Effects of supercritical CO2 extraction parameters on chemical composition and free radical-scavenging activity of pomegranate (Punica granatum L.) seed oil

Pomegranate (Punica granatum L.) seed oil (PSO) was prepared by supercritical CO₂ (SC-CO₂) extraction technology. Changes in the yield, chemical composition and free radical-scavenging activity of PSO under different extraction parameters were investigated. The results of SC-CO₂ extraction revealed that extraction pressure was the dominant factor to affect the oil yield. PSO was characterized by a high content of punicic acid (approximately 60%) and γ-tocopherol (more than 300 mg/100 g oil). A slight increase in the contents of punicic acid, arachidic acid and gadoleic acid was observed under higher extraction pressure and temperature. At lower pressure or shorter extraction time, PSO with high amount of total tocopherols was obtained. PSO extracted by SC-CO₂ showed strong free radical-scavenging activity towards 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azinobis-(3-ethylbenz-thiazoline-6-sulfonic) diammonium salt (ABTS) radicals and its scavenging ability was correlated with the level of tocopherols in extracted oils.     

Supercritical extraction of carob kibbles (Ceratonia siliqua L.)

Carob pulp kibbles, a by-product of carob been gum production, was studied as a source of bioactive agents. Firstly, the carob kibbles were submitted to an aqueous extraction to extract sugars, and supercritical fluid extraction (SFE) was applied to the solid residue of that aqueous extraction, by using compressed carbon dioxide (SC-CO₂) as the solvent and a mixture of ethanol and water (80:20, v/v) as a co-solvent. Pressure and temperature were studied in the ranges 15–22 MPa, and 40–70 °C. Particle diameter, and co-solvent percentage in ranges of 0.27–1.07 mm, and 0–12.4%, respectively, were also studied, as well as the flow rate of SC-CO₂ between 0.28 and 0.85 kg h⁻¹, corresponding, respectively, to 0.0062 and 0.0210 cm s⁻¹ of superficial velocity. The extracts were characterised in terms of antioxidant capacity by DPPH method, and total phenolics content by the Folin–Ciocalteu method. The central composite non-factorial design was used to optimise the extraction conditions, using the Statistica, version 6 software (Statsoft). The best results, in terms of yield and antioxidant capacity, were found at 22 MPa, 40 °C, 0.27 mm particle size, about 12.4% of co-solvent and a flow rate of 0.29 kg h⁻¹ of SC-CO₂. The phenolics profile of the extracts obtained at these conditions was qualitatively evaluated by HPLC-DAD. The solid residue of the supercritical extraction was also studied showing to be a dietary fiber, which can be compared to Caromax™, a carob fiber commercialised by Nutrinova Inc.     

Characterization of wheat bran oil obtained by supercritical carbon dioxide and hexane extraction

Supercritical carbon dioxide (SC-CO₂) and soxhlet extraction using was carried out to extract oil from wheat bran oil. For SC-CO₂, the pressure and temperature were ranging from 10 to 30 MPa and 313.15–333.15 K. The extraction was performed in a semi batch process with a CO₂ flow rate of 26.81 g/min for 2 h. Wheat bran oil was characterized to investigate the quality. Acid value (AV) and peroxide value (POV) were higher in hexane extracted oil compared to SC-CO₂ extracted oil. Induction period was measured by rancimat test. The oil obtained by SC-CO₂ extraction had higher capability to delay the oxidation by surrounding environment. The DPPH radical scavenging activity was also measured. The SC-CO₂ extracted oil showed higher radical scavenging activity compared to hexane extracted oil.     

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.     

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%).     

A bioactivity based comparison of Echinacea purpurea extracts obtained by various processes

Echinacea species is provided as dietary supplements for various infectious and immune related disorders and has a potential role in cancer prevention. The aim of this study was to optimize the extraction of total flavonoids using different extraction methods and investigate the cytotoxic effects on various cancer cell lines (CaCo-2, MCF-7, A549, U87MG, and HeLa) and VERO (African green monkey) as a non-cancerous cell line. Box-Behnken statistical design was used to evaluate the effect of pressure (100–200 bar), temperature (40–80 °C) and ethanol as co-solvent (6–20 wt%) at a flow rate of 15 g/min for 60 min in supercritical CO₂ extraction and the effect of temperature (60–100 °C), time (5–15 min) and power (300–900 W) in microwave-assisted extraction. Optimum extraction conditions were elicited as 300 bar, 80 °C and 13% co-solvent yielding 0.472 mg rutin equivalent total flavonoids/g extract in SC-CO₂ extraction, whereas 60 °C, 10 min and 300 W yielded the highest (0.202 mg rutin equivalent) total flavonoids in microwave-assisted extraction. Additional trials with subcritical water (0.022 mg/g) and Soxhlet extraction with methanol (0.238 mg/g) yielded lower flavonoid contents. The exposures upto 50 μg/ml of extracts revealed no significant inhibition on the proliferation of both tested cancer cells and healthy VERO cells.     

