Efficient new method for extraction and isolation of three flavonoids from Patrinia villosa Juss. by supercritical fluid extraction and high-speed counter-current chromatography

Supercritical fluid extraction (SFE) of orotinin, orotinin-5-methyl ether and licoagrochalcone B from Patrinia villosa was performed. The optimization of parameters including pressure, temperature, modifier and sample particle size on yield was carried out using an analytical-scale SFE system. The process was then scaled up by 100 times using a preparative SFE system under the optimized conditions of 25 MPa, 45 degrees C, a sample particle size 40-60 mesh and modified CO2 with 20% methanol. The yield of the preparative SFE was 2.82% (crude extract I) and the combined yield of orotinin, orotinin-5-methyl ether and licoagrochalcone B was 0.82 mg/g of dry sample mass. Then the crude extract I was re-dissolved in methanol and methanol soluble fraction (crude extract II, 0.17%) was obtained, which was successfully isolated and separated by a preparative high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (5:6:6:6, v/v/v/v) by increasing the flow-rate of the mobile phase stepwise from 1.0 to 2.0 ml/min after 3 h. The target compounds isolated and purified by HSCCC were analyzed by high performance liquid chromatography. The separation produced total of 38.2 mg of orotinin at 99.2% purity, 19.8 mg of orotinin-5-methyl ether at 98.5% purity and 21.5 mg of licoagrochalcone B at 97.6% purity from 400 mg of the crude extract in a one-step separation. The recoveries of orotinin, orotinin-5-methyl ether and licoagrochalcone B were 91.1, 91.6 and 90.3%, respectively, and the chemical structure identification was carried out by UV, IR, MS, 1H NMR and 13C NMR.     

Orthogonal test design for optimization of supercritical fluid extraction of daphnoretin, 7-methoxy-daphnoretin and 1,5-diphenyl-1-pentanone from Stellera chamaejasme L. and subsequent isolation by high-speed counter-current chromatography

Supercritical fluid extraction (SFE) of daphnoretin, 7-methoxy-daphnoretin and 1,5-diphenyl-1- pentanone from Stellera chamaejasme L. was performed. An orthogonal L9 (3)4 test design was applied to select the optimum extraction parameters including pressure, temperature, modifier and sample particle size on yield using an analytical-scale SFE system. The process was then scaled up by 100 times using a preparative SFE system under the optimized conditions of 25 MPa of pressure, 45 degrees C of temperature, 40-60 mesh of sample particle size and modified CO2 with 20% methanol. The yield of the crude extract from preparative SFE was 2.65%, which contained daphnoretin 25.2%, 7-methoxy-daphnoretin 22.8% and 1,5-diphenyl-1-pentanone 21.1%, respectively. Then the crude extract was successfully isolated and separated by preparative high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (10:13:13:10, v/v) by increasing the flow-rate of the mobile phase stepwise from 1.0 to 2.0 ml/min after 90 min. The target compounds isolated and purified by HSCCC were analyzed by high-performance liquid chromatography (HPLC). The separation produced total of 69.2mg of daphnoretin at 99.2% purity, 63.4 mg of 7-methoxy-daphnoretin at 98.7% purity and 58.3 mg of 1,5-diphenyl-1-pentanone at 98.1% purity from 300 mg of the crude extract in one-step separation. The recoveries of daphnoretin, 7-methoxy-daphnoretin and 1,5-diphenyl-1-pentanone were 90.8, 91.5 and 90.4%, respectively, in HSCCC isolation step and the chemical structure identification was carried out by MS, 1H NMR and 13C NMR.     

Supercritical fluid extraction of grape seed oil and subsequent separation of free fatty acids by high-speed counter-current chromatography

Supercritical fluid extraction (SFE) of grape seed oil was performed to study the effect of various parameters such as pressure, temperature and the particle size of the sample on the yield and composition of oil using an analytical-scale SFE system. Then the extraction was scaled up by 125 times using a preparative SFE system under the optimized conditions of high pressure (30-40 MPa) and low temperature (35-40 degrees C) with medium particle size (20-40 mesh). The maximum yield of the oil can reach 6.2% with pure supercritical CO2 and 4.0% more oil can be obtained by adding 10% of ethanol as modifier. The unsaturated fatty acids (UFSs) make up about 70% in the oil on the basis of free fatty acids. The grape seed oil was then subjected to separation and purification for free fatty acids after saponification by high-speed counter-current chromatography coupled with evaporative light scattering detection (ELSD). The separation of 1.0 g of oil can yield about 430 mg pure linoleic acid at 99% purity. The fatty acids were analyzed by HPLC-ELSD.     

