发酵液中多拉菌素的提取和萃取条件研究

采用单因素实验,分别研究提取试剂、发酵液放置时间、pH值和温度对发酵液中多拉菌素提取效果的影响;然后以乙酸乙酯为萃取试剂,研究萃取次数及萃取体积对多拉菌素萃取效果的影响。结果显示,甲醇为最佳提取试剂;发酵液在pH为3～11、温度为20～80℃的条件下放置144 h,多拉菌素均能稳定存在,提取得到的多拉菌素的质量浓度没有显著变化;浓缩提取液液经2倍体积乙酸乙酯萃取2次即可。该条件下多拉菌素的质量浓度和萃取率分别为151.78μg/mL和98.00%。     

Acetic acid removal from corn stover hydrolysate using ethyl acetate and the impact on Saccharomyces cerevisiae bioethanol fermentation

Acetic acid is introduced into cellulose conversion processes as a consequence of composition of lignocellulose feedstocks, causing significant inhibition of adapted, genetically modified and wild‐type S. cerevisiae in bioethanol fermentation. While adaptation or modification of yeast may reduce inhibition, the most effective approach is to remove the acetic acid prior to fermentation. This work addresses liquid–liquid extraction of acetic acid from biomass hydrolysate through a pathway that mitigates acetic acid inhibition while avoiding the negative effects of the extractant, which itself may exhibit inhibition. Candidate solvents were selected using simulation results from Aspen Plus?, based on their ability to extract acetic acid which was confirmed by experimentation. All solvents showed varying degrees of toxicity toward yeast, but the relative volatility of ethyl acetate enabled its use as simple vacuum evaporation could reduce small concentrations of aqueous ethyl acetate to minimally inhibitory levels. The toxicity threshold of ethyl acetate, in the presence of acetic acid, was found to be 10 g L^?1. The fermentation was enhanced by extracting 90% of the acetic acid using ethyl acetate, followed by vacuum evaporation to remove 88% removal of residual ethyl acetate along with 10% of the broth. NRRL Y‐1546 yeast was used to demonstrate a 13% increase in concentration, 14% in ethanol specific production rate, and 11% ethanol yield. This study demonstrated that extraction of acetic acid with ethyl acetate followed by evaporative removal of ethyl acetate from the raffinate phase has potential to significantly enhance ethanol fermentation in a corn stover bioethanol facility. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:929–937, 2016     

Simultaneous quantification of carteolol and dorzolamide in rabbit aqueous humor and ciliary body by liquid chromatography/atmospheric pressure chemical ionization mass spectrometry

A rapid, sensitive and selective method for the simultaneous quantification of carteolol and dorzolamide in rabbit aqueous humor (AH) and ciliary body (CB) has been developed and validated using reversed phase-high performance liquid chromatography (RP-HPLC) with isocratic elution coupled with atmospheric pressure chemical ionization mass spectrometry/mass spectrometry (APCI-MS/MS). The analytes and nadolol (used as internal standard, IS) were purified from AH by protein precipitation. The sample preparation from CB was based on a two steps extraction procedure at different pH, utilizing a liquid–liquid extraction with a mixture of ethyl acetate, toluene and isopropanol 50:40:10 (v/v) at pH 8, followed by a second extraction with ethyl acetate at pH 11. The combined organic extracts were then back extracted into 0.1% aqueous trifluoroacetic acid (TFA). The accuracy and precision values, calculated from three different sets of quality control samples analyzed in sestuplicate on three different days, were within the generally accepted criteria for analytical methods (<15%). The assay proved to be accurate and precise when applied to the in vivo study of carteolol and dorzolamide in rabbit AH and CB after single administration of an eye drops containing both drugs.     