Supercritical carbon dioxide extraction of microalgae lipid: Process optimization and laboratory scale-up

Supercritical carbon dioxide (SC-CO₂) extraction of lipid from Scenedesmus sp. for biodiesel production was investigated and compared to conventional extraction methods. The effect of biomass pre-treatment prior to extraction and extracting conditions, namely pressure in the range of 200–500 bar, temperatures in the range of 35–65 °C and CO₂ flow rate in the range of 1.38–4.02 g min⁻¹, on SC-CO₂ extraction yield and quality of lipid were investigated. Three levels full factorial design of experiments and response surface methodology was used to model the system. A second order polynomial model was developed and used to predict the optimum conditions. Scaling up to a laboratory larger scale was also tested. The results indicated that SC-CO₂ extraction was superior to other extraction techniques, but exhibited significant variations in yield with changes in operating parameters. In the developed model, it was found that the linear and quadratic terms of the temperature, as well as the interaction with pressure had a significant effect on lipid yield; whereas, their effect on lipid quality was insignificant. The best operating conditions, in the tested range, were 53 °C, 500 bar and 1.9 g min⁻¹, in which lipid extraction yield of 7.41% (dry weight basis) was obtained. Negligible differences were observed when the fatty acid composition of SC-CO₂ extracted lipid was compared to that extracted by the conventional methods. At the optimum conditions, SC-CO₂ extraction was successfully scaled-up by eight-folds and the extracted lipid yield dropped by 16%.     

Phenolic and flavonoid content and antioxidants capacity of pressurized liquid extraction and perculation method from roots of Scutellaria pinnatifida A. Hamilt. subsp alpina (Bornm) Rech. f.

The extract was separated from roots of Scutellaria pinnatifida using perculation and pressurized liquid extraction (PLE). A circumscribed central composite (CCC) was used to optimize the effective extraction variables. For achieving maximum extraction yield via PLE the temperature, pressure, static time, dynamic time, and the solvent flow rate were adjusted 65.8 °C, 39.2 bar, 12.9 min, 18.9 min, and 0.76 mL/min, respectively. Ferric reducing antioxidant power (FRAP) (mmol/g) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity (mg/mL) were evaluated and the highest antioxidant activity was observed from the PLE extract. The total phenolic and flavonoid content was calculated and a good correlation founded between phenolic content and antioxidant activity. The results indicated the root of this plant is a potential source of natural antioxidants and flavonoids. The PLE method is quicker and it has more extraction yield than perculation.     

Response surface optimization of Smyrnium cordifolium Boiss (SCB) oil extraction via supercritical carbon dioxide

In this study, the essential oil of aerial parts of a species of a plant called Smyrnium cordifolium Boiss (SCB) was extracted by supercritical CO₂. The essence was analyzed by the method of GC/MS. Design of experiments was carried out with response surface methodology by Minitab 16 software to optimize four operating variables of supercritical carbon dioxide (SC-CO₂) extraction (pressure, temperature, CO₂ flow rate and extraction dynamic time). This is the first report announcing optimization of the operation of supercritical extraction of SCB in laboratorial conditions. Optimizing process was done to achieve maximum yield extraction. Independent variables were dynamic time (t_d), pressure (P), temperature (T) and flow rate of SC-CO₂ (Q) in the range of 30–150 min, 10–30 MPa, 40–60 °C and 0.5–1.7 ml/min, respectively. The experimental optimal recovery of essential oil (0.8431, w/w%) was obtained at 13.43 MPa, 40 °C, 150 min (dynamic) and 1.7 ml/min (CO₂ flow rate).     

Modelling of the inactivation kinetics of Escherichia coli,Saccharomyces cerevisiae and pectin methylesterase in orange juice treated with ultrasonic-assisted supercritical carbon dioxide

The combined effect of supercritical carbon dioxide (SC-CO₂) and high power ultrasound (HPU) on the inactivation kinetics of Escherichia coli, Saccharomyces cerevisiae and pectin-methyl esterase (PME) in orange juice was studied in order to select models that can predict their inactivation behaviour based on process parameters. Experiments were performed at different temperatures (31–41 °C, 225 bar) and pressures (100–350 bar, 36 °C). The inactivation rate of E. coli, S. cerevisiae and PME increased with pressure and temperature during SC-CO₂ + HPU treatments. The SC-CO₂ + HPU inactivation kinetics of E. coli, S. cerevisiae and PME were represented by models that included temperature, pressure and treatment time as variables, based on the Biphasic, the Peleg Type B, and the fractional models, respectively. The HPU-assisted SC-CO₂ batch system permits the use of mild process conditions and treatment times that can be even shorter than those of continuous SC-CO₂ systems.     

