Supercritical carbon dioxide extraction of marigold at high pressures: comparison of analytical and pilot-scale extraction

The effects of pressure and co-solvent on the extraction of anti-inflammatory faradiol esters in marigold (Calendula officinalis L.) were investigated by supercritical fluid extraction at laboratory and pilot scales. Pressures higher than 300 bar and modifier (ethanol) concentrations ranging from 0 to 20% (v/v) were used at an extraction temperature of 50 degrees C. With an analytical extractor, exhaustive extraction of the drug and highest concentrations in the extracts were achieved with 0.5% ethanol at the maximum pressure of 689 bar. Increased modifier concentrations improved the extractability at lower pressure, but the higher amount of total extractables led to a lower concentration of faradiol esters in the extracts. The HPLC fingerprints of the extracts, the yields of total extract and the concentration of faradiol esters obtained with analytical and pilot scale extractors under the same conditions were comparable.     

Extraction of fucoxanthin and polyphenol from <Emphasis Type="Italic">Undaria pinnatifida</Emphasis> using supercritical carbon dioxide with co-solvent

Extraction of brown seaweed (Undaria pinnatifida) oil was carried out by using supercritical carbon dioxide (SCO₂) and ethanol as co-solvent. The flow rate of ethanol was 3.0% (v/v) as compared to that of SCO₂. Experiments were performed in a semi-batch flow apparatus on dried samples at temperatures from 303 to 333 K and pressures from 80 to 300 bar. Fucoxanthin and polyphenol were quantitatively analyzed by using HPLC and UV-spectrometer. The highest yields of fucoxanthin and polyphenol were shown at 200 bar, 323 K and 250 bar, 333 K, respectively. The solubility of fucoxanthin in SCO₂ agreed well with the Chrastil model.     

Batch solvent extraction of flavanolignans from milk thistle (Silybum marianum L. Gaertner)

Seeds of milk thistle (Silybum marianum L. Gaertner) contain silymarins and ca. 25% (w/w) of oil. A pre-treatment step involving refluxing with petroleum ether is usually performed before extraction of the silymarins using organic solvents. This paper compares the extraction of whole and defatted milk thistle seeds in various solvents as a function of temperature. The extraction of whole seeds of milk thistle with water at 50, 70 and 85 degrees C was also examined: the yield of silymarin increased with increasing water temperature. In most cases, ethanol at 60 degrees C recovered the largest quantities of silymarins. However, boiling water proved to be an efficient extraction solvent for the more polar silymarins such as taxifolin and silychristin, even when using whole seeds. Extractions of defatted seed meal with boiling ethanol returned maximum yields of 0.62, 3.89, 4.04, and 6.86 mg/g defatted seed of taxifolin, silychristin, silybinin A and silybinin B, respectively. When extracting defatted seed meal with ethanol, yields of taxifolin, silybinin A and silybinin B were, respectively, 6.8-, 0.95-, 1.7- and 1.6-fold higher than when extracting whole seeds. When extracting with boiling water, the yields of silychristin, silybinin A, and silybinin B were 380, 47 and 50% higher for whole seeds compared with defatted seeds.     

Supercritical carbon dioxide extraction of chamomile flowers: extraction efficiency, stability, and in-line inclusion of chamomile-carbon dioxide extract in beta-cyclodextrin

The extraction of chamomile flowers using supercritical carbon dioxide was investigated with respect to extraction efficiency and compared with solvent extraction. The stability of matricine, a sensitive constituent of the essential oil of chamomile, in these extracts was studied during storage at different temperatures over 6 months. Matricine was stable at -30 degrees C. A slight decrease (80-90% recovery) occurred at +5 degrees C, whereas complete decomposition of matricine took place within 3-4 months at room temperature and at +30 degrees C, respectively. An in-line inclusion of chamomile constituents in beta-cyclodextrin (beta-CD) during the extraction process was assessed and inclusion rates between 40 and 95% were obtained depending on the amount of beta-CD and the type of chamomile constituent. No further stabilization of matricine in the carbon dioxide extract/beta-CD complexes was achieved. High residual water contents in the complexes even after freeze-drying were identified as accelerating the decomposition. In addition, the extractability of flavonoids, such as apigenin and apigenin-7-glucoside, was determined. Apigenin-7-glucoside, the more hydrophilic substance, was not extractable with pure carbon dioxide and showed a recovery of 11% using methanol modified carbon dioxide (18%, w/w) at 60 degrees C and 380 bar. Extraction conditions in the two-phase region of the binary mixture carbon dioxide-methanol (70 degrees C, 100 bar) led to a drastic change in fluid polarity and hence extractability increased to 92-95%.     

