Purification of algal anti-tyrosinase zeaxanthin from Nannochloropsis oculata using supercritical anti-solvent precipitation

This work investigated the changes in content of algal zeaxanthin in submicronized precipitates generated from the supercritical anti-solvent (SAS) process of extracting microalgae Nannochloropsis oculata. Following a reverse phase elution chromatography, the particulates were successfully generated from feed solutions containing zeaxanthin that ranged from 0.4 to 0.8 mg/mL by a SAS process. The precipitation condition was set at 323 K and pressures ranged from 10 to 20 MPa. Experimental results of a three-factor center composite response surface method for the SAS process indicated that the size of the precipitates was significantly affected by the flow rate of carbon dioxide. The purity of zeaxanthin increased with increasing solvent flow rate and with reducing solution concentration. The recovery of zeaxanthin and the morphology of the precipitates was also examined. The content of zeaxanthin in submicronsized precipitates increased from 485.9 (48.6%) to 673.7 mg/g (67.4%). This work demonstrates that elution chromatography coupled with a SAS process is an environmentally benign method to recover anti-tyrosinase zeaxanthin from Nannochloropsis oculata as well as to generate submicrosized precipitates of the purest zeaxanthin from algal solutions.     

Supercritical CO2 extraction of Persea indica: Effect of extraction parameters, modelling and bioactivity of its extracts

The objective of the work was to optimize the extraction of Persea indica L. bioactive compounds by means of supercritical fluid extraction (SFE) and analyze their insecticidal effects. P. indica L. is one of the dominant species of the Canarian laurel forest, a relict of the Tertiary flora. Different extraction conditions (pressure, plant material particle size, temperature, CO₂ flow) and the influence of entrainer were tested and the evolution of the extracted compounds was screened by HPLC-MS. A comparison with conventional techniques such as hydrodistillation (HD) or organic solvent extraction (OSE) was also presented. Particularly, four CO₂ densities ranging from 628.61 kg/m³ to 839.81 kg/m³ were studied in the range of 10.0-20.0 MPa and 40-50 °C. The extracts contained insecticidal ryanodanes of great interest, previously described as insecticidal components of P. indica. The insecticidal antifeedant activity of selected extracts was inspected. A model based on mass transfer equations, the Sovová model, was successfully applied to correlate the experimental data.     

Supercritical fluid extraction of flavonoids from Maydis stigma and its nitrite-scavenging ability

Supercritical fluid carbon dioxide (SF-CO₂) extraction (SFE) of flavonoids from Maydis stigma and its nitrite-scavenging ability were investigated. The effects of extraction time, particle size and co-solvent composition in terms of water content in ethanol were first optimized. Then, a Box-Behnken design combined with response surface methodology (RSM) was employed to study the effects of three independent variables (temperature, pressure and co-solvent amount) on the extraction yield of flavonoids. A maximal extraction yield of flavonoids of approximately 4.24 mg/g of M. stigma by SFE was obtained under optimal conditions (a temperature of 50.88 °C, a pressure of 41.80 MPa, a co-solvent amount of 2.488 mL/g and an extraction time of 120 min with 0.4-mm particle sizes and 20% aqueous ethanol as the co-solvent). Furthermore, the nitrite-scavenging ability of the flavonoid-enriched SFE extracts was assessed using the Griess reagent. The flavonoid-enriched SFE extracts exhibited the highest scavenging ability on nitrite (88.1 ± 3.04%) at the concentration of 500 μg/mL and at pH 3.0. The nitrite-scavenging ability of the extracts appeared to be concentration dependent but negatively correlated with the pH.     

Supercritical CO2 extraction of oil, carotenoids, squalene and sterols from lotus (Nelumbo nucifera Gaertn) bee pollen

The high-quality oil, abundant in carotenoids, squalene and sterols (mainly consisting of campesterol, stigmasterol, β-sitosterol and β-amyrin), was extracted by supercritical CO₂ from lotus bee pollen for its potential nutraceutical use. The effects of extraction pressure and temperature on the yields and the compositions of the extracts were investigated by using a two-factor central composite rotatable design experiment. ANOVA for response surface model demonstrated that the data were adequately fitted into four polynomial models. The yields of the oil, carotenoids, squalene and sterols were significantly influenced by the experimental variables. It was predicted that maximum oil yield obtained at the extraction pressure of 38.2 MPa and temperature of 49.7 °C contained the maximum amount of carotenoids, squalene and sterols. GC-FID analysis of the fatty acid composition of lotus bee pollen oil showed that polyunsaturated fatty acids accounted for approximately 22% of the total fatty acids.     

