Efficient purification and morphology characterization of paclitaxel from cell cultures of Taxus chinensis

Paclitaxel was purified from cell cultures of Taxus chinensis by a combination of extraction, low‐pressure chromatography, precipitation and HPLC. A crude extract (purity 6.9%) was obtained by methanol extraction of plant cell cultures, followed by liquid–liquid extraction using dichloromethane. The extract was purified to greater than 32% with a 97% step yield by low‐pressure chromatography. After acetone/pentane precipitation, the resulting purity and step yield were 75.8% and 97.4%, respectively. High performance liquid chromatography steps, which were composed of an HPLC step with column‐packed ODS and an HPLC step with column‐packed silica, were applied to give over 99.5% purity with high yield. Amorphous paclitaxel with a fine particle size, which has a solubility advantage compared with the stable crystalline form, was obtained by dissolving in dichloromethane, followed by spray‐drying. Copyright © 2004 Society of Chemical Industry     

Purification of homoharringtonine and removal of residual solvents by spray drying

Homoharringtonine was purified from Cephalotaxus koreana by a combination of extraction, synthetic adsorbent treatment, low-pressure chromatography, and high performance liquid chromatography (HPLC). A crude extract was obtained by methanol extraction of biomass, followed by liquid-liquid extraction using chloroform. The waxy compounds were efficiently removed by adsorbent (active clay P-1) treatment. The extract was purified to greater than 52% with an 86.4% step yield by silica gel low-pressure chromatography. High performance liquid chromatography steps, which were composed of an HPLC step with silica column and an HPLC step with ODS column, were applied to give 98% purity with high yield. Amorphous homoharringtonine, with a fine particle size, was simply made by dissolving homoharringtonine in methylene chloride/methanol (98/2, v/v), followed by spray drying. Residual solvents, methylene chloride and methanol, could be reduced to 250 ppm and 1,160 ppm by spray drying and successive drying in a vacuum oven.     

Extraction of pollen lipids by SFE‐CO2 and determination of free fatty acids by HPLC

Rape bee pollen lipids obtained by petrol ether extraction (PEE) or supercritical fluid (carbon dioxide) extraction (SFE) were compared with regard to their free fatty acid (FFA) components. Optimal SFE conditions were selected by carrying out the Taguchi method with an OA9 (3³) matrix design, and are as follows: extraction pressure at 35 MPa, temperature at 45 °C, and dynamic extraction time at 90 min. The lipid yield based on PEE was 7.42 wt‐% and the extracts of the desired analytes based on SFE varied in the range of 3.23–5.58 wt‐% under different conditions. With the optimized procedure, the lipid yield was 6.09 wt‐%. The FFA in the lipids were separated with a pre‐column derivation method and 1‐[2‐(p‐toluenesulfonate) ethyl]‐2‐phenylimidazole [4,5‐f]9,10‐phenanthrene as labeling regent, followed by high‐pressure liquid chromatography (HPLC) with fluorescence detection. HPLC analysis shows that the lipids contain abundant unsaturated fatty acids (UFA) in high to low concentrations as follows: linolenic acid (18:3), oleic acid (18:1), linoleic acid (18:2), nervonic acid (24:1), and lignoceric acid (20:4). The UFA contents in the SFE extracts were higher than those after PEE. The results indicated that SFE under suitable conditions is more selective than conventional PEE with regard to lipid extraction and preservation of their quality.     

Comparison of Supercritical Fluid Extraction and Liquid Solvent Extraction on Antitumor Diterpenoid from Pteris semipinnata L.

