高速逆流色谱法分离纯化青皮中六种多甲氧基黄酮

应用高速逆流色谱法(HSCCC)分离制备了青皮中6种多甲氧基黄酮类(Polymethoxyflavones,PMFs)化合物。以正己烷-乙酸乙酯-甲醇-水(体积比为4∶6∶4∶6)为两相溶剂系统,在主机转速800 r/min、流动相流速2mL/min、检测波长254 nm条件下进行分离制备,460 min内从270 mg青皮粗提物中一步分离制备得到甜橙素(1,sinensetin)3.5 mg,5,7,8,4-四甲氧基黄酮(2)13.9 mg,川陈皮素(3,Nobiletin)51.3 mg,3,5,6,7,8,3′,4′-七甲氧基黄酮(4)9.5 mg,橘皮素(5,Tangeretin)44.7 mg,5-去甲川陈皮素(6,5-O-DesmethylNobiletin)11.2 mg,纯度均达97%以上,各化合物结构经质谱和核磁共振氢谱、碳谱鉴定。利用该方法可以对青皮中的黄酮类化合物进行快速的分离和纯化。     

聚酰胺色谱结合高速逆流色谱分离制备萹蓄黄酮类化合物

采用聚酰胺色谱结合高速逆流色谱法分离纯化了萹蓄中3种黄酮类化合物,建立了快速分离制备萹蓄中3种黄酮类化合物的方法。通过聚酰胺柱色谱富集黄酮类成分,再经过高速逆流色谱分离,以乙酸乙酯-甲醇-水-甲酸(体积比为4∶1∶5∶0.1)组成的二相系统作为固定相与流动相,在主机转速为850 rpm,流速为2.0m L/min,检测波长为254 nm的条件下制备样品。从150 mg富集黄酮成分的馏分中,一次性分离制备得到纯度为94.86%的杨梅树皮苷(myricitrin)7.5 mg,94.28%的黄芪苷(astragalin)13.8 mg,91.86%的合欢草素1(desmanthin-1)20.6 mg。所得馏分经高效液相色谱法(HPLC)检测纯度,并经MS和NMR鉴定化合物的结构。该方法简便、快速,所得产物纯度高,适合于黄酮类化合物的制备分离。     

高速逆流色谱技术分离纯化红葱中的萘醌类化合物

采用高速逆流色谱(HSCCC)技术从红葱中快速分离纯化得到红葱乙素和异红葱乙素,建立了快速分离制备红葱中萘酚类化合物的方法。首先采用95%乙醇加热回流提取得红葱提取物,再用乙酸乙酯萃取富集萘醌类成分,然后用高速逆流色谱分离纯化,以石油醚-乙酸乙酯-甲醇-水(6∶4∶5∶5,v/v)组成二元溶剂系统,上相为固定相,下相为流动相,仪器转速为850 rpm,流速为2.0 m L/min,检测波长为254 nm。从200 mg富集萘醌类成分的粗提物中,一次性分离制备得到60 mg异红葱乙素和49 mg红葱乙素,经高效液相色谱法(HPLC)分析,其纯度分别为97.3%和98.6%。通过核磁共振氢谱(~1H NMR)和核磁共振碳谱(~(13)C NMR)鉴定化合物为红葱乙素和异红葱乙素。研究结果表明,该方法快速、高效,适用于红葱中萘酚类化合物的分离纯化。     

高速逆流色谱分离制备川西獐牙菜中两种苷类化合物

采用高速逆流色谱从川西獐牙菜中分离制备了两种高纯度苷类化合物.以正丁醇-氯仿-甲醇-水(3.4∶8∶5∶6,v/v)为溶剂系统,主机转速为800 rpm,流速:O～210 min,1.5mL/min;210 ～360m in,2.5 mL/min,检测波长254 nm的条件下进行分离制备,在360 min内从100 mg样品中一步分离制备得到1-O-樱草糖-3,7,8-三甲氧基(口山)酮(Ⅰ,11 mg)和异荭草苷(Ⅱ,24 mg).经HPLC检测,两个化合物的纯度均在99％以上,结构由UV、1H和13C NMR鉴定.     