Supercritical fluid extraction of wormwood (Artemisia absinthium L.)

The objective of the work was to optimize the extraction of wormwood oil by supercritical fluid extraction (SFE) of growth-controlled plant material. Different extraction conditions, two growth techniques and various crops were tested and the evolution of the extracted oil composition was screened chromatographically. A comparison with conventional techniques such as hydrodistillation (HD) or organic solvent extraction (OSE) was also presented. Particularly, six CO₂ densities ranging from 285.0 kg/m³ to 819.5 kg/m³ were studied in the range of 9.0-18.0 MPa and 40-50 °C. A systematic study was carried out with plant material from 2005, while SFE of 2006, 2008 and aeroponically grown crops was performed for comparative purposes. The effect of ethanol as a modifier of the supercritical fluid extraction was also studied. The major compounds found in the SFE extracts as well as in the HD essential oils were Z-epoxyocimene, chrysanthenol and chrysanthenyl acetate. A model based on mass transfer equations, the Sovová model, was successfully applied to correlate the experimental data.     

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.     

Supercritical CO2 extraction of glycosides from Stevia rebaudiana leaves: Identification and optimization

The aim of this work was to optimize the glycoside composition of Stevia rebaudiana leaves using supercritical fluid extraction (SFE). A Box-Behnken statistical design was used to evaluate the effect of various values of pressure (150–350 bar), temperature (40–80 °C) and concentration of ethanol-water mixture (70:30) as co-solvent (0–20%) by CO₂ flow rate of 15 g min⁻¹ for 60 min. The most effective variables were co-solvent concentration (P < 0.005) and temperature (P ≤ 0.005). Evaluative criteria for both dependent variables (stevioside and rebaudioside A yields) in the model was assigned maximum. Optimum extraction conditions were elicited as 211 bar, 80 °C and 17.4% which yielded 36.66 mg/g stevioside and 17.79 mg/g rebaudioside A. Total glycosides composition were close to those obtained using conventional water extraction (64.49 mg/g) and a little higher than ethanol extraction (48.60 mg/g) demonstrating challenges for industrial scale application of SFE.     

The analysis of simultaneous clove/oregano and clove/thyme supercritical extraction

The goal of present work was to investigate and explain kinetics and mass transfer phenomena occurring during the SFE from the mixture of two plants with different initial composition. The extractions from pure clove, oregano and thyme as well as from clove/oregano (C/O) and clove/thyme (C/T) mixtures with various initial compositions of plant material were carried out using supercritical CO₂ at 10 MPa and 40 °C. The results indicated that presence of light compounds in supercritical CO₂ originated from the oregano leaves or thyme at the beginning of extraction process increases the extraction rate of compounds from clove bud. Only small added amounts of oregano or thyme to clove bud (C/O - 90:10%, w/w; or C/T - 84:16%, w/w) in the starting plant mixture had the same effect resulted in the similar and the highest increase of the extraction rate and had negligible influence on total extraction yield compared to extract isolated from pure clove. Different mathematical models were used for simulation of experimental data which showed that the highest increase of the solubility of extractable compounds in supercritical CO₂ as well as the highest mass transfer rate in the solid phase during extractions existed during extraction from C/O (90:10, w/w) and C/T (84:16, w/w) mixtures. Decrease of SC CO₂ consumption or shorter time of extraction necessary for achieving desired extract yield in the case of SFE of the clove buds could be important for industrial-scale application.     