Extraction techniques using Supercritical Fluid Extraction (SFE) and Liquid Solvent Extraction (LSE) were evaluated for the extraction of Ent-11α-hydroxy-15-oxo-kaur-16-en-19-oic-acid (5 F), the antitumor diterpenoid from Pteris semipinnata L. The extracts were analyzed by high performance liquid chromatography (HPLC). SFE experiments showed that many factors had a great impact on the yield and purity of the diterpenoid, such as extraction temperature, pressure, fluid flow rate, extraction time, and modifier. For the SFE process, the optimum operation conditions were as follows: extraction temperature of 328.15 K, extraction pressure of 30 MPa, supercritical CO₂ flow rate of 160 kg/h, extraction time of 4 h, and 10% ethanol as the modifier. Under such a condition, the diterpenoid was almost completely extracted from the material and the yield was approximately 0.504 g/kg dry herb by HPLC analysis. The yield was approximately 3 fold higher than that by liquid solvent extraction. The purity of 5F was 5.148 g/kg dried extract with SFE, it was about 9 fold higher than that by LSE. Mass spectrum data indicated there were two correlative compounds, 5F and its derivative with glycose, in both the extracts, and the ratio of the signal strength of 5F and its derivative was about 3:1 in the SFE extract while that ratio was 1:3 in the LSE extract. The results demonstrated that the supercritical fluid extraction was selective, highly efficient, and with less consumption of organic solvents.     

Preparative Purification of the Major Flavonoid Glabridin from Licorice Roots by Solid Phase Extraction and Preparative High Performance Liquid Chromatography

This study investigated a novel, simple, and economical method for the preparation and purification of glabridin from licorice roots. Glabridin was initially obtained from ethyl acetate extraction of licorice, followed by using solid phase extraction (SPE) and preparative high performance liquid chromatography (HPLC). The content of glabridin increased from 0.23% to 35.2% after SPE, and then a 16 mg product at a high purity of over 95% was obtained from 10 g licorice roots after purification by preparative HPLC. The purity was assessed by analytical HPLC, and the purified compound was characterized by LC-MS/MS and ¹H NMR.     

An Efficient Strategy Based on Macroporous Resins and Semi-Preparative High Performance Liquid Chromatography for Rapid Separation of Five Flavonoids Components From Flaveria Bidentis(L.) Kuntze

In this study, a simple, rapid, and efficient purification method of five major flavonoids from crude Flaveria bidentis extracts was established. Five flavonoids components were initially obtained by ethanol-water extraction of Flaveria bidentis (L.) Kuntze, followed by using D4020 resin-based column chromatography and semi-preparative high performance liquid chromatography. 9.3 mg hyperoside, 6.5 mg patuletin-3-O-glucoside, 1.7 mg isorhamnetin 3-sulphate, 8.4 mg astragalin, and 4.9 mg 6-methoxykaempferol-3-O-galactoside at high purity of over 94% were obtained from 580 mg Flaveria bidentis extracts. This method is more efficient than HSCCC comparing with the results of these two methods regarding analysts’ purity, recovery, and running time.     

甘油磷酰胆碱的分析、制备及纯化研究进展

甘油磷酰胆碱（GPC）作为重要神经递质乙酰胆碱的生物合成前体,能够促进乙酰胆碱在脑部的合成,增强人体的记忆力和认知能力,预防老年痴呆类疾病,已引起医药界的高度关注。为进一步促进GPC相关产品的研究与应用,本文重点介绍了近几年GPC的分析、制备及纯化技术的研究进展,并比较了不同方法的优缺点。指出高效液相色谱法（HPLC）是分析GPC的主流方法,但没有一种检测器能检测到所有关键的杂质,需要多种检测器联用才可判断GPC的质量是否达到要求。全化学合成和水解卵磷脂（PC）是制备GPC的主要方法。文中指出全化学合成法具有收率高、产品纯度高和制备工艺完善的优点,但起始原料昂贵,全化学合成的GPC中会残留基因毒性杂质,严重影响产品品质。水解PC法能够制备出食品级GPC,所得产品无毒无害,但水解法收率低,产品纯度低,纯化难度大,难以规模化生产。结晶法和柱色谱法是纯化GPC粗品的主要方法。结晶法得到的产品纯度高,但收率低,生产成本高。柱色谱法能除去大量杂质,除杂效果好,但生产过程中会产生大量“三废”,纯化周期长。文中分析表明,HPLC和多种检测器联用是定量分析GPC及其相关杂质的最有效方法,水解PC以制备食品级GPC是如今的研究热点,开发出高效的纯化技术是工业化生产GPC的关键和难点。     