Removal of detergents from protein digests for mass spectrometry analysis

Detergents are commonly used for the extraction of hydrophobic proteins and must be removed for sensitive detection of peptides by mass spectrometry. We demonstrate that ethyl acetate is able to extract octylglycoside from a protease digest without loss of peptides or interference with the peptide mass spectral profile. Ethyl acetate extraction was also found to reduce interference by sodium dodecyl sulfate, Nonidet P-40, or Triton X-100 in the mass spectrometry analysis.     

Distribution and radical scavenging activity of phenols in Ascophyllum nodosum (Phaeophyceae)

Phlorotannins have been purified and fractionated in the brown alga Ascophyllum nodosum using successively differential extraction, liquid-liquid separation and dialysis. Both the phenol content and the radical scavenging capacity of the resulting fractions were assayed by the Folin-Ciocalteu test and the DPPH method, respectively, whilst purity of the fractions was assessed by ¹H NMR analysis. The purification process resulted in the isolation of six fractions from each crude extract with only minor losses. High levels of phenols, up to 97-99%, were measured in semi-purified fractions containing phlorotannins more than 50 kDa in average molecular size, accounting for more than 95% of the ethyl acetate phenol pool. As a consequence, purity decreased in ethyl acetate fractions together with the molecular size of compounds. The importance of differential extraction based on the polarity of phenols is highlighted by the fact that most of these compounds were found in the ethyl acetate fraction after the first extraction step in 100% methanol, whilst two thirds of phenols extracted by 50% methanol remained in the aqueous phase. The radical scavenging activity of the fractions was correlated with the phenol content and was maximal in complete ethyl acetate fractions and in dialysis concentrates containing molecules more than 50 kDa in size. The specific activity of phenols was found to be maximal for molecules smaller than 2 kDa when isolated from the 100% methanol extract and 1-4 times smaller in the water phase separated from the same extract. The distribution of radical-scavenging potentials in the phenol pool of A. nodosum supports the idea that physiological roles and putative uses of phlorotannins are under the control of a polarity-molecular size complex.     

Analysis of mycotoxins in cereals and animal feed using a multi-toxin gel permeation chromatography clean-up method

Gel permeation chromatography has been used to clean up extracts from cereals and animal feeds containing a range of mycotoxins. A mixture of dichloromethane: IM hydrochloric acid, 10:1 by volume is used as the extraction solvent and clean-up is carried out on a Bio — Beads S-X3 column using dichloromethane: ethyl acetate containing a small amount of formic acid as the elution solvent. Chromatographic separation and detection was by HPLC with fluorescence or ultraviolet detection although the choice of detection method is left to the user. The method has been tested for 14 mycotoxins and results are presented for cereals fortified with mycotoxins and for samples naturally contaminated with aflatoxins, citrinin, zearalenone, and ochratoxin A.     

响应面分析法优化大蒜蒜氨酸提取工艺

以新鲜大蒜为原料,微波灭酶后,通过乙酸乙酯打浆除去大蒜中脂溶性成分,以蒸馏水为提取液,采用响应面法研究料液比、提取温度、提取时间对蒜氨酸提取率的影响。结果表明回归模型能较好的预测各因素与响应值之间的关系,所优化的最佳工艺为:料液比为1∶5,提取温度为32℃,提取时间为70 min。此时蒜氨酸的提取率为92.85±0.63%。     

Evaluation of free radical scavenging activity of Butea monosperma Lam

In the present study, ethyl acetate, butanol and aqueous fractions derived from total methanol extract of Butea monosperma flowers were evaluated for radical scavenging activities using different in vitro models like reducing power assay, scavenging of 2,2 diphenyl-1-picrylhydrazyl (DPPH) radical, nitric oxide radical, superoxide anion radical, hydroxyl radical and inhibition of erythrocyte hemolysis using 2, 2' azo-bis (amidinopropane) dihydrochloride (AAPH). Methanol extract along with its ethyl acetate and butanol fractions showed potent free radical scavenging activity, whereas aqueous fraction was found to be devoid of any radical scavenging properties. The observed activity could be due to the higher phenolic content in the extracts (16.1, 25.29, and 17.74% w/w in methanol extract, ethyl acetate and butanol fractions respectively). HPTLC fingerprint profile of the ethyl acetate and butanol fractions were developed which would serve as reference standard for quality control of the extracts.     