Brazilian Ginseng extraction via LPSE and SFE: Global yields, extraction kinetics, chemical composition and antioxidant activity

Brazilian Ginseng extracts of two species, Pfaffia paniculata and Pfaffia glomerata, were obtained by supercritical fluid extraction (SFE) with CO₂ and by low-pressure solvent extraction (LPSE) with methanol, hexane and ethanol. The SFE assays were conducted at pressures of 100, 200 and 300 bar, and temperatures of 30 and 50 °C. The qualitative chemical compositions of the extracts were determined by thin layer chromatography (TLC). One of the active principles of interest from P. glomerata extract, β-ecdysone, was identified and quantified by HPLC. The antioxidant activities of Brazilian Ginseng extracts were determined by the coupled reaction of linolenic acid and β-carotene. For P. paniculata, the highest SFE yield was obtained at 200 bar/50 °C (0.22%, dry basis—d.b.), while the best extraction condition for P. glomerata was obtained at 200 bar/30 °C (0.18%, d.b.). The higher extract yields obtained by LPSE were 2.0% and 5.8% (w/w, d.b.) for P. paniculata and P. glomerata, respectively, both obtained with methanol as extraction solvent. From the overall extraction curve of P. glomerata, it was possible to obtain the kinetic parameters of extraction; the duration of the CER (constant extraction rate) period was determined as 134 min. The TLC plates showed the possible presence of flavonoids in the ethanolic extract for both Pfaffia species. The antioxidant activity analysis detected that LPSE extracts had higher activity than SFE extracts.     

Supercritical fluid extraction of piceid, resveratrol and emodin from Japanese knotweed

In the present study, the use of supercritical fluid extraction was investigated for selected compounds from the plant Japanese knotweed (Polygonum cuspidatum Siebold & Zucc.). The effects of parameters such as type of modifier, pressure, temperature and time on the extraction efficiency of piceid, resveratrol and emodin were studied. The optimal conditions were found as follows: modifier acetonitrile, 40 MPa, 100 °C and 45 min. SFE results were compared with those obtained by conventional Soxhlet extraction carried out for 4 h. The extracts obtained using these two techniques were analysed by liquid chromatography coupled with UV detection. LiChrospher^® 100, RP-18 column (125 mm × 4 mm, 5 μm) coupled with gradient elution acetonitrile in acidified water was used for the separation of compounds at flow rate 0.5 mL min⁻¹. Detection was carried out at 306 nm. Limits of detection were 21, 8 and 52 μg L⁻¹ for piceid, resveratrol and emodin, respectively. The linear range was 0.5-10 mg L⁻¹ for piceid and resveratrol, and 1-50 mg L⁻¹ for emodin with correlation coefficients above 0.9981. Based on the comparison of both methods extracted amount of piceid by Soxhlet extraction is approximately 10 times higher than by SFE method, while the extraction yield of emodin by Soxhlet extraction in approx. 2.5 times lower than by SFE. The advantage of SFE over Soxhlet extraction method is more than 5 times shorter extraction time period.     

Supercritical fluid extraction of lipids from spent coffee grounds

Supercritical fluid extraction of lipids from spent coffee grounds was studied in this work. Extraction experiments were carried out with supercritical carbon dioxide at different pressure and temperature conditions to study the influence of those process parameters on the extraction rate and oil composition. Supercritical carbon dioxide extracted up to 85% of the total amount of oil of spent coffee grounds after 3 h of extraction (corresponding to a maximum yield of 15.4 g_oil/100 g_{dry spent coffee}). The fatty acid composition of the extracted oil showed the presence of fatty acids of C14, C16, C18, and C20 carbon chains. Palmitic (C16:0) and linoleic (C18:2) acids were the major fatty acids and comprise about 35% each of the total fatty acid content of the oil. A soxhlet extraction with n-hexane was done for comparison resulting in a maximum yield of oil of 18.3 g_oil/100 g_{dry spent coffee}. Finally, a diffusional model that takes into account the properties of the substrate, the solute partition between the solid and the supercritical phase, and the mass transfer coefficient and axial dispersion in the fluid phase was applied to this system and a good agreement with experimental results was obtained.     

Ultrasonic extraction of ferulic acid from Ligusticum chuanxiong

The extraction of ferulic acid, a pharmacologically active ingredient from the root of Ligusticum chuanxiong, with ultrasonic extraction was investigated. Percolation and supercritical fluid extraction (SFE) were employed to make comparisons with ultrasonic extraction. Three variables, which included the concentration of solvent, the ratio of solvent volume/sample (mL/g), and extraction time, were found to have a great influence on ultrasonic extraction. The optimum extraction was with pure ethanol with a solvent volume/sample ratio 8:1 (mL/g) and extraction time of 30 min. Under the optimum extraction conditions, the extraction yield could reach 8.8% which was higher than that using SFE with ethanol as co-solvent and a content of ferulic acid of 0.7%; both the yield and the content were higher than those obtained by percolation. Ultrasonic extraction significantly shortened the time required for the extraction process. Overall, ultrasonic extraction was shown to be highly efficient in the extraction of ferulic acid from Ligusticum chuanxiong.     

