Optimisation of supercritical fluid extraction of indole alkaloids from Catharanthus roseus using experimental design methodology--comparison with other extraction techniques

Response surface modelling, using MODDE 6 software for Design of Experiments and Optimisation, was applied to optimise supercritical fluid extraction (SFE) conditions for the extraction of indole alkaloids from the dried leaves of Catharanthus roseus. The effects of pressure (200-400 bar), temperature (40-80 degrees C), modifier concentration (2.2-6.6 vol%) and dynamic extraction time (20-60 min) on the yield of alkaloids were evaluated. The extracts were analysed by high-performance liquid chromatography and the analytes were identified using ion trap-electrospray ionisation-mass spectrometry. The method was linear for alkaloid concentration in the range 0.18-31 microg/mL. The limits of detection and quantification for catharanthine, vindoline, vinblastine and vincristine were 0.2, 0.15, 0.1 and 0.08 microg/mL and 2.7, 2.0, 1.3 and 1.1 microg/g, respectively. The dry weight content of major alkaloids in the plants were compared using different extraction methods, i.e. SFE, Soxhlet extraction, solid-liquid extraction with sonication and hot water extraction at various temperatures. The extraction techniques were also compared in terms of reproducibility, selectivity and analyte recoveries. Relative standard deviations for the major alkaloids varied from 4.1 to 17.5% in different extraction methods. The best recoveries (100%) for catharanthine were obtained by SFE at 250 bar and 80 degrees C using 6.6 vol% methanol as modifier for 40 min, for vindoline by Soxhlet extraction using dichloromethane in a reflux for 16 h, and for 3',4'-anhydrovinblastine by solid-liquid extraction using a solution of 0.5 m sulphuric acid and methanol (3:1 v/v) in an ultrasonic bath for 3 h.     

Investigation of the pharmaceutical and pharmacological equivalence of different Hawthorn extracts

Seven Hawthorn extracts were tested in isolated guinea pig aorta rings. The effect on noradrenaline- (10 microM) induced contraction was investigated. The extracts were prepared using ethanol (40 to 70% v/v), methanol (40 to 70% v/v), and water as the extraction solvents. The aqueous-alcoholic extracts displayed similar spectra of constituents. They were characterised by similar procyanidin, flavonoid, total vitexin and total phenols content and by similar TLC fingerprint chromatograms. The aqueous extract, however, showed a different fingerprint and a noticeably lower concentration of procyanidins, flavonoids and total phenols but a similar total vitexin content. All 7 extracts had a relaxant effect on the aorta precontracted by noradrenaline and led to relaxations to 44 until 29% of the initial values. The EC50 values of the aqueous-alcoholic extracts varied between 4.16 and 9.8 mg/l. The aqueous extract produced a similarly strong maximal relaxation as the other extracts, but the EC50, at 22.39 mg/l, was markedly higher. The results show that Hawthorn extracts with comparable quality profiles were obtained by using aqueous-alcoholic extraction solvents (40 to 70% ethanol or methanol). The extracts exerted comparable pharmacological effects. When using water as the extraction solvent, both, the spectrum of constituents and the pharmacological effect, deviated remarkably. It is thus possible to obtain bioequivalent extracts with comparable effect profiles by using 40 to 70% ethanol or methanol as the extraction solvent.     

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.     

Supercritical carbon dioxide extraction of selected medicinal plants--effects of high pressure and added ethanol on yield of extracted substances

The possibilities and limitations of supercritical fluid extraction of natural products of low, medium and high polarity under very high pressure and with polar modifiers has been investigated. The medicinal herbs marigold (Calendula officinalis), hawthorn (Crataegus sp.) and chamomile (Matricaria recutita) were used as models in this study. Extraction profiles and the spectra of extractable metabolites were recorded following extraction with mixtures of carbon dioxide:ethanol of varying proportions (0-20% ethanol) and at various pressures in the range 300-689 bar. Components were identified by HPLC-PAD-MS or GC-MS and quantified by HPLC or GC as appropriate. Extraction yields under the varying conditions depended to a large extent on the profiles of secondary metabolites present in the three drugs. Whereas the extractability of lipophilic compounds increased substantially at pressures above 300 bar, the yields of polyphenolic and glycosidic compounds remained low even at 689 bar and with 20% modifier in the extraction fluid.     