Global metabolite analysis: the influence of extraction methodology on metabolome profiles of Escherichia coli

The global pool of all metabolites in a cell, or metabolome, is a reflection of all the metabolic functions of an organism under any particular growth condition. In the absence of in situ methods capable of universally measuring metabolite pools, intracellular metabolite measurements need to be performed in vitro after extraction. In the past, a variety of cell lysis methods were adopted for assays of individual metabolites or groups of intermediates in pathways. In this study, metabolites were extracted from Escherichia coli using six different commonly used procedures including acid or alkaline treatments, permeabilization by freezing with methanol, high-temperature extraction in the presence of ethanol or methanol, and by lysis with chloroform-methanol. Metabolites were extracted by the six methods from cells grown under identical conditions and labeled with [14C]glucose. The metabolomes were compared after 2-dimensional thin-layer chromatography of labeled compounds. For global analysis, extraction with cold (-40 degrees C) methanol showed the greatest promise, allowing simultaneous resolution of more than 95 metabolite spots. In contrast, 80 or less spots were obtained with other extraction methods. Extraction also influenced quantitative analysis of particular compounds. Metabolites such as adenosine exhibited up to 20-fold higher abundance after cold methanol extraction than after extraction with acid, alkali, or chloroform. The simplicity, rapidity, and universality of cold methanol extraction offer great promise if a single method of lysis is to be adopted in metabolome analysis.     

Optimization of Ethanol Production From Microfluidized Wheat Straw by Response Surface Methodology

In this study, wheat straw was pretreated with a microfluidizer to improve its enzymatic hydrolysis and ethanol yields. The pretreatment was performed at various pressures (500, 1000, and 1500 bar) and solid loadings (1, 2, and 3%). The microfluidized biomass was then subjected to hydrolysis and simultaneous saccharification and co-fermentation (SSCF) experiments at different enzyme loadings (5, 10, and 15 FPU/g dry wheat straw) using a mutant yeast. The results indicated that the microfluidization method alters the structure of biomass and leads to a reduction in lignin content. The samples pretreated at 1% solid loading contained the minimum lignin concentration and provided the maximum sugar and ethanol yields. These results signified that the microfluidization method is more effective on biomass at low solid loadings. The process conditions were optimized for higher ethanol and sugar yields using response surface methodology (RSM). The optimum pressure and solid and enzyme loadings were found as 1500 bar, 1%, and 15 FPU/g dry wheat straw, respectively. The yields obtained at this condition were 82%, 94%, and 65% for glucose, xylose, and ethanol, respectively. High sugar yields implied that microfluidization is an effective pretreatment method for cellulosic ethanol production. On the other hand, low ethanol yield may indicate that the microorganism was sensitive to inhibitory compounds present in the fermentation medium.     

Quantification of seed oil from species with varying oil content using supercritical fluid extraction

Introduction: The quantity and composition of seed oil affects seed viability and storability and hence the value of a species as a resource for nutrition and plant conservation. Supercritical fluid extraction with carbon dioxide (SFE‐CO₂) offers a rapid, environmentally friendly alternative to traditional solvent extraction. Objective: To develop a method using SFE‐CO₂ to quantify the seed oil content in a broad range of species with high to low oil contents. Methodology: Seed oil was extracted using SFE‐CO₂ from four crop species representing high, medium and low oil content: Helianthus annuus, Asteraceae, with ca. 55% oil; Brassica napus, Brassicaceae, with ca. 50% oil; Glycine max, Fabaceae, with ca. 20% oil; and Pisum sativum, Fabaceae, with ca. 2% oil. Extraction pressures of 5000, 6000 and 7500 psi and temperatures of 40, 60 and 80°C were examined and a second step using 15% ethanol as a modifier included. Oil yields were compared with that achieved from Smalley Butt extraction. The optimised SFE‐CO₂ method was validated on six species from taxonomically distant families and with varying oil contents: Swietenia humilis (Meliaceae), Stenocereus thurberi (Cactaceae), Sinapis alba (Brassicaceae), Robinia pseudoacacia (Fabaceae), Poa pratensis (Poaceae) and Trachycarpus fortunei (Arecaceae). Results: The two‐step extraction at 6000 psi and 80°C produced oil yields equivalent to or higher than Smalley Butt extraction for all species, including challenging species from the Brassicaceae family. Conclusion: SFE‐CO₂ enables the rapid analysis of seed oils across a broad range of seed oil contents. Copyright © 2008 John Wiley & Sons, Ltd.     