Purification of fatty acid ethyl esters by solid-phase extraction and high-performance liquid chromatography

We have developed a two-step method to purify fatty acid ethyl esters (FAEE) using solid-phase extraction (SPE), with a recovery of 70±3% (mean±S.E.M.) as assessed using ethyl oleate as a recovery marker from a standard lipid mixture in hexane. The first step of the SPE procedure involves application of a lipid mixture to an aminopropyl-silica column with simultaneous elution of FAEE and cholesteryl esters from the column with hexane. Gas chromatographic analysis of FAEE without interference from cholesteryl esters may be performed using the eluate from the aminopropyl-silica column, thus eliminating the need for an octadecylsily (ODS) column in this case. The FAEE can then be separated from the cholesteryl esters, if necessary, by chromatography on an ODS column and elution with isopropanol-water (5:1, v/v). Both the aminopropyl-silica and ODS columns were found to be effective for up to four uses. To permit isolation of specific FAEE species following isolation of total FAEE by the two-step SPE method, we have also developed a purification scheme for individaal FAEE by high-performance liquid chromatography (HPLC). Thus, this simple method allows for reproducible isolation of total FAEE by SPE and isolation of individual FAEE species by HPLC.     

Dynamic changes in size distribution of emulsion droplets during ethyl acetate-based microencapsulation process

This study investigated the dynamic effect of the emulsification process on emulsion droplet size in manufacturing microspheres using ethyl acetate as an organic solvent. A dispersed phase consisting of poly(lactide-co-glycolide) and ethyl acetate was emulsified in a poly(vinyl alcohol) aqueous solution for a predetermined time ranging from 2 to 9, 16, 23, 30, 40, 50, or 60 minutes. Ethyl acetate was then quickly extracted to transform emulsion droplets into solidified microspheres, and their size distribution was determined. This experimental design allowed quantification of the size distribution of emulsion droplets over the course of emulsification. When emulsification time was extended from 2 to 60 minutes, the emulsion droplets decreased in size from 98.1 to 50.3 microm and their surface area increased from 0.07 to 0.29 m2/g. Overall, prolonging emulsification time up to 60 minutes resulted in the progressive evolution of smaller emulsion droplets (1-60 microm) and the simultaneous disappearance of larger ones (> 81 microm). Increases in the total number of microspheres and their surface area were caused mainly by continuous fragmentation of emulsion droplets before ethyl acetate extraction. The increase in the smaller microsphere population might also be due in part to shrinkage of microspheres. These results show that the onset of ethyl acetate extraction influenced the kinetics of the breakup and formation of emulsion droplets, thereby affecting to a great extent the size distribution of microspheres.     

超声波及脱蛋白处理对猴头菌子实体多糖提取效果的影响

本研究以猴头菌子实体为供试材料,在采用水提醇沉法进行多糖提取的过程中,系统研究了超声波和脱蛋白处理两个关键工艺环节对多糖提取效果的影响。研究结果表明：采用超声波辅助处理可提高猴头菌多糖提取率,降低多糖的纯度,使部分由蒸馏水洗脱获得的HEFP-α组分与全部0.1mol/L NaCl洗脱获得的HEFP-β组分经Sephacryl-S400洗脱后的曲线的小峰面积增加,也使主峰处的吸光值出现偏移。3种脱蛋白方法中,复合法对蛋白的除去效率最高,但多糖的保留率最低;亚铁氰化钾-乙酸锌法操作简便且蛋白除去率与多糖保留率均较高,但会使HEFP-β-2组分的含量和回收率降低;Sevage法蛋白脱除效率最低,多糖组分的保留率相对较高,且柱洗脱后发现经Sephacryl-S400凝胶柱分离后获得的主峰整体峰值较高,该方法较为适合多糖活性组分未知时的蛋白脱除。这一研究结果将会为该类大型真菌子实体的大分子生物活性物质的提取以及相关产品的开发提供重要的参考依据。     