The effect of solvent extraction on antioxidant properties of apricot fruit

Two solvent extraction procedures were used to investigate the extraction efficiency in terms of total antioxidant capacity and total phenols in apricot fruit. Samples were either sequentially extracted with aqueous ethanol (ethanol/water 80% v/v) and tetrahydrofuran or directly extracted with tetrahydrofuran. Each extract was analyzed for total antioxidant capacity by the Trolox Equivalent Antioxidant Capacity (TEAC) assay and total phenols by the Folin-Ciocalteu assay. The results showed that using sequential solvent extraction, the majority (85%) of the total antioxidant capacity and total phenols was due to hydrophilic compounds. In tetrahydrofuran direct extractions, the total antioxidant capacity and total phenols were higher than values obtained with aqueous ethanol and the sum of results obtained from sequential extracts for either total antioxidant capacity or total phenols was similar to the tetrahydrofuran-extract antioxidant values. A linear correlation between total antioxidant capacity and total phenols was found and was independent of the solvent extraction method. In conclusion, the choice of solvent is related to the antioxidant potential of fruit and depends on the food hydrophilic/lipophilic composition.     

Influence of the extraction solvent on antioxidant activity of Althaea officinalis L. root extracts

Althaea officinalis (Malvaceae) is a well-known plant that is widely distributed throughout the world. Aqueous and hydroalcoholic extracts from A. officinalis root are used mainly because of their antitussive and expectorant activity. It is well known that these activities are based on the polysaccharide composition, but little is known about the possible antioxidant activity of root extract. The present study evaluated antioxidant activity of root extracts prepared with different extraction solvents applying ABTS^·+ (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), hypochlorous acid scavenging assay and iron-induced lipid peroxidation. The results showed that the extract prepared with water as extraction solvent did not possess antioxidant activity, whereas the extracts obtained using ethanol:water as extraction agent showed well pronounced antioxidant activity. In particular, the extracts obtained at low concentration of ethanol in the mixed solvent (50:50 and 70:30, v/v) showed higher scavenging activity for ABTS^·+ radicals and hypochlorite ions than the extract obtained with the higher ethanol concentration (90:10, v/v). These results correlated very well with phenolic and flavonoid content of the extracts. The extracts did not show cytotoxic effect on human BV-173 leukemic cells but may have immunomodulating effects due to their antioxidant properties.     

Determination of the structure and degree of polymerisation of fructans from Echinacea purpurea roots

Highly water soluble fructans have been isolated from Echinacea purpurea (L.) Moench. roots by hot water extraction and precipitation at three different ethanol concentrations (80% v/v, 60% v/v and 40% v/v). The structure of the fructans has been characterised by three analytical methods: GC of silylated oxime derivatives and partially methylated alditol acetates, respectively, as well as 13C NMR analysis. The mean degree of polymerisation (mean DP) of each fructan has been determined by the glucose/fructose ratio. E. purpurea fructans represent linear inulin-type fructans with almost exclusively beta-(2-->1)-linked fructosyl units, terminal glucose and terminal fructose. Small proportions of beta-(2-->1,2-->6)-linked branch point residues were detected. The mean DP of the fructan fractions depends on the ethanol concentration used for precipitation: the lower the ethanol concentration the higher the mean DP. Corresponding results were found with all of the three analytical methods: 80% ethanol-insoluble fructan from E. purpurea shows an average mean DP of 35, 60% ethanol-insoluble fructan of 44 and 40% ethanol-insoluble fructan of 55. The applied methods provide sufficient sensitivity to determine not only the composition and structure but also the mean degree of polymerisation of fructans.     