Supercritical fluid extraction of aurentiamide acetate from Patrinia villosa Juss and subsequent isolation by silica gel and high-speed counter-current chromatography

Supercritical fluid extraction (SFE) of aurentiamide acetate from Patrinia villosa Juss was performed. The optimization of parameters was carried out using an analytical-scale supercritical fluid extraction (SFE) system. Then the extraction was scaled up by 100 times using a preparative SFE system under the optimized conditions of 55 degrees C, 35 MPa and modified CO2 with 10% methanol. Then, the crude extract I obtained by SFE was chromatographed on silica gel and the solvent system composed of petroleum ether-ethyl acetate (5:1, v/v) was used to produce the crude extract II, which was further isolated and purified by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (1:1.2:1.2:1, v/v/v/v). One hundred fifty-five milligrams of aurentiamide acetate was obtained from 400 mg crude extract II (contained 42% target) with a purity of 99.3% determined by HPLC and 92.3% recovery in one-step elution, and identification was performed by UV, MS, 1H NMR and 13C NMR. As far as we know, this is the first report of discovering aurentiamide acetate from the plant of Patrinia genius.     

Preparative isolation and purification of psoralen and isopsoralen from Psoralea corylifolia by high-speed counter-current chromatography

Psoralen and isopsoralen were separated from Psoralea corylifolia by high-speed counter-current chromatography (HSCCC). A two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (5:5:4.5:5.5, v/v) was used for HSCCC separation, and yielded, from 100 mg of crude extract, 39.6 mg of psoralen and 50.8 mg of isopsoralen each at over 99% purity as determined by high performance liquid chromatography (HPLC). The identification of psoralen and isopsoralen were performed with 1H NMR and 13C NMR.     

An efficient strategy for the extraction and purification of lignans from Schisandra chinensis by a combination of supercritical fluid extraction and high‐speed counter‐current chromatography

An efficient strategy for extracting and separating five lignans from Schisandra chinensis (Turcz.) Baill has been developed using supercritical fluid extraction (SFE) and high‐speed counter‐current chromatography (HSCCC) in the present study. First, the extraction was performed by a preparative SFE system under 15 MPa of pressure at 36°C for 4 h. Then, the SFE extract was successfully separated and purified by HSCCC with a two‐phase solvent system composed of n‐hexane/ethyl acetate/methanol/water (6:4:5:5, 6:4:6:4, 6:4:8:2, v/v) in a stepwise elution mode. The fractions were analyzed by HPLC, and the chemical structures of the products were identified by ESI‐MS and ¹H NMR spectroscopy. As a result, a total of 12.5 mg of schisandrin at 98.0% purity, 7.1 mg of gomisin A at 98.1% purity, 1.8 mg of schisantherin B at 93.3% purity, 4.4 mg of deoxyschisandrin at 92.9% purity, and 6.8 mg of γ‐schisandrin at 89.1% purity were obtained from 300 mg crude extract in a one‐step purification.     

Supercritical fluid extraction of catechins from Cratoxylum prunifolium dyer and subsequent purification by high-speed counter-current chromatography

Supercritical fluid extraction of tea catechins including epigallocatechin-3-O-gallate (EGCG) and epicatechin-3-O-gallate (ECG) from Cratoxylum prunifolium Dyer was performed. The optimization of parameters was carried out using an analytical-scale supercritical fluid extraction (SFE) system designed in our laboratory. Then the extraction was scaled up by 100 times using a preparative SFE system under a set of optimized conditions of 40 degrees C, 25 MPa and modified CO2 with 80% ethanol aqueous solution. The combined yield of EGCG and ECG reached about 1 mg per 1 g of tea leaves where the solubility was near 1.4 x 10(-4) mass fraction of CO2 fluid. EGCG and ECG of high purity (>98%) were obtained from the crude preparative extract by high-speed counter-current chromatography.     