Application of continuous supercritical anti-solvents for rapid recrystallization and purification of zeaxanthin dipalmitates from de-glycosides of Lycium barbarum fruits

This study employed column elution fractionation coupled with supercritical anti-solvent recrystallization to isolate the purest zeaxanthin dipalmitates (ZP), one of the eye-protecting compounds in the Lycium barbarum fruits. The amount of ZP increased to 983 mg/g in liquid anti-solvent precipitates with 29.1% recovery and increased to 929 mg/g in supercritical anti-solvent precipitates with 59.6% recovery, respectively from the ultrasonic acetone extract. Experimental data showed that supercritical anti-solvent recrystallization is feasible in producing platelet type precipitates within a few minutes. These ZP compounds exhibit anti-oxidative abilities by proliferating human adult retinal pigment epithelial cells (from 22% to 31%); though it was a meager increase when compared to those of algal 95% zeaxanthin (from 43% to 94%).     

Supercritical carbon dioxide extraction of triglycerides from Jatropha curcas L. seeds

This study examines the effects of pressure, temperature and solvent to solid ratio (SSR) on extraction efficiency of triglycerides from powdered Jatropha seeds by using supercritical carbon dioxide extraction. Supercritical extractions were designed for pressures ranging from 250 to 350 bar, temperatures ranging from 313 to 333 K and SSR values ranging from 65:1 to 125:1. All values were selected using response surface methodology in order to determine their effects on the concentration of triglycerides from the extracted oil. Using 3750 g of carbon dioxide over 5 h, a supercritical carbon dioxide extraction (at 350 bar, 333 K and an SSR value of 125:1) yielded 43.51% oil. The concentration and extraction efficiency (i.e. recovery) of triglycerides in the extract reached 657.1 mg/g and 97.62%, respectively. Changes in pressure presented more effective in increasing the recovery of triglycerides, but both temperature and the SSR value are important in obtaining high concentration of triglycerides from the Jatropha seeds that are useful for biodiesel materials.     

SFE from Bidens pilosa Linné to obtain extracts rich in cytotoxic polyacetylenes with antitumor activity

Bidens pilosa L. is a plant considered medicinal by some South American cultures. It contains polyacetylenes which may be the constituents responsible for its antitumor activity. Extracts obtained by hydroethanol maceration (HCE) and supercritical fluid extraction (SFE) were monitored for antitumor activity and the presence of polyacetylenes in the constitution. Both extracts killed concentration-dependently the MCF-7 cells in culture, although the SFE extract presented superior cytotoxic activity. The SFE presented IC₅₀ = 437 (428-446) μg/mL in 24 h of incubation, decreasing to IC₅₀ = 291 (282-299) μg/mL at 48 h. The HCE started causing DNA cleavage at 160 μg/mL while the SFE extract started at 40 μg/mL, a concentration enough to initiate the in vitro cleavage. The presence of polyacetylenes as the major compounds in SFE was confirmed by TL chromatography combined with UV-vis analyses. Ehrlich ascites carcinoma-bearing mice were used for the antitumor study. Animals were divided in five groups: normal, negative control, positive control (Doxorubicin 0.06 mg/kg), test group HCE and test group SFE (100 mg/kg b.w. per day). After 9 days of treatment, 50% of randomly chosen animals from each group were sacrificed for the study. The parameters evaluated were: body weight, abdominal circumference, volume of ascitic fluid and tumor cells, viable and nonviable tumor cell count, determination of mean survival time and increased life-span. Both extracts presented antitumor activity, but SFE reduced more the volumes of ascites fluid and the tumor cells (4 ± 1 and 1 ± 0.4 mL, respectively), while caused higher mean survival time (17 days) and increased life span (∼31%). The results suggest the importance of the polyacetylenes from B. pilosa as leader molecules to contribute to a new anticancer drug by using the supercritical technology.     