益智的抗氧化作用

本文探讨了益智(Alpinia oxyphylla Miquel)超临界CO2提取物及其渣的水提物、正丙醇提取物和乙酸乙酯提取物的抗氧化作用,测定了总酚含量、黄酮含量、抗氧化力、还原能力、DPPH清除率.结果表明,益智超临界CO2提取物和正丙醇提取物的总酚含量最高,均为5.53%,乙酸乙酯提取物的总酚含量为4.04%,水提物总酚含量最低,为O.89%.抗氧化力与酚含量相关(R2=0.703).四种提取物中黄酮含量顺序为:乙酸乙酯提取物(6.29%)＞丙醇提取物(5.81%)＞水提取物(4.85%)＞超临界CO2提取物(4.70%).在还原能力、清除DPPH自由基和羟自由基方面,乙酸乙酯提取物表现出了很强的抗氧化能力,呈现剂量依赖关系.     

Simultaneous quantification of alpha-/beta-diastereomers of arteether, sulphadoxine and pyrimethamine: a promising anti-relapse antimalarial therapeutic combination, by liquid chromatography tandem mass spectrometry

A rapid, sensitive, selective and specific HPLC/ESI-MS/MS assay method was developed and validated for the simultaneous quantitation of alpha-/beta-diastereomers of arteether (AE), sulphadoxine (SDX) and pyrimethamine (PYR) in rat blood plasma using propyl ether analogue of beta-arteether as internal standard. The method involved a single-step, liquid-liquid extraction with ethyl acetate and the analytes were chromatographed on a C18 chromatographic column by isocratic elution with methanol:ammonium acetate buffer (10 mM, pH 4) (90:10%, v/v) and analyzed by tandem mass spectrometry. The run time was 4.5 min and the weighted (1/x2) calibration curves were linear over a range of 0.78-400 ng ml-1. The method was validated fully and the lower limit of quantification (LLOQ) in plasma was 0.78 ng ml-1 for all the analytes. The intra- and inter-day precision and accuracy were found to be well within the acceptable limits (<15%) and the analytes were stable after three freeze-thaw (f-t) cycles. The absolute recoveries were consistent and reproducible. The assay method was applied to pre-clinical pharmacokinetic interaction studies of alpha-/beta-AE, SDX and PYR in rats.     

Assay of succinate dehydrogenase activity by the tetrazolium method: evaluation of an improved technique in skeletal muscle fractions

An improved spectrophotometric method for measuring succinate dehydrogenase (EC 1.3.99.1) activity with the use of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT) is described. The procedure has been evaluated in mitochondrial fractions and homogenates of frog skeletal muscle. For mitochondrial suspensions, extraction of formazan with alcohol was found to be superior to extraction with ethyl acetate. For homogenates, complete extraction of formazan required sequential treatment with alcohol and ethyl acetate; the generally employed procedure of extracting once with ethyl acetate alone led to serious underestimation of the amount of formazan in the tissue. Observations of mitochondrial suspension incubated with various concentrations of INT led to the selection of 0.8 mM INT for optimal results. Higher concentrations, although commonly used, can exert undesirable inhibitory effects on succinate dehydrogenase activity, especially at low concentrations of mitochondria and after longer periods of incubation. The problem of instability of succinate dehydrogenase was solved by the addition of buffer at pH 7.5.     