Optimization of cordycepin extraction from cultured Cordyceps militaris by HPLC‐DAD coupled with uniform design

BACKGROUND: Uniform design was used to optimize the ultrasonic‐assisted extraction of cordycepin from cultured Cordyceps militaris. The peak area of cordycepin identified using high performance liquid chromatography (HPLC) at a detection wavelength of 260 nm was considered the detection index. Three factors, ethanol concentration, extraction time and volume ratio of solvent to sample were studied. Optimal quadric polynomial step by step regression was applied to process the experimental results. RESULTS: Results show that the main factors affecting cordycepin extraction yield were the volume ratio of solvent to sample, extraction time and ethanol concentration, in that order. Cordycepin extraction yield reached a peak with ethanol concentration 20.21%, extraction time 101.88 min, and volume ratio of solvent to sample 33.13 mL g^?1. An extraction model was developed based on the findings. CONCLUSION: A direct, reliable and accurate assay has been developed for the quantification of cordycepin in cultured Cordyceps militaris by a HPLC‐DAD method. The validation procedure confirmed that this method is appropriate for the quality control of cordycepin. Results show that the uniform experimental design approach is useful for finding polynomial functions describing the relationships between variables and responses and to find the best experimental conditions for the extraction of cordycepin in the experimental domain considered. Copyright © 2007 Society of Chemical Industry     

Large‐scale isolation and purification of C‐phycocyanin from the cyanobacteria Anabaena marina using expanded bed adsorption chromatography

BACKGROUND: Phycobiliproteins are water soluble proteins useful as fluorescent markers of cells and macromolecules, and as natural colorants, and are anticarcinogenic. Although phycobiliproteins have many applications, their use is limited by the high cost of the purified macromolecules, mainly related with the cost of extraction and purification. In this study a fast and scalable method for preparative extraction and purification of C‐phycocyanin (C‐PC) from Anabaena marina is developed. RESULTS: The method developed consists in the extraction of phycobiliproteins using repeated single contact strategy, separation being performed by expanded bed adsorption (EBA) chromatography using Streamline‐DEAE. Optimal conditions for EBA were obtained at small scale, using a 15 mm internal diameter column, these being a sample load of 0.9 mg C‐PC mL^?1 adsorbent, an expanded bed volume twice the settled bed volume and a sample viscosity of 1.109 mP. The process was then scaled up 36 times, the success of the scale‐up process being verified. Finally, to obtain pure C‐PC conventional ion‐exchange chromatography was utilized. CONCLUSION: Small diameter columns was shown to be useful to simulate the behavior of larger diameter columns for use in scaled up systems. Expanded bed adsorption was demonstrated to be a scalable technology allowing large quantities of C‐PC to be obtained, maintaining high protein recovery while reducing both processing cost and time. The proposed methodology allows recovery of more than 62% of the C‐PC contained in the biomass in the form of pure C‐PC concentrates. Copyright © 2010 Society of Chemical Industry     

Low‐Temperature Chemical Synthesis of High‐Purity Diacylglycerols (DAG) from Monoacylglycerols (MAG)

A chemical method was developed for low‐temperature synthesis of DAG from MAG followed by an easy purification procedure in order to obtain high‐purity DAG. Solvent‐assisted and solvent‐free reaction conditions were used, combined with different catalysts (sodium methoxide, p‐toluenesulfonic acid, methanesulfonic acid, and sulfuric acid). All reactions were performed at 35 and 70 °C. By increasing both acidity and polarity of the catalyst the equilibrium shifts towards the formation of DAG. When using sulfuric acid in solvent‐assisted condition at 70 °C, 88 % conversion was obtained after 20 min of reaction (77 % w/w DAG in the reaction mixture after evaporation of the solvent). After purifying by means of column chromatography, 96 % pure DAG were obtained. The overall yield of DAG was 81 %.     