超临界二氧化碳萃取秋水仙碱（英文）

 利用超临界二氧化碳对秋水仙块根（经粉碎）中的秋水仙碱进行了萃取,采用高效液相色谱法对萃取出的秋水仙碱的质量分数进行了测定。实验选择40℃20～40MPa作为超临界苹取的操作条件,采用体积分数为95％的乙醇在索氏提取器中对样品进行了对比苹取实验。结果表明:不加浸泡剂进行浸泡处理的样品中的秋水仙碱很难被超临界二氧化碳萃取,在40℃,35MPa条件下,消耗1．28mol的二氧化碳只得到3％的萃取率。加入极少量的有机溶剂浸泡处理样品15min后再进行超临界萃取,可以极大程度地提高秋水仙碱的萃取率。     

Supercritical fluid extraction of quinolizidine alkaloids from Sophora flavescens Ait. and purification by high-speed counter-current chromatography

Supercritical fluid extraction (SFE) was used to extract quinolizidine alkaloids from Sophora flavescens Ait. (Kushen). An orthogonal test L(9)(3)(4) including pressure, temperature, flow rate of CO(2) and the amount of modifier was performed to get the optimal conditions. The process was then scaled up by 30 times with a preparative SFE system under 25 MPa, 50 degrees C and a flow rate of CO(2) (2l/min) and the amount of modifier (0.04 ml/min). The crude extracts were separated and purified by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of chloroform-methanol-2.3 x 10(-2)M NaH(2)PO(4) (27.5:20:12.5, v/v), and the collected fractions were analyzed by high-performance liquid chromatography (HPLC). Three kinds of quinolizidine alkaloids were obtained, yielding 10.02 mg of matrine, 22.07 mg of oxysophocarpine and 79.93 mg of oxymatrine with purities of 95.6, 95.8, 99.6% in one-step separation, respectively.     

Supercritical CO2 extraction of emodin and physcion from Polygonum cuspidatum and subsequent isolation by semipreparative chromatography

Emodin and physcion are abundant anthraquinone compounds found in the traditional Chinese medicinal herb Polygonum cuspidatum Sied. et Zucc. In this paper, emodin and physcion were successfully extracted with supercritical CO2 plus ethanol modifier after the extraction conditions were optimized with uniform design-sequential optimization. Results showed that the ethanol modifier concentration was the main factor for the effective extraction of the emodin. The optimal extraction condition was obtained: 20 MPa, 30 degrees C, and 95% ethanol, at which the yields of emodin and physcion were 0.616 and 0.178 g/100 g, respectively. The yield obtained by supercritical fluid extraction (SFE) was a little lower than that obtained by sonication extraction (SE). The crude extract obtained by SFE was further isolated and purified by semipreparative chromatography with the mobile phase composed of methanol-water (90:1, v/v). Emodin and physcion were obtained with purity 98.6 and 99.1%, respectively, when determined by HPLC, and identification was performed by retention time and UV spectra of the standards. The result suggested that SFE is an alternative and promising method for extraction of the two compounds from P. cuspidatum owing to its environment-friendly properties and fewer coextracts.     

超临界流体萃取法对有机锡化合物的选择性萃取

 研究了用超临界流体萃取法直接从脂肪基质的固体样品（大豆粉）中选择性地萃取有机锡化合物的方法。模拟试样萃取结果表明：用较低压力和较高温度的超临界态ＣＯ２作流动相时，有机锡达到最大萃取率，而脂肪类物质仅被少量萃取，从而消除了脂肪类物质对超临界流体色谱法测定有机锡的干扰。     

Modeling of supercritical fluid extraction of flavonoids from Calycopteris floribunda leaves

The aim of this study was to obtain flavonoids extracts from Calycopteris floribunda leaves using supercritical fluid extraction (SFE) with CO₂ and a co-solvent. Pachypodol, a potential anticancer drug lead compound separated from the extracts, was examined. Classical organic solvent extraction (CE) with ethanol was performed to evaluate the high pressure method. HPLC analysis was introduced to interpret the differences between SFE and CE extracts in terms of antioxidant activity and the concentration of pachypodol. SFE kinetics and mathematical modeling of the overall extraction curves (OEC) were investigated. Evaluation of the models against experimental data showed that the Sovová model performs the best. The supercritical fluid extraction process was optimized using a central composite design (CCD), where temperature and pressure were adjusted. The optimal conditions of SFE were: pressure of 30 MPa and temperature of 35°C.     