泥胡菜等8种草本植物提取物除草活性的生物测定

采用顺序提取法制备了泥胡菜（Hemistepta lyrata Bunge）等8种草本植物全草的石油醚、乙酸乙酯和乙醇提取物,并以高粱（Sorghum vulgare Pers．）、黄瓜（Cucumis sativus L．）、小麦（Triticum aestivum L．）和油菜（Brassica campestris L．）为供试对象,用种子萌发法对提取物的除草活性进行了生物测定。结果表明,所有提取物对4种作物幼苗根和茎的生长均有一定的抑制作用,但抑制率有一定差异。泥胡菜和獯草[Humulus scandens（Lour．）Merr．]的乙酸乙酯提取物对作物幼苗根和茎的抑制作用最强,抑制率随提取物浓度的提高逐渐增高,且对作物幼苗根生长的抑制强度高于茎。在低浓度（12．5g·L^-1）条件下,泥胡菜乙酸乙酯提取物对小麦幼苗根和茎生长的抑制作用最强;蓓草乙酸乙酯提取物对高粱幼苗根的生长及油菜幼苗根和茎的生长抑制作用最强。研究结果显示,泥胡菜及蓰草的乙酸乙酯提取物具有潜在的除草活性。     

Enantiomeric determination of amlodipine in human plasma by liquid chromatography coupled to tandem mass spectrometry

A sensitive method for the separation and determination of amlodipine enantiomers in plasma has been developed based on solid-phase extraction (SPE) with disposable extraction cartridges (DECs) in combination with chiral liquid chromatography (LC). The SPE technique is used to isolate the drug from the biological matrix and to prepare a cleaner sample before injection and analysis by HPLC coupled to mass spectrometry. The DEC is filled with ethyl silica (50 mg) and is first conditioned with a 2.5% ammonia in methanol solution and then with ammonium acetate buffer. A 1.0-ml volume of plasma is then applied on the DEC. The washing step is first performed with ammonium acetate buffer and secondly with a mixture of water and methanol (65:35, v/v), while the final elution step is obtained by dispensing methanol containing 2.5% of ammonia. The eluate is then collected and evaporated to dryness before being dissolved in the LC mobile phase and injected into the LC system. The stereoselective analysis of amlodipine is achieved on a Chiral AGP column containing alpha(1)-acid glycoprotein as chiral selector by using a mobile phase consisting of a 10-mM acetate buffer (pH 4.5) and 1-propanol (99:1, v/v). The LC system is coupled to tandem mass spectrometry with an APCI interface in the positive-ion mode. The chromatographed analytes are detected in the selected reaction monitoring mode (SRM). The MS/MS ion transitions monitored are 409 to 238 for amlodipine, and 260 to 116 for S-(-)-propranolol used as internal standard (IS). The method was validated considering different parameters, such as linearity, precision and accuracy. The limit of quantitation was found to be 0.1 ng/ml for each amlodipine enantiomer.     

飞机草提取物对红脉穗螟的产卵忌避及杀卵活性

为探讨飞机草提取物对红脉穗螟Tirathaba rufivena Walker的防控潜力,采用室内生物测定法,研究了飞机草提取物对红脉穗螟的产卵忌避作用和杀卵活性。结果表明,飞机草乙酸乙酯提取物对红脉穗螟的忌避效果最好,选择性和非选择性忌避率分别为41.85%和46.84%;不同提取物处理红脉穗螟的卵后,均造成卵孵化率降低和初孵幼虫的死亡,其中乙酸乙酯提取物对红脉穗螟卵的影响最大,孵化率和1龄幼虫死亡率分别达到65.89%和40.37%。对飞机草乙酸乙酯提取物进行分级萃取测试各萃取物的活性发现,正己烷萃取物对红脉穗螟的产卵忌避作用和杀卵活性最强。田间试验表明,采用正己烷萃取物稀释50倍和100倍浓度进行田间喷雾,药后15 d对红脉穗螟的种群控制率可达到50%以上。     

Pulsed electric field (PEF) as an intensification pretreatment for greener solvent lipid extraction from microalgae

