethanol = 9%) in order to analyze the influence of pressure, temperature and sample pre-treatment on the extraction yield and bioactivity of the extracts. The addition of 9% of ethanol to the supercritical solvent enhanced both the extraction yield and the biological activity of the extracts. The best conditions were a pressure of 400 bar and a temperature of 55 °C. In an effort to improve the bioactivity of the extract, a cascade fractionation of the extracts was carried out and this gave different results in terms of biological activities and extraction yields. The phytochemical compositions of the extracts were analyzed by thin layer chromatography. The fractionation that gave the best results was carried out at 90 bar and 40 °C in the first separator. Finally, the effect of extracts on the growth of seeds from different plants was analyzed.     

Supercritical fluid extracts with antioxidant and antimicrobial activities from myrtle (Myrtus communis L.) leaves. Response surface optimization

Supercritical Fluid Extraction (SFE) was used to obtain myrtle leaf extracts, and to study the antioxidant capacity (AOC) and in vitro antimicrobial activity of those extracts. To optimize the SFE operational conditions, the response surface methodology (RSM) was adopted. The parameters studied were: pressure (P), within the range 10 to 30 MPa; temperature (T), between 35 °C and 60 °C and supercritical carbon dioxide (SCCO₂) flow rate (Q) within the range 0.15 to 0.45 kg h⁻¹. The results show a good fit to the proposed model and the optimal conditions obtained (23 MPa, 45 °C, and SCCO₂ flow rate of 0.3 kg h⁻¹) were within the experimental range. The predicted values agreed with experimental ones, thus indicating the suitability of the RSM model for the optimization of the extraction conditions being investigated. With those values remaining constant, ethanol as a co-solvent was then studied. There was an observed rise in AOC as the amount of ethanol increased, within the range studied (0–30 wt% ethanol). The extract with the highest AOC was tested for its antimicrobial activity against gram-positive and gram-negative bacteria. The minimum inhibitory concentration (MIC) values obtained showed significant inhibitory effect against gram-positive bacteria.     

Supercritical fluid extraction of volatile oil from Lippia alba (Mill.) cultivated in Aragón (Spain)

The objective of this study is the supercritical extraction of the volatile oil from Lippia alba (Mill.) cultivated in Aragón. The influence of extraction pressure and temperature and cosolvent percentage on overall yield and volatile oil composition was studied. The supercritical extraction conditions were optimized using the 2007 crop; operating at 35.0 MPa, 40 °C and 5% of ethanol as cosolvent, 2009 and 2011 crops were also tested. Supercritical fluid extract compositions and overall yields were compared with the extracts obtained by conventional extraction techniques such as hydrodistillation (HD) and organic solvent extraction (OSE). Four terpenoids (linalool, 1,8-cineole, β-caryophyllene and β-caryophyllene oxide) were selected as target compounds and their content in the different extracts was monitored by gas chromatography. Linalool was selected as indicator of the plant adaptation to the commercial purposes. HD is the technique that provides the highest percentage of linalool in all the extracts, but showed always the lowest overall yields; whereas, SFE extracts provides a good balance between the overall yield and presence of the four target compounds.     

Extraction of essential oil from Cyperus articulatus L. var. articulatus (priprioca) with pressurized CO2

The main goal of this study was to assess the yield and the antimicrobial activity of extracts from Cyperus articulatus L. var. articulatus obtained by pressurized carbon dioxide based on their system phase diagram behavior. The extractions were carried out at 313, 323, 333 K temperatures and, 13 and 25 MPa pressures. The extracts were quantified and chemically characterized by using gas chromatography coupled to mass spectrometry technique. The extracts obtained at the following experimental conditions: 333 K and 13 MPa, showed antifungal activity against Cladosporium sphaerospermum ATCC 4464. At 323 K – 25 MPa, and 333 K – 25 MPa, the extracts showed antibacterial activity against Staphylococcus aureus ATCC 25923. To describe the kinetics of extraction with a packed bed, a mathematical model was employed highlighting the transference mechanisms for masses in the pseudo-binary system as follows (1) carbon dioxide and (2) priprioca extract, the monophasic and multiphasic regions.