Isolation of vindoline from Catharanthus roseus by supercritical fluid extraction.

Vindoline was extracted from the leaves of Catharanthus roseus over the ranges of 35-70 degrees C and 100-300 bar using supercritical carbon dioxide with and without the addition of 3 wt % ethanol as a cosolvent. The vindoline contents in the extracts were determined by HPLC and identified by LC/MS. The remarkable highest vindoline concentration, 58 wt %, was obtained at the lowest temperature, 35 degrees C, and the highest pressure, 300 bar, of this study. The use of a cosolvent only slightly improved the extraction yields or selectivities at some experimental conditions.     

Effects of ethanol on the growth and elongation of Escherichia coli under high pressures up to 40 MPa

The effects of ethanol on growth, viability and cell length were studied in Escherichia coli cultured under pressures up to 40 MPa (400 bar). A pressure of 10 MPa reversed the effect of ethanol in retarding cell growth. Cells cultured in the absence of ethanol became about seven times longer at 40 MPa than at atmospheric pressure, and some cells showed incomplete cell division. Ethanol also increased cell length but these effects were not seen at pressures of 20 MPa or more.     

Evaluation of metabolite extraction strategies from tissue samples using NMR metabolomics

Metabolomic analysis of tissue samples can be applied across multiple fields including medicine, toxicology, and environmental sciences. A thorough evaluation of several metabolite extraction procedures from tissues is therefore warranted. This has been achieved at two research laboratories using muscle and liver tissues from fish. Multiple replicates of homogenous tissues were extracted using the following solvent systems of varying polarities: perchloric acid, acetonitrile/water, methanol/water, and methanol/chloroform/water. Extraction of metabolites from ground wet tissue, ground dry tissue, and homogenized wet tissue was also compared. The hydrophilic metabolites were analyzed using 1-dimensional (1D) ¹H nuclear magnetic resonance (NMR) spectroscopy and projections of 2-dimensional J-resolved (p-JRES) NMR, and the spectra evaluated using principal components analysis. Yield, reproducibility, ease, and speed were the criteria for assessing the quality of an extraction protocol for metabolomics. Both laboratories observed that the yields of low molecular weight metabolites were similar among the solvent extractions; however, acetonitrile-based extractions provided poorer fractionation and extracted lipids and macromolecules into the polar solvent. Extraction using perchloric acid produced the greatest variation between replicates due to peak shifts in the spectra, while acetonitrile-based extraction produced highest reproducibility. Spectra from extraction of ground wet tissues generated more macromolecules and lower reproducibility compared with other tissue disruption methods. The p-JRES NMR approach reduced peak congestion and yielded flatter baselines, and subsequently separated the metabolic fingerprints of different samples more clearly than by 1D NMR. Overall, single organic solvent extractions are quick and easy and produce reasonable results. However, considering both yield and reproducibility of the hydrophilic metabolites as well as recovery of the hydrophobic metabolites, we conclude that the methanol/chloroform/water extraction is the preferred method. C. Y. Lin and H. Wu contributed equally.     

Dynamic superheated liquid extraction of anthocyanins and other phenolics from red grape skins of winemaking residues

Grape skins from a grape pomace were subject to extraction with superheated ethanol-water mixtures for quantitative extraction of anthocyans and other phenolic compounds. The variables affecting dynamic extraction of these compounds were studied and identification and quantification of the extracted compounds were performed by both direct spectrophotometry or after HPLC separation using UV or MS detectors. The optimal working conditions for total extraction of anthocyans were: 1:1 (v/v) ethanol-water acidified with 0.8% (v/v) HCl, 120 degrees C, 30 min, 1.2 ml/min and 80 bar. The yields of anthocyanins, total phenolics and flavanols thus obtained were much higher (3 times for anthocyanins, 7 times for total phenolics and 11 times for flavanols) than those provided by dynamic conventional solid-liquid extraction. Several sample preparation procedures for skins as alternatives to free-drying were also investigated and drying at 40 degrees C for 24h provided the best results. Extraction with acidified water provides similar composition and poorer efficiency than 1:1 ethanol-water; also similar to two commercial grape skin extracts used as natural colorants.     