Microalgae, with their high lipid content, are a promising feedstock for renewable fuels. Traditionally, human and environmentally toxic solvents have been used to extract these lipids, diminishing the sustainability of this process. Herein, pulsed electric field technology was utilized as a process intensification strategy to enhance lipid extraction from Ankistrodesmus falcatus wet biomass using the green solvent, ethyl acetate. The extraction efficiency for ethyl acetate without PEF was lower (83–88%) than chloroform. In addition, the ethyl acetate exhibited a 2‐h induction period, while the chloroform showed no time dependence. Utilizing PEF technology resulted in 90% of the cells being lysed and a significant enhancement in the rate of lipid recovery using ethyl acetate. The increase in lipid recovery was due to the presence of the electric field and not due to temperature effects. The PEF technology uses less energy than other PEF systems reported in the literature. Biotechnol. Bioeng. 2013; 110: 1605–1615. © 2013 Wiley Periodicals, Inc.     

Simultaneous determination of dexamethasone and 6 beta-hydroxydexamethasone in urine using solid-phase extraction and liquid chromatography: applications to in vivo measurement of cytochrome P450 3A4 activity

It has been demonstrated that the formation of the hydrophilic metabolites of dexamethasone, 6 alpha- and 6 beta-hydroxydexamethasone, correlated with cytochrome P450 (CYP) 3A4 enzyme levels. So, the 6 beta-hydroxydexamethasone/dexamethasone urinary ratio could be a specific marker for human CYP3A4 activity. We have developed a sensitive and specific high-performance liquid chromatographic method for the simultaneous quantification of urinary free dexamethasone and 6 beta-hydroxydexamethasone using 6 alpha-methylprednisolone as internal standard. This method involved a solid phase extraction of the three compounds from urine using Oasis HLB Waters cartridges with an elution solvent of ethyl acetate (2 ml) followed by diethyl ether (1 ml). Separation of the three analytes was achieved within 24 min using a reversed-phase Nova-Pak C(18) analytical column (4 microm, 300 mm x 3.9 mm i.d.). An ultraviolet detector operated at 245 nm was used with a linear response observed from 10 to 100 ng/ml for dexamethasone and from 25 to 1000 ng/ml for 6 beta-hydroxydexamethasone. Obtained from the method validation, inter-assay precision was below 15% and accuracy ranged from 95.7 to 110%. The extraction efficiency of the assay was approximately of 99% and was constant across the calibration range. The lower limit of quantitation was 10 ng/ml for dexamethasone and 25 ng/ml for 6 beta-hydroxydexamethasone; at these levels, precision was below 16% and accuracy was 99-109%. This method was applied to in vivo measure of the CYP3A4 activity.     

Isolation and purification of series bioactive components from Hypericum perforatum L. by counter-current chromatography

Counter-current chromatography (CCC) combined with pre-separation by ultrasonic solvent extraction was successively used for the separation of series bioactive compounds from the crude extract of Hypericum perforatum L. The petroleum ether extract was separated by the solvent system of n-heptane-methanol-acetonitrile (1.5:0.5:0.5, v/v) and n-heptane-methanol (1.5:1, v/v) in gradient elution, yielding a phloroglucinol compound, hyperforin with HPLC purity over 98%. The ethyl acetate extract was separated by using the solvent system composed of hexane-ethyl acetate-methanol-water (1:1:1:1 and 1:3:1:3, v/v) in gradient through both reverse phase and normal phase elution mode, yielding a naphthodianthrone compound, hypericin with HPLC purity about 95%. The n-butanol extract was separated with the solvent system composed of n-butanol-ethyl acetate-water (1:4:5 and 1.5:3.5:5, v/v) in elution and back-extrusion mode, yielding two of flavones, rutin and hyperoside, with HPLC purity over 95%. HPLC-MS, reference sample and UV spectrum were selectively used in separation to search for target compounds from HPLC-DAD profiles of different sub-extracts. The structures of isolated compounds were further identified by ESI-MS, 1HNMR and 13CNMR.