油菜花粉总黄酮的微波辅助提取及其抗氧化活性研究

采用微波辅助提取法提取油菜花粉总黄酮,通过单因素和正交试验,得到最佳提取条件：80％的乙醇、1：20的料液比、提取时间140s、微波功率242W、提取3次,总黄酮质量分数为（2．64±0．05）％。同时还研究提取物对羟自由基、DPPH自由基的清除作用,以及对小鼠肝组织脂质过氧化的抑制作用,自由基半清除质量浓度和脂质过氧化丰抑制盾奄浓度分剐为0．115、0．325和0．065mg／mL。     

Microwave-assisted extraction of glycyrrhizic acid from licorice root

In the present study, a microwave-assisted extraction (MAE) technique has been developed for the extraction of glycyrrhizic acid (GA) from licorice root. Various experimental conditions, such as extraction time, different ethanol and ammonia concentration, liquid/solid ratios, pre-leaching time before MAE and material size for the MAE procedure were investigated to optimize the efficiency of the extraction. Under appropriate MAE conditions, such as extraction times of 4-5min, ethanol concentrations of 50-60% (v/v), ammonia concentrations of 1-2% (v/v) and liquid/solid ratios of 10:1(ml/g), the recovery of GA from licorice root with MAE was equivalent with conventional extraction methods. Those methods include extraction at room temperature (ERT), the traditional Soxhlet extraction, heat reflux extraction and ultrasonic extraction. Due to the considerable savings in time and solvent, MAE was more effective than the conventional methods. This novel method is suitable for fast extraction of GA from licorice root.     

High anti-inflammatory activity of harpagoside-enriched extracts obtained from solvent-modified super- and subcritical carbon dioxide extractions of the roots of Harpagophytum procumbens

Solvent-modified carbon dioxide extractions of the roots of Harpagophytum procumbens have been investigated with respect to extraction efficiency and content of harpagoside, and compared with a conventional extract. The effects of pressure, temperature, type and concentration of the modifier have been examined. Two extraction steps were necessary in order to achievehigh anti-inflammatory harpagoside-enriched extracts. The first extraction step was carried out in the supercritical state using carbon dioxide modified with n-propanol to remove undesired lipophilic substances. The main extraction was performed either in the supercritical or in the subcritical state with carbon dioxide modified with ethanol. The supercritical fluid extraction resulted in extracts containing up to 30% harpagoside. The subcritical extracts showed a harpagoside content of ca. 20%, but the extraction yield was nearly three times greater compared with supercritical conditions. The total harpagoside recovery resulting from the sum of the extract and the crude drug residue was greater than 99% in all experiments. The conventional extract and two carbon dioxide extracts were tested for in-vitro inhibition of 5-lipoxygenase or cyclooxygenase-2 biosynthesis. Both carbon dioxide extracts showed total inhibition on 5-lipoxygenase biosynthesis at a concentration of 51.8 mg/L. In contrast, the conventional extract failed to show any inhibition of 5-lipoxygenase biosynthesis.     

β-Carotene extraction from Dunaliella salina by supercritical CO2

This paper reports the results of supercritical carbon dioxide (scCO₂) extraction of β-carotene from Dunaliella salina as potential alternative to conventional organic solvent extraction. In pilot-scale scCO₂ experiments, the pressure, temperature, and co-solvent concentration were varied. The supercritical extraction at 500 bar, 70 °C, and 10 wt% ethanol as co-solvent yielded in the highly efficient pigment recovery of over 90%. Techno-economic assessment demonstrated higher energy consumption for the scCO₂ extraction that was compensated by lower solvent costs. Thus, comparable pigment production costs to the reference extraction with n-hexane were estimated for the scCO₂ process. Due to the green solvent properties of scCO₂ and ethanol, this approach is highly promising for extraction of algal biomass in industrial scale.

     

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.     

Efficient extraction of intracellular reduced glutathione from fermentation broth of Saccharomyces cerevisiae by ethanol

Reduced glutathione (GSH) from fermentation broth of Saccharomyces cerevisiae was extracted with ethanol without disruption of the cells. The effects of ethanol concentration, extraction temperature and extraction time were assessed by using 2(3) full factorial designs (FFD). Preliminary studies showed that ethanol concentration had the most influence on GSH yield by ethanol extraction, based on the first order regression coefficients derived using MINITAB software, and an optimal ethanol concentration (25%, v/v) was obtained. However, compared to the conventional extraction technique (hot water extraction), there was no significant advantage in yield of GSH from yeast cells using ethanol extraction under these optimized conditions. But ethanol extraction has several advantages, such as lower energy consumption and lower protein concentration of extraction broth, which may reduce the complexity and cost of the purification process. Hence, ethanol extraction which does not disrupt yeast cells could be an inexpensive, simple and efficient alternative to conventional extraction techniques in the GSH industry.     