Optimization of subcritical water extraction of antioxidants from Coriandrum sativum seeds by response surface methodology

Subcritical water extraction (SWE) of antioxidants from Coriandrum sativum seeds (CSS) was optimized by simultaneous maximization of the total phenolics (TP) and total flavonoids (TF) yield and antioxidant activity, using IC₅₀ value. Box–Behnken experimental design (BBD) on three levels and three variables was used for optimization together with response surface methodology (RSM). Influence of temperature (100–200 °C), pressure (30–90 bar) and extraction time (10–30 min) on each response was investigated. Experimentally obtained values were fitted to a second-order polynomial model and multiple regression. Analysis of variance (ANOVA) was used to evaluate model fitness and determine optimal conditions. Moreover, three-dimensional surface plots were generated from employed mathematical model. The optimal SWE conditions obtained in simultaneous optimization were temperature of 200 °C, pressure of 30 bar and extraction time of 28.3 min, while obtained values of TP and TF yields and IC₅₀ value at this experimental point would be 2.5452 g GAE/100 g CSS, 0.6311 g CE/100 g CSS and 0.01372 mg/ml, respectively. Moreover, good and moderate linear correlation was observed between antioxidant activity (IC₅₀ value) and total phenolics content (R² = 0.965), and total flavonoids content (R² = 0.709) which indicated that these groups of compounds are responsible for antioxidant activity of C. sativum extracts.     

Cosolvent-modified supercritical carbon dioxide extraction of phenolic compounds from bamboo leaves (Sasa palmata)

This study highlights the possibility of supercritical carbon dioxide for extracting phenolic compounds from bamboo leaves that have shown antioxidant and anticancer activities. The CO₂ extraction solvent was modified by adding ethanol–water mixture cosolvent of different concentrations to allow extraction of both polar and non-polar compounds. Conventional Soxhlet extraction was also done to investigate the advantages of supercritical extraction over the conventional extraction method. For addition of 5% (mol) of a 25:75 (mol:mol) ethanol–water mixture solvent to CO₂, the highest amount of polyphenols (7.31 ± 0.06 mg/g bamboo leaves in catechin equivalents) and radical scavenging activity (3.65 ± 0.05 mg/g bamboo leaves in BHA equivalents) at 20 MPa and 95 °C, could be obtained among the mixture cosolvents studied. For Soxhlet extraction with a 25:75 (mol:mol) ethanol–water mixture, 1.48 times the amount of phenolic compounds (10.85 ± 0.52 mg/g bamboo leaves in catechin equivalents), could be isolated compared with the supercritical extraction method, however, the radical scavenging activity (3.30 ± 0.05 mg/g bamboo leaves in BHA equivalents) was 0.90 times lower than the extract obtained from the supercritical extraction method. The seven major antioxidative compounds identified from the SC-CO₂ extraction method were: (1) dl-alanine, (2) gluconic acid, (3) phosphoric acid, (4) ß-siosterol, (5) β-amyrene, (6) α-amyrin acetate and (7) friedelin.     

Supercritical anti-solvent micronization of marigold-derived lutein dissolved in dichloromethane and ethanol

This work aims to study supercritical anti-solvent (SAS) micronization of lutein derived from marigold flowers. Lutein solution in dichloromethane (DCM) or ethanol was atomized into the stream of supercritical carbon dioxide (SC-CO₂) through a concentric nozzle in a pressurized vessel. The effects of pressure and SC-CO₂ flow rate on morphology, mean particle size (MPS) and particle size distribution (PSD) were investigated. The reduction in lutein MPS from 202.3 μm of unprocessed lutein to 1.58 μm and 902 nm could be achieved by SAS micronization using DCM and ethanol, respectively. In both solvent systems, no significant effects of pressure and SC-CO₂ flow rate on particle morphology were observed. However, pressure was found to have a significant effect on MPS and PSDs of lutein particles.     

Digestive enzymes characterization of krill (Euphausia superba) residues deoiled by supercritical carbon dioxide and organic solvents

In this study, Enzyme activities of krill were characterized before and after lipid extraction by supercritical carbon dioxide (SC-CO₂) and organic solvent, n-hexane and acetone. Krill SC-CO₂ extraction was performed under the conditions of temperature range from 35 to 45 °C and pressure, 150–250 bar for 2.5 h with a constant flow rate of 22 g/min. Extraction yields of lipids increased with pressure and temperature. The digestive enzyme activities of protease, lipase and amylase of SC-CO₂ treated krill residues were slightly decreased comparing to organic solvent, n-hexane and acetone treated residues. In SC-CO₂ treated samples, all of the digestive enzymes showed slightly higher temperature stability. In the other hand the crude extracts of SC-CO₂ and n-hexane treated krill samples showed almost same optimum pH and pH stability for each of the digestive enzymes. It was also found in SDS-PAGE that there are no significant differences in protein patterns of the crude extracts of untreated, SC-CO₂, n-hexane and acetone treated krill indicating no denaturation of proteins.     