Supercritical fluid extraction and high performance liquid chromatographic determination of benzopyrans and phloroglucinol derivative in Hypericum polyanthemum

The aerial parts of Hypericum polyanthemum Klotzsch ex Reichardt (Guttiferae) were successively extracted with supercritical carbon dioxide (SC CO₂) under pressures of 90, 120, 150 and 200 bar at different temperatures (40, 50 and 60 °C), and compared with the n-hexane extract obtained by ultrasound-assisted extraction. The samples obtained were examined regarding extraction yield and HPLC quantification of the main secondary metabolites, the benzopyrans HP1 (6-isobutyryl-5,7-dimethoxy-2,2-dimethylbenzopyran), HP2 (7-hydroxy-6-isobutyryl-5-methoxy-2,2-dimethyl-benzopyran) and HP3 (5-hydroxy-6-isobutyryl-7-methoxy-2,2-dimethyl) and the phloroglucinol derivative, uliginosin B. SFE presented higher selectivity than the n-hexane maceration, and the best condition to extract the target metabolites has been determined to be at 50 °C and for the high molecular-weight compound, uliginosin B, higher pressures were required.     

Toward Arabidopsis thaliana hydrophilic metabolome: assessment of extraction methods and quantitative 1H NMR

Our goal was to establish the hydrophilic metabolome of heterotrophic Arabidopsis thaliana cells grown in suspension, a cellular model of plant sink tissues. Water‐soluble metabolites were extracted using four protocols: perchloric acid, boiling ethanol, methanol and methanol/chloroform (M/Chl). They were detected and quantified using ¹H nuclear magnetic resonance (NMR) spectroscopy at 400 MHz. Extraction yields and reproducibility of the extraction methods were investigated. The effects of cell harvest protocol, cell grinding and lyophilization and storage conditions on the measured metabolic profiles were also studied. These quantitative studies demonstrated for the first time that the four extraction protocols commonly used do lead to quite similar molecular compositions as analyzed by ¹H NMR. The M/Chl method proved effective and reliable to prepare series of physiologically significant extracts from plant cells for ¹H NMR analysis. Reproducibility of the detected metabolome was assessed over long periods of time by analyzing a large number of separate extracts prepared from independent cultures. Larger variations in the NMR metabolite profiles could be correlated to changes in physiological parameters of the culture medium. Quantitative resolved ¹H NMR of cell extracts proved to be robust and reliable for routine metabolite profiling of plant cell cultures.     

Effects of pressure and temperature of supercritical carbon dioxide on the extraction of triacylglycerols from plant tissue

The rate of extraction of triacylglycerols with supercritical carbon dioxide can be greatly enhanced by raising the pressure of the fluid to 600 bar, or higher, and its temperature to 60°C, or higher. Both the amount of carbon dioxide and the time required for complete extraction are reduced at such high pressures and temperatures.     

Sampling techniques and comparative extraction procedures for quantitative determination of intra- and extracellular metabolites in filamentous fungi

Two methods for rapid sampling and three procedures for extraction of metabolites from the filamentous fungus Monascus ruber were compared. It is shown that arrest of metabolism by either dropping the mycelial cultures in liquid nitrogen or by spraying them on a 60% solution of methanol kept at −40°C followed by rapid centrifugation at −10°C were equally effective. Metabolites were extracted from mycelia using different procedures including acid and alkaline treatments, permeabilization by cold chloroform and extraction by boiling buffered ethanol, to demonstrate that the latter method gave the best results both in terms of recovery and stability of metabolites. In addition, this method is very simple to handle and allows the use of very low amounts (i.e. 10–20 mg dry mass) of cellular material since the removal of ethanol by evaporation after extraction results in a concentration step of metabolites.     

Enzyme-assisted extraction of fucoxanthin and lipids containing polyunsaturated fatty acids from Undaria pinnatifida using dimethyl ether and ethanol

A novel method for the efficient extraction of fucoxanthin and lipids containing polyunsaturated fatty acids (PUFAs) from the brown seaweed Undaria pinnatifida was developed and demonstrated at a laboratory scale. U. pinnatifida, also known as Wakame, contains a number of biologically active lipophilic compounds, particularly fucoxanthin, which has anti-oxidant, anti-cancer, anti-obesity and anti-inflammatory properties. The yield of fucoxanthin and lipids containing PUFAs was determined by extraction from wet and freeze-dried seaweed using dimethyl ether (DME) and ethanol and from enzyme-pretreated seaweed using the same solvents. The highest yields of fucoxanthin (94%) and lipids (94%) rich in PUFAs were obtained from fresh (wet) U. pinnatifida by enzyme pre-processing, followed by extraction using DME with ethanol as a co-solvent. In comparison, ethanol extraction resulted in lower extraction yields for both fucoxanthin (86%) and lipids (73%) under the conditions described. Enzyme pre-processing using alginate lyase resulted in the hydrolysis of cell wall polysaccharides, resulting in high extraction yields. The hydrolysis time, pH and temperature were found to be the most important parameters for the enzyme pre-processing step and for minimizing fucoxanthin losses due to oxidative degradation. The removal of water-soluble compounds (polysaccharides) following the enzyme pre-treatment prior to DME extractions doubled the throughput and maximized the yield. The residual biomass was colorless or a pale-brown color after the DME extraction, which indicated the highly effective extraction of fucoxanthin. The PUFA content and fucoxanthin levels were not affected by the enzyme or extraction using the described enzyme-assisted DME + ethanol co-solvent process.     