Free radical scavenging of grape pomace extracts from Cabernet sauvingnon (Vitis vinifera)

Pressed grape pomace obtained from the wine production of Cabernet sauvignon (Vitis vinifera) vintage was dried until 9.8% moisture content, ground and submitted to extraction of soluble components from different extraction techniques. Low pressure extractions were performed with ethanol maceration followed by fractionation with n-hexane, dichloromethane, butanol and ethyl acetate. These solvents were furthermore applied for soxhlet extraction. Supercritical fluid extraction (SFE) was also performed to obtain grape pomace extracts by using pure CO(2) and CO(2) with ethanol as co-solvent in concentrations of 10, 15 and 20%w/w. The operating condition used in high pressure extractions was 150bar and 40 degrees C. The antioxidant activity of the grape pomace extracts was determined considering the free radical scavenging assay using 1,1-Diphenyl-2-picrylhydrazyl (DPPH) and was correlated with the total phenol content determined according to the Folin-Ciocalteu method. The results obtained in DPPH tests indicate the highest antioxidant activity of 96.6+/-0.3%AA, with an IC(50) value of 13+/-1, for the extracts obtained with ethyl acetate in solid-liquid extraction. The highest yield values were achieved in soxhlet extraction with ethanol (13.2%w/w) and with butanol (12.2%w/w), and also by SFE with 15% ethanol (9.2%w/w). The lipophilic composition of grape pomace extracts was evaluated by gas chromatography-mass spectrometry with the identification of components like linoleic acid and ethyl linoleate, with important therapeutic activities.     

Combined effects of ethanol, high homogenization pressure, and temperature on cell fatty acid composition in Saccharomyces cerevisiae

The specific aims of this research were to evaluate the combined effects of ethanol and high-pressure homogenization at different temperatures on cell viability in Saccharomyces cerevisiae and to study the induced modification of fatty acid composition. The decrease in viability was weak at 10 degrees C while a homogenization pressure over 1000 bar (1 bar = 100 kPa) induced a significant reduction in viability when the cells were incubated at 20 and 30 degrees C. The cell tolerance to pressure decreased with an increase in ethanol concentration and temperature. Ethanol, particularly intracellular ethanol accumulated by S. cerevisiae, played an important role in the response to homogenization pressure and in modification of the cell fatty acid composition. In fact, an unusually elevated accumulation of ethyl esters in lipid extracts of yeast cells subjected to high homogenization pressure, especially in the presence of exogenous ethanol and at 30 degrees C, was observed. Moreover, only unsaturated and traces of short chain fatty acids were esterified with ethanol.     

Determination of ximelagatran, melagatran and two intermediary metabolites in plasma by mixed-mode solid phase extraction and LC-MS/MS

An analytical method was developed for the determination of ximelagatran, an oral direct thrombin inhibitor, its active metabolite melagatran, and the two intermediate metabolites, OH-melagatran and ethyl-melagatran in human plasma. Extraction of plasma was carried out on a mixed mode bonded sorbent material (C8/SO(3)(-)). All four analytes, including their isotope-labelled internal standards, were eluted at high ionic strength with a mixture of 50% methanol and 50% buffer (0.25 M ammonium acetate and 0.05 M formic acid, pH 5.3) with an extraction recovery above 80%. The extracts were demonstrated to be clean in terms of a low concentration of albumin and lysoPC. The sample extraction was fully automated and performed in 96-well plates using a Tecan Genesis pipetting robot. Analysis of the extracts were performed with liquid chromatography followed by positive electrospray ionization mass spectrometry. The low organic content and the low pH of the extracts allowed for, after dilution 1:3 with buffer, direct injection onto the LC-column. The four analytes were separated on a C18 analytical LC-column using gradient elution with the acetonitrile concentration varying from 10 to 30% (v/v) and the ammonium acetate and acetic acid concentration kept constant at 10 and 5 mmol/L, respectively, at a flow rate of 0.75 mL/min. Linearity was achieved over the calibrated range 0.010-4.0 micromol/L with accuracy and relative standard deviation in the range 96.9-101.2% and 6.6-17.1%, respectively at LLOQ, and in the range 94.7-102.6% and 2.7-6.8%, respectively at concentrations above 3 x LLOQ. The method replaces a manual method, and displays the advantages of having a fully automated sample clean-up, no evaporation/reconstitution step, high recovery, and complete LC-separation of all four analytes.     