Modeling the inactivation of Staphylococcus aureus and Serratia marcescens in clinical solid waste using supercritical fluid carbon dioxide

The aim of the present study was to determine an effective sterilization method for safe handling and recycle-reuse of clinical solid waste materials. Supercritical fluid carbon dioxide (SC-CO₂) was applied in the inactivation of gram positive Staphylococcus aureus (S. aureus) and gram negative Serratia marcencens (S. marcescens) in clinical solid waste. The colony forming activity of the bacteria was completely lost at pressures 10–40 MPa, temperatures 35–80 °C and treatment period between 5 and 120 min. An increase in pressure at constant temperature and vice versa with the increasing treatment time enhanced the SC-CO₂ inactivation efficiency. The inactivation process was illustrated by the modified Gompertz equation. The SC-CO₂ inactivation of bacteria was compared with the steam autoclaved bacteria. Regrowth of the bacteria was observed in the autoclaved sample while no re-growth was detected in the SC-CO₂ treated clinical solid waste. Results from SEM image analysis, cellular protein and enzymatic activity of untreated, autoclaved and SC-CO₂ treated S. marcescens and S. aureus cells confirmed that SC-CO₂ is an effective sterilization method.     

Effects of supercritical CO2 extracts of Melia azedarach L. on the control of fall armyworm (Spodoptera frugiperda)

This work reports the use of Melia azedarach L. extracts obtained from supercritical carbon dioxide extraction (SC-CO₂) as an insecticidal agent against fall armyworm (Spodoptera frugiperda). For this purpose, SC-CO₂ extractions were performed, varying the pressure (150–250 bar), temperature (313–333 K), sample particle size and extraction time. Secondary metabolites from the classes of coumarins, sterols and terpenes were identified in the extracts, with the triterpene melianone being the major constituent. For the biological activity tests, diets were prepared with different SC-CO₂ extract concentrations (100, 500, 1000 and 5000 mg/kg) and offered to S. frugiperda (Lepidoptera: Noctuidae). The results indicated that mortality increased with increasing extract concentrations with 50% mortality (LC₅₀) at a concentration of 376.74 mg/kg and reaching 100% mortality at 5000 mg/kg. The inhibition of insect growth was observed at higher concentrations due to the antifeedant action of the extract. At the lowest extract concentration (100 mg/kg), ingestion caused low pupal viability and adults presenting morphological deformities, which thus indicated a chronic toxicity effect.     

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.     

Optimization of supercritical carbon dioxide removal of lipid and cholesterol from goat placenta using response surface methodology

Supercritical CO₂ (SC-CO₂) extraction was applied to remove lipid and cholesterol from freeze-dried goat placenta. A response surface methodology (RSM) was employed to optimize the extraction parameters. The effects of pressure, temperature, flow rate of CO₂ and extraction time on the yields of lipid and cholesterol were investigated. Response surface analysis showed that the data were adequately fitted to second-order polynomial model. The independent variables, quadratics of pressure and extraction time, and the interaction between pressure and temperature had significant effects on the yields of lipid and cholesterol, respectively. The optimum parameters within the experimental range of the variables were 34.6 MPa, 35.3 °C, 29.1 min with a CO₂ flow rate of 18.2 L/h. Under such condition, the yields of lipid and cholesterol were predicted to be 21.02% and 8.46 mg/g, respectively. Furthermore, the removal efficiency of cholesterol by SC-CO₂ was higher than those achieved by Soxhlet and Folch extraction methods.     

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).     

Optimization of multi-stage countercurrent extraction of antioxidants from Ginkgo biloba L. leaves

Multi-stage countercurrent extraction (MCE) as a novel extraction technique was used to extract antioxidants from Ginkgo biloba leaves. Orthogonal array design (OAD) was employed to optimize the ratio of 60% ethanol to raw material (8–16 mL/g), extraction time (30–60 min) and extraction temperature (60–80 °C) to obtain a high yield of antioxidants from G. biloba leaves by MCE. The optimum conditions were a ratio of 60% ethanol to raw material of 16 mL/g and extraction time of 30 min at 80 °C. Under these conditions, the yields of flavonoids and total phenolics were 1.74% and 2.42%, respectively, and DPPH radicals scavenging activity of the extract was 89.97%. Compared with heat-reflux extraction, MCE had obvious advantages of less extraction time and lower solvent and energy consumption. It may be used as a promising technique for the extraction of bioactive compounds from plant materials.