Flowsheet simulation of aqueous two‐phase extraction systems for protein purification

BACKGROUND: Aqueous two‐phase extraction (ATPE) has many advantages as an efficient, inexpensive large‐scale liquid–liquid extraction technique for protein separation. However, the realization of ATPE as a protein separation technology at industrial scales is rather limited due to the large, multidimensional design space and the paucity of design approaches to predict phase and product behavior in an integrated fashion with overall system performance. This paper describes a framework designed to calculate suitable flowsheets for the extraction of a target protein from a complex protein feed using ATPE. The framework incorporated a routine to set up flowsheets according to target protein partitioning behavior in specific ATPE systems and a calculation of the amounts of phase‐forming components needed to extract the target protein. The thermodynamics of phase formation and partitioning were modeled using Flory‐Huggins theory and calculated using a Gibbs energy difference minimization approach. RESULTS: As a case study, suitable flowsheets to recover phosphofructokinase from a simple model feedstock using poly(ethylene glycol)‐dextran (PEG6000‐DxT500) and poly(ethylene glycol)‐salt (PEG6000‐Na₃PO₄) two‐phase systems were designed and the existence of feasible solutions was demonstrated. The flowsheets were compared in terms of product yield, product purity, phase settling rate and scaled process cost. The effect of the mass flowrates of phase‐forming components on product yield and purity was also determined. CONCLUSION: This framework is proposed as a basis for flowsheet optimization for protein purification using ATPE systems. Copyright © 2010 Society of Chemical Industry     

Purification and characterization of recombinant human erythropoietin from milk of transgenic pigs

BACKGROUND: Human erythropoietin (hEPO), a hydrophobic acidic glycoprotein responsible for the regulation of red blood cell production in mammals, is used for the treatment of anemia. In general, the purification of transgenic animal‐derived therapeutic proteins is not easy due to their low titer concentrations and abundant contaminant proteins. For the first time, here the purification and characterization of rhEPO from the milk of transgenic pigs are described. RESULTS: The rhEPO was purified by heparin chromatography, reverse‐phase chromatography, and gel filtration chromatography, resulting in a 16.5% yield and > 98% purity. The rhEPO purified from the milk of transgenic pigs contained less acidic isoforms and was underglycosylated in contrast to CHO‐derived rhEPO. Cell proliferation of the F‐36/EPO‐dependent cell line was proportional to the dose of transgenic pig‐derived rhEPO. CONCLUSION: Transgenic pig‐derived rhEPO with high purity was achieved after three‐step chromatography following two‐step precipitation. The transgenic pig‐derived rhEPO was demonstrated to have comparable potency with CHO‐derived rhEPO. Transgenic pig‐derived rhEPO may not be therapeutically feasible because of different glycosylation, and thus further studies are required to elucidate the effect of this aberrant glycosylation on the biological activity and stability in vivo. Copyright © 2008 Society of Chemical Industry     

Industrial high-performance liquid chromatography purification of docosahexaenoic acid ethyl ester and docosapentaenoic acid ethyl ester from single-cell oil

The use of polyunsaturated fatty acids (PUFA) as medicine or in functional diets requires high purity. An industrial purification method for PUFA from Schizochytrium sp. SR21 oil was investigated. This oil contains fewer unwanted components than fish oils. Docosahexaenoic acid and docosapentaenoic acid ethyl esters (DHA-E and DPA-E) were prepared by treatment of this oil with ethanol and 1 N potassium hydroxide in hexane. DHA-E and DPA-E were purified by an industrial high-performance liquid chromatography (HPLC) plant. The separation plant consists of two columns (400 mm i.d., 1,000 mml) with temperature-controlled water jackets and double-plunger (four heads) injection and eluent pumps. This plant was computer-controlled and equipped with an explosion-prevention system. The packed material was octadecylsilica (reverse-phase ODS), and the eluent was methyl alcohol/water (98:2). DHA-E and DPA-E from single-cell oil were highly purified by this industrial HPLC method in a one-step process. The DHA-E and DPA-E obtained were better than 99% purity.     