Comparison of solid-phase microextraction, supercritical fluid extraction, steam distillation, and solvent extraction techniques for analysis of volatile consituents in Fructus Amomi

Four sampling techniques, solid-phase microextraction (SPME), supercritical fluid extraction (SFE), steam distillation (SD), and solvent extraction (SE), were compared for the analysis of volatile constituents from a traditional Chinese medicine (TCM) of the dried ripe fruit of Fructus Amomi (Sha Ren). A total of 38 compounds were identified by gas chromatography/mass spectrometry. Different SFE and SPME parameters (modifier content, extraction pressure, and temperature for SFE and fibers, extraction temperature, and time for SPME) were studied. The results by SFE and SPME were compared with those obtained by conventional SD and SE methods. The results showed that SFE and SPME are better sample preparation techniques than SD and SE. Due to SFE's requirement for expensive specialized instrumentation, the simplicity, low cost, and speed of SPME make it a more appropriate technique for extraction of volatile constituents in TCMs.     

Supercritical fluid extraction of the volatile oil from Santolina chamaecyparissus

Supercritical fluid extraction (SFE) of the volatile oil from Santolina chamaecyparissus L. flower heads was performed under different conditions of pressure, temperature, mean particle size and CO₂ flow rate. This oil was compared with the essential oil isolated by hydrodistillation (HD). The SFE volatile and essential oils were analysed by GC and GC‐MS. The range of the main volatile components obtained with HD and SFE were, respectively: 1,8‐cineole (25–30% and 7–48%), camphor (7–9% and 8–14%), borneol (7–8% and 2–11%), terpinen‐4‐ol (6–7% and 1–4%), terpinolene (1–4% and 1–7%) and isobornyl acetate (1–2% and 1–11%). The chemical composition of the extracts was greatly influenced by the conditions of pressure and temperature used. In fact, it was possible to enrich the sesquiterpene fraction by increasing the pressure from 8 to 9 MPa, while changing the temperature from 40 to 50°C at 90 bar enriched of the volatiles in n‐alkanes.     

Supercritical fluid extraction of isoflavones from biological samples with ultra-fast high-performance liquid chromatography/mass spectrometry

An efficient method of modifier addition for supercritical fluid extraction (SFE) of polar isoflavones was developed and yielded extraordinarily high recoveries. To find the optimal extraction conditions, a temperature and pressure optimization and modifier impact study was performed in naturally contaminated and spiked samples. Ultra-fast high-performance liquid chromatography/mass spectrometry (HPLC/MS) was used for the determination of isoflavones on an Atlantis dC18 high-speed reversed phase chromatographic column (20 x 2.1 mm, 3 microm particle size). A newly elaborated supercritical fluid extraction (SFE) procedure allowed more accurate (< 5%) and precise (< 4-7%) determination of isoflavones in biological materials. The HPLC/MS method significantly reduced analysis time with simultaneous improvement of sensitivity and detection limits. The on-column limits of detection LOD (S/N = 3) for isoflavone glycosides (daidzin, genistin, glycitin, ononin, and sissotrin) were 1.3-3.6 fmol and 0.2-1.0 fmol for aglycones (daidzein, glycitein, genistein, formononetin, and biochanin A), respectively.     

超临界流体萃取法测定补骨脂中的主要成分

 采用超临界流体萃取(SFE)技术和CGC技术测定了 中药补骨脂中的补骨脂素和异补骨脂素。对超临界流体萃取过程中影响萃取效率的主要因素 采用正交设计法和方差分析法进行了考察,确定了主效应和适宜的操作条件。与传统的萃取 法比较,SFE 具有经济、快速、简便、选择性好、环境污染小等优点。     

Determination of sunscreen agents in cosmetic products by supercritical fluid extraction and high-performance liquid chromatography

A rapid supercritical fluid extraction (SFE) procedure for the isolation of five of the most common sunscreen agents (2-ethylhexyl-p-dimethylaminobenzoate, 2-hydroxy-4-methoxybenzophenone, 2-ethylhexyl-p-methoxycinnamate, 4-methylbenzylidene camphor and 4-tert.-butyl-4′-methoxydibenzoylmethane) from cosmetic products is described. Investigation of the factors affecting the extraction efficiency in SFE indicated that sunscreen recoveries were affected mainly by the supercritical CO₂ pressure and by the trapping method. The sunscreens were analyzed by reversed-phase high-performance liquid chromatography after a 10-min extraction of the cosmetic product with CO₂ at 250 bar and 40°C, using sequential glass surface and C₁₈ sorbent as collection system. A quantitative comparison of SFE with a liquid extraction procedure was performed on commercial cosmetics. The SFE method yielded recoveries higher than 94.8% compared with conventional liquid extraction and exhibited a precision better than 5.3% relative standard deviation. Moreover, SFE minimized sample handling, reduced the consumption of harmful solvents and afforded a more effective purification of the cosmetic matrices.     