猴头菌子实体超临界流体技术萃取活性物质初探

以猴头菌子实体为原料,萃取物得率作为指标,采用CO₂超临界流体萃取技术,以萃取压力和CO₂流量等参数为考察因素,结合正交试验获得优化的萃取工艺：萃取压力为30MPa,温度为45℃,时间为1.5h,CO₂流量为20g/min,夹带剂（乙醇）与猴头菌子实体的物料比为5:1（mL:g）,在此条件下,萃取物得率为2.78%。与猴头菌子实体的醇提物相比,猴头菌子实体超临界萃取物具有更好的体外抗氧化能力和抗肿瘤活性,本研究结果为合理地开发和利用猴头菌子实体超临界萃取物产品提供科学的数据。     

Separation of cashew (Anacardium occidentale L.) nut shell liquid with supercritical carbon dioxide

Cashew nut shell liquid (CNSL) represents the largest readily available bioresource of alkenyl phenolic compounds. In this work, separation of CNSL from the pericarp of the cashew nut with supercritical carbon dioxide was studied. In the initial extractions with CO(2) at 40-60 degrees C and at pressures from 14.7 to 29.4 MPa, low yields were obtained. However, when the extractions were performed with one or more intermediate depressurization steps, the yield of CNSL increased to as high as 94%. Most of the oil did not separate from the shell during the depressurization step, but was obtained during the subsequent repressurization. The CNSL extract had a clear light brownish pink color and exhibited no evidence of polymerization or degradation. The pressure profile extraction method proposed in this work increases the possible CNSL extraction yields and greatly reduces the amount of CO(2) required for CNSL separation.     

Determination of Parameters for the Supercritical Extraction of Antioxidant Compounds from Green Propolis Using Carbon Dioxide and Ethanol as Co-Solvent

The aim of this study was to determine the best processing conditions to extract Brazilian green propolis using a supercritical extraction technology. For this purpose, the influence of different parameters was evaluated such as S/F (solvent mass in relation to solute mass), percentage of co-solvent (1 and 2% ethanol), temperature (40 and 50°C) and pressure (250, 350 and 400 bar) using supercritical carbon dioxide. The Global Yield Isotherms (GYIs) were obtained through the evaluation of the yield, and the chemical composition of the extracts was also obtained in relation to the total phenolic compounds, flavonoids, antioxidant activity and 3,5-diprenyl-4-hydroxicinnamic acid (Artepillin C) and acid 4-hydroxycinnamic (p-coumaric acid). The best results were identified at 50°C, 350 bar, 1% ethanol (co-solvent) and S/F of 110. These conditions, a content of 8.93±0.01 and 0.40±0.05 g/100 g of Artepillin C and p-coumaric acid, respectively, were identified indicating the efficiency of the extraction process. Despite of low yield of the process, the extracts obtained had high contents of relevant compounds, proving the viability of the process to obtain green propolis extracts with important biological applications due to the extracts composition.     

Validated methods for the quantification of biologically active constituents of poplar-type propolis

The validation of rapid, low-cost spectrophotometric procedures for the quantification of the three main groups of bioactive substances (flavones and flavonols, flavanones and dihydroflavonols, and total phenolics) in poplar-type propolis has been performed. A spectrophotometric assay based on the formation of an aluminium chloride complex was applied for the quantification of total flavones and flavonols using galangin as standard. Because of the high amount of flavanones and dihydroflavonols in "poplar type" propolis, the introduction of a distinct procedure for their quantification was considered of special significance and the DAB9 colorimetric method was applied for the purpose. Total phenolic content was measured by the Folin-Ciocalteu procedure using a mixture of pinocembrin and galangin as a reference. The procedures were validated using a model mixture of compounds representing the poplar-type propolis composition as found in previous studies. The accuracy (recovery) varied in the range 84-109%, and the relative standard deviation was 0.5-6.2%. The developed spectrophotometric procedures were applied to six poplar type propolis samples. The results were verified independently by a HPLC procedure. The two sets of results agreed satisfactory, as proven by Student's t-test.