Precipitation of pharmaceuticals using a supercritical anti‐solvent (SAS) dechnique: A preliminary study

The aims of this research were to investigate the applicability of the supercritical anti‐solvent (SAS) process on the precipitation of pharmaceuticals (andrographolide and acetaminophen). In particular, the goal of this research was to study the influence of pressure at 10 and 24 MPa on particle characteristics (morphology, crystalline structure, polymorphic form, size, size distribution, and precipitation yield), and to compare the precipitation efficiency of SAS process and evaporation process. Scanning electron microscope (SEM), X‐ray diffraction (XRD), and high performance liquid chromatography (HPLC) showed a significant change in particle size, size distribution, morphology, and precipitation yield, respectively. From an analysis of the results it was found that the crystal size of andrographolide and acetaminophen decreased with increasing pressure. The morphology of andrographolide particles changed from slice‐like to column‐like when the pressure was increased. On the other hand, the acetaminophen particles obtained were found to be monoclinic form (I) under both operating pressures. The SAS process produced small uniform shaped crystals, with a narrow size distribution, high precipitation yield and selective precipitation were also observed.     

Improved recovery of B‐phycoerythrin produced by the red microalga Porphyridium cruentum

A simplified process for the primary recovery and purification of B‐phycoerythrin (BPE) from Porphyridium cruentum exploiting aqueous two‐phase systems (ATPS) and isoelectric precipitation was developed in order to reduce the number of unit operations and benefit from increased purity and yield of the protein product. Evaluation of the partitioning behaviour of BPE in polyethylene glycol (PEG)/sulphate, PEG/dextran and PEG/phosphate ATPS was carried out to determine under what conditions the BPE and contaminants concentrated into opposite phases. An additional stage of isoelectric precipitation at pH 4.0 after cell disruption resulted in an increase in purity of the target protein from the BPE crude extract and enhanced the performance of the subsequent ATPS. PEG1000/phosphate ATPS proved to be suitable after isoelectric precipitation for the recovery of highly purified (defined as absorbance ratio A_{545 nm}/A_{280 nm} > 4.0) BPE with a potential commercial value as high as US$ 50/mg. An ATPS extraction stage comprising 29.5% (w/w) PEG1000, 9.0% (w/w) phosphate, a volume ratio (V_r) equal to 1.0, a system pH of 7.0 and loaded with 40% (w/w) of the BPE extract generated by precipitation allowed BPE recovery with a purity of 4.1±0.2 and an overall product yield of 72% (w/w). The purity of BPE from the crude extract increased 5.9‐fold after isoelectric precipitation and ATPS. The results reported herein demonstrate the benefits of the practical application of isoelectric precipitation together with ATPS for the recovery and purification of BPE produced by P. cruentum as a first step in the development of a commercial purification process. Copyright © 2006 Society of Chemical Industry     

A rapid procedure for screening and isolating oxygenated free fatty acid metabolites from plants and natural waters

A rapid method of isolating a relatively pure fraction of oxygenated fatty acids from plants and natural waters is described. These metabolites were isolated from aqueous extracts using octadecylsilyl silica in a reverse-phase batch extraction method. The extraction method, together with reverse-phase analytical high pressure liquid chromatography (HPLC), was used to establish a routine screening method for the presence of these compounds in a variety of natural sources. A reverse-phase preparative HPLC purification method is also described.     

羟基红花黄色素A的提纯新工艺研究

为制备高纯度、高附加值的羟基红花黄色素A(hydroxy - safflor yellow A,以下简称HSYA)产品奠定基础,以HSYA的纯度作为考察指标,采用高效液相色谱(HPLC)仪测定不同工艺路线得到的HSYA产品的纯度,采用紫外分光光度计在线监测每一步产品的浓度.选取HPD200A型号的大孔吸附树脂、PES1...     