Determination of fenpyroximate in apples by supercritical fluid extraction and packed capillary liquid chromatography with UV detection

A method using off-line supercritical fluid extraction (SFE) and micro liquid chromatography (μLC) with UV detection at 260 nm, was developed for selective determination of fenpyroximate in apple samples. The packed capillary liquid chromatography method utilises 20 μl injection volumes with on-column focusing. A 350×0.32 mm capillary column packed with Kromasil 100-C₁₈ of 5 μm particle size was used with a mobile phase of acetonitrile–10 mM ammonium acetate (85:15, v/v) at a flow of 5 μl/min. A two-step SFE procedure was used to extract fenpyroximate selectively in 2 g apple samples, with Hydromatrix (HMX) added as a water absorbent at a 1:1 (w:w) ratio. Fenpyroximate was extracted at 200 bar and 90°C for 15 min using carbon dioxide at a flow of 2 ml/min, and solvent trapping collection in 10 ml acetonitrile. The volume of the acetonitrile extract was reduced by evaporation and water was added to a final composition of acetonitrile–water (40:60, v/v). The resulting 2.0 ml solution was filtered using a 0.45 μm poly(vinylidene difluoride) syringe filter before μLC analysis. Validation of the method was accomplished with apple samples spiked with fenpyroximate, covering the range of 0.1 to 1.0 μg/kg. The within-day and between-day repeatabilities were in the range 4–18% relative standard deviation. Accuracy, measured as recovery, was found to be approximately 60%. Apple samples from a field treated with fenpyroximate were analysed. None of the samples contained fenpyroximate above the quantification level.     

Optimization of supercritical fluid extraction of saikosaponins from Bupleurum falcatum with orthogonal array design

Supercritical fluid extraction (SFE) was used to extract saikosaponins a, c and d from the root of Bupleurum falcatum. An orthogonal array design L₉(3)⁴ was employed as a chemometric method for the optimization of the SFE conditions. The effects of four factors including pressure (30–40 MPa), temperature (40–50°C), ethanol concentration (60–100%) and time (2.5–3.5 h) on the yields of saikosaponins were investigated by a preparative SFE system in the SFE mode. Under the optimized conditions, namely 35 MPa of pressure, 45°C of temperature, 80% of ethanol concentration and 3.0 h of time, the yields of saikosaponin c, saikosaponin a, saikosaponin d, total saikosaponins and SFE extract were 0.16, 0.12, 0.96, 1.24 and 16.48 mg/g, respectively. Determinations of the saikosaponins were performed by HPLC.     

Composition and antioxidant activity of Thymus vulgaris volatiles: Comparison between supercritical fluid extraction and hydrodistillation

Supercritical fluid extraction (SFE) of the volatile oil from Thymus vulgaris L. aerial flowering parts was performed under different conditions of pressure, temperature, mean particle size and CO₂ flow rate and the correspondent yield and composition were compared with those of the essential oil isolated by hydrodistillation (HD). Both the oils were analyzed by GC and GC‐MS and 52 components were identified. The main volatile components obtained were p‐cymene (10.0–42.6% for SFE and 28.9–34.8% for HD), γ‐terpinene (0.8–6.9% for SFE and 5.1–7.0% for HD), linalool (2.3–5.3% for SFE and 2.8–3.1% for HD), thymol (19.5–40.8% for SFE and 35.4–41.6% for HD), and carvacrol (1.4–3.1% for SFE and 2.6–3.1% for HD). The main difference was found to be the relative percentage of thymoquinone (not found in the essential oil) and carvacryl methyl ether (1.0–1.2% for HD versus t?0.4 for SFE) which can explain the higher antioxidant activity, assessed by Rancimat test, of the SFE volatiles when compared with HD. Thymoquinone is considered a strong antioxidant compound.