Measurement of ethanol concentration using solvent extraction and dichromate oxidation and its application to bioethanol production process

A method for measuring the ethanol concentration in a yeast culture broth was developed using both microtubes and a 96-deepwell microplate. The strategy involved first the solvent extraction of ethanol from the yeast culture broth and measurements of the ethanol concentration using the dichromate oxidation method. Particular focus was made on selecting the extraction solvent as well as determining the measurable range of ethanol concentrations using this solvent extraction-dichromate oxidation method. This method was developed as an assay format in 2.0-ml microtubes and 1.2-ml 96-deepwell microplates, and the ethanol concentration in the batch cultures and fed-batch fermentations was measured. Tri-n-butyl phosphate [non-alcoholic solvent, density = 0.9727, solubility in water = 0.028% (w/v)] was used for solvent extraction when measuring the ethanol concentration from the yeast culture broth. The maximum detectable ethanol concentration was 8% (v/v) when 10 g potassium dichromate in 100 ml of 5 M sulfuric acid was used. The concentrations determined from the solvent extraction-dichromate oxidation methods were remarkably similar to those of gas chromatography in which samples were prepared from seven experiments, such as four batch cultures and three fed-batch fermentations.     

Application of centrifugal force to the extraction and separation of parasorboside and gerberin from Gerbera hybrida

A method for the separation of parasorboside and gerberin from the ornamental plant Gerbera hybrida (Asteraceae) has been developed. The two closely related glucosides were extracted using an Extrachrom instrument, a prototype multi-functional separation tool equipped with an extraction chamber. The rotation planar extraction procedure was compared with that of a medium pressure solid-liquid extraction system. The resulting extracts were pre-purified using rotation planar chromatography and the results compared with those obtained using medium pressure liquid chromatography with silica gel as the stationary phase and a mobile phase of methanol:ethyl acetate:tetrahydrofuran at selectivity point Ps = 111 with 1% formic acid as modifier. The title compounds were isolated from the purified extracts by TLC and their structures confirmed by 1H- and 13C-NMR spectroscopy.     

Optimized ultra-high-pressure-assisted extraction of procyanidins from lychee pericarp improves the antioxidant activity of extracts

To establish optimal ultra-high-pressure (UHP)-assisted extraction conditions for procyanidins from lychee pericarp, a response surface analysis method with four factors and three levels was adopted. The optimum conditions were as follows: 295 MPa pressure, 13 min pressure holding time, 16.0 mL/g liquid-to-solid ratio, and 70% ethanol concentration. Compared with conventional ethanol extraction and ultrasonic-assisted extraction methods, the yields of the total procyanidins, flavonoids, and phenolics extracted using the UHP process were significantly increased; consequently, the oxygen radical absorbance capacity and cellular antioxidant activity of UHP-assisted lychee pericarp extracts were substantially enhanced. LC-MS/MS and high-performance liquid chromatography quantification results for individual phenolic compounds revealed that the yield of procyanidin compounds, including epicatechin, procyanidin A2, and procyanidin B2, from lychee pericarp could be significantly improved by the UHP-assisted extraction process. This UHP-assisted extraction process is thus a practical method for the extraction of procyanidins from lychee pericarp.     

盾叶薯蓣总皂苷超声提取及动力学

考察了乙醇体积分数、溶剂用量、超声时间、超声功率和超声频率对盾叶薯蓣总皂苷提取率的影响,研究了以体积分数70%乙醇溶液或水作溶剂从盾叶薯蓣中超声提取总皂苷的动力学模型。结果表明,在扩散过程中超声提取薯蓣总皂苷的动力学模型满足非定常扩散方程,相关系数为r=0.95,最佳超声时间为40min。