Squalene from Fish Livers Extracted by Ultrasound-Assisted Direct In Situ Saponification: Purification and Molecular Characteristics

An ultrasound-assisted direct in situ saponification (U-DS) process was developed to extract unsaponifiable matter (rich in squalene) from the livers of four fish species, sea bass, skipjack tuna, gray bamboo shark, and spot-tail shark. The conventional solvent extraction method was used for comparison. Box–Behnken experimental design (BBD) was adopted to optimize the independent variables including the biomass/methanol ratio (1:3 to 1:9 w/v), 50% KOH volume (1–12 mL), and sonication time (0–30 min) over the unsaponifiable yield. With the conventional process, the yield of squalene was 0.10 ± 0.02 to 5.52 ± 0.06 g (100 g)⁻¹, whereas the U-DS process rendered the yield of 0.13 ± 0.03 to 6.86 ± 0.05 g (100 g)⁻¹, depending on the fish species. After extraction, squalene was further concentrated (purity ≥60%) from all fish species via fractional crystallization (yield of squalene concentrate ranged from 48.35% to 74.49%) and purified using a silica gel column with a maximum recovery up to 98%. For all the fractions, components were examined using thin layer chromatography (TLC). Squalene was qualitatively and quantitatively analyzed using reversed-phase high-performance liquid chromatography (RP-HPLC). Entire extraction processes yielded squalene with a purity of ≥94%. Fourier transform infrared (FTIR) analysis confirmed the native structure of squalene with six nonconjugated bonds, suggesting no degradation of squalene taken place during the U-DS process. Thus, the U-DS process along with fractionation and purification could be used for recovering the squalene from fish livers.     

高纯胸腺肽α1的制备

为了获得高纯产品,在制备胸腺肽α1(Tα1)工艺中分别对合成、纯化两步骤进行控制。在固相多肽合成过程中,以TBTU/HOBt/DIEA体系作为缩合试剂,在易形成α螺旋和β转角的肽序位置,通过采用HOBt/DIC补投的方法促使偶联反应完全。在分离纯化阶段,结合反相色谱和离子色谱的优势,以反相离子对液相色谱法为主要分离手段,通过多步分离实现纯化。获得的粗肽纯度为67.2%,胸腺肽α1产品纯度达99.5%以上,总收率为17.2%。此法可作为制备高纯多肽药物的有效手段。     

Hyphenated Flash Chromatographic Separation and Isolation of Coumarins and Polymethoxyflavones from Byproduct of Citrus Juice Processing Industry

The present study describes an efficient hyphenated flash chromatographic (FC) for the purification of coumarins and polymethoxy flavones (PMF's) from cold pressed grapefruit peel oil (GFO), a byproduct from citrus juice processing plant. GFO was subjected to silica column chromatography using sequential isocratic elution by hexane and chloroform to separate the waxes and phytochemicals enriched fractions, respectively. The chloroform extract was used for hyphenated FC separation using a gradient mobile phase of hexane and acetone. The peak separations were monitored at dual wavelengths of λ₂₁₀ nm and λ₃₄₀ nm. The individual fractions were analyzed by HPLC, pooled and concentrated resulting in isolation of two coumarins (auraptene and bergapten) and three PMF's (tangeretin, heptamethoxyflavone, and nobiletin). The purity and identity of the isolated compounds was confirmed by spectral analysis using high performance liquid chromatography, matrix-assisted laser desorption/Ionization-time of flight-mass spectrometry, and nuclear magnetic resonance. The hyphenated FC separation method was further validated by test of repeatability resulting in low RSD for the yield of the phytochemicals. Utilization of the developed separation method for isolation of value added products from grapefruit byproducts could be beneficial to the citrus processing industry. Supplemental materials are available for this article. Go to the publisher's online edition of Separation Science & Technology to view the supplemental file.