Study on supercritical extraction of lipids and enrichment of DHA from oil-rich microalgae

The present study aims to isolate the lipids from microalgae by supercritical CO₂ (SC-CO₂) extraction followed by a further enrichment of crude lipids to produce high-purity docosahexenoic acid (DHA) by an urea complexation method. Our systematic approach indicates the optimum conditions of supercritical CO₂ extraction were obtained as follows: 35 MPa, 40 °C, ethanol (95%, v/v) as the co-solvent, and the mass ratio of material to co-solvent 1:1. Under these conditions, 33.9% of lipid yield and 27.5% of DHA content were achieved. Despite the relatively low lipid yield, supercritical CO₂ extraction has exhibited many advantages over the Soxhlet extraction for the DHA enrichment such as high DHA purity and superb product quality. Furthermore, urea complexation method on DHA enrichment considerably increased the DHA purity from 29.7% to 60.4% with an enrichment ratio of 60.6%, under the optimum complexation conditions of urea/fatty acid 2:1, complexation time 8 h, and the complexation temperature of −10 °C.     

Supercritical fluid extraction of Prunus persica leaves and utilization possibilities as a source of phenolic compounds

Mediterranean countries contribute highly on world peach production and tonnes of waste leaves are released due to pruning. The aim of this study was to investigate the utilization possibilities of the leaves by supercritical fluid extraction. A statistical design was used to evaluate the effect of temperature (40–80 °C), pressure (150–300 bar) and concentration of ethanol as co-solvent (6–20%) at a flow rate of 15 g/min and for a duration of 60 min. The most effective variables were found as pressure and co-solvent ratio (p < 0.005). Optimum extraction conditions were elicited as 60 °C, 150 bar and 6% co-solvent yielding a total phenol content of 79.92 mg GAE/g extract, EC₅₀ value of 232.20 μg/ml and a radical scavenging activity of 53.25% which was higher than the value obtained by conventional solvent extraction method (32.23%). Consequently, Prunus persica L. leaves were found as a potential phenolic source for industrial applications.     

超临界流体在环境保护中的应用和展望

超临界流体技术广泛应用于化工、医药、生物、食品、陶瓷等领域。将超临界流体技术用于环境保护则是一个新的研究方向,由于该技术对废物处理具有经济、快速、高效等特点,近几年来发展异常迅速。综述了超临界流体的特性以及超临界流体技术（超临界萃取、超临界色谱和超临界水氧化）在环境保护方面的应用现状。     

Supercritical extraction of Hypericum caprifoliatum using carbon dioxide and ethanol + water as co-solvent

Hypericum caprifoliatum is a natural source of phloroglucinol derivatives such as hyperbrasilol B, uliginosin B and japonicin A, compounds with pharmaceutical interest due to principally their antidepressant properties. This work studies supercritical fluid extraction to obtain non-volatile compounds from the aerial parts of H. caprifoliatum as well as the influence of the co-solvent as agent of process intensification. The experimental procedures were carried out in a pilot unity. The mathematical simulation of the experimental data was performed by mean of three mathematical models aiming to promote the improvement of the knowledge about this technology. The feasibility of the mathematical model was investigated by mean of fitting of the experimental data obtained. The co-solvents tested were ethanol, water, and water–ethanol mixtures.     

Enzyme and high pressure assisted extraction of carotenoids from tomato waste

Enzyme (EA) and high pressure (HP) assisted extraction of carotenoids, especially lycopene, from tomato waste using various organic solvents was examined. Total carotenoid and lycopene extraction yields were increased by the use of pectinase and cellulase enzymes, when compared to the non enzyme treated solvent extraction process. The increase of extraction yield depended on the solvent. Maximum total carotenoid (127 mg/kg d.w.) and lycopene (89.4 mg/kg d.w.) extraction yields were obtained in enzyme treated samples extracted with ethyl lactate (solvent:solid = 10:1 mL:g), corresponding to almost 6-fold and 10-fold increase, respectively, with respect to non enzyme treated samples. HP assisted extraction led to higher extraction yields (from 2 to 64% increase depending on the solvent used) compared to conventional solvent extraction process performed at ambient pressure for 30 min. HP assisted solvent extraction was successfully performed at 700 MPa by using significantly (P < 0.05) lower ratios of solvent:solid (6:1 and 4:1 mL:g) and reduced processing time (10 min), compared to solvent extraction performed at ambient pressure, solvent:solid ratio 10:1 mL:g and 30 min extraction time.