高速逆流色谱分离制备蛹虫草中虫草素和N6-(2-羟乙基)-腺苷

采用高速逆流色谱（HSCCC）技术从蛹虫草子实体粗提物中分离制备高纯度虫草素和N⁶-(2-羟乙基)-腺苷。利用高效液相色谱（HPLC）测定目标产物在溶剂体系中的分配系数,优化HSCCC分离虫草素和N⁶-(2-羟乙基)-腺苷的溶剂体系,确定了以乙酸乙酯-正丁醇-1.5%氨水（1:4:5,V/V/V）为HSCCC的两相溶剂体系,并运用此溶剂体系,上相为固定相,下相为流动相,主机转速850r/min,流动相流速为1.5mL/min,检测波长为254nm条件下进行分离制备,在250min内从200mg蛹虫草子实体粗提物中一步分离得到10.8mg纯度99%的虫草素和6.1mg 纯度98%的N⁶-(2-羟乙基)-腺苷。该方法简便、快速,为虫草素和N⁶-(2-羟乙基)-腺苷的大量制备建立了基础。     

海南黄芩中黄酮类化合物的分离与鉴定

目的:分离纯化海南黄芩中黄酮类化合物,并对其进行结构鉴定。方法:采用95%乙醇回流提取,醇提物用石油醚和氯仿萃取获得黄酮类化合物;采用岛津LC-10AD二元低压半制备液相色谱仪和Agilent半制备色谱柱(Phenyl,2.5 mm×25.0 cm,5μm)对氯仿萃取物进行分离纯化;采用高效液相色谱法对分离纯化获得的化合物进行纯度测定,并采用1H-NMR、13C-NMR波谱技术对其进行结构鉴定。结果:从海南黄芩中分离得到了5种化合物,纯度均大于95%,分别鉴定为5-羟基-7,8,2',6'-四甲氧基二氢黄酮、5,6,7-三羟基-2'-甲氧基黄酮、5,2'-二羟基-6,7-二甲氧基黄酮、黄芩素和黄芩素-7甲醚。结论:5种化合物均为首次从海南黄芩中分离得到,它们的获得丰富了海南黄芩化学成分组成,为其深入研究与开发提供了科学依据。     

离子液体优化高速逆流色谱法分离槐花中的黄酮类化合物

应用高速逆流色谱法首次从槐花中一步分离出2种黄酮类化合物,并利用离子液体提高分离效果。以正己烷-乙酸乙酯-乙醇-水-冰醋酸(1∶1∶1∶1∶0.05,v/v)为两相溶剂体系,从50 mg槐花粗提物中一步分离得到芦丁18.2 mg,槲皮素9.6 mg,其纯度均在97%以上。加入离子液体1-丁基-3-甲基咪唑六氟化硼酸([BMIM][PF6]),使出峰时间由原来的85 min提前到55 min,分离度由0.9提高到1.8,达到完全分离,分离效果得到明显提高,为离子液体在高速逆流色谱中的进一步应用提供依据。     

高速逆流色谱法(HSCCC)分离制备香水莲花中的柚皮素

采用高速逆流色谱法对香水莲花中的黄酮类化合物进行了分离,溶剂系统为正己烷-醋酸乙酯-甲醇-水(体积比1∶1.2∶2∶1),上层为固定相,下层为流动相,流速2.0 mL/min,转速830 r/min,检测波长280 nm,进样量300 mg,分离出80 mg化合物C,经HPLC测定纯度98.6%,根据UV光谱、MS分析及1H-NMR、13C-NMR图谱分析结果,化合物C鉴定为柚皮素,该化合物为首次从该植物中分离得到,为香水莲花的开发应用提供了理论依据。     

溶剂系统极性在高速逆流色谱分离油橄榄叶多酚类化合物过程中的影响

为探讨目标化合物极性和溶剂系统极性在HSCCC分离过程中的相关性,本文以油橄榄叶中的2种主要有效成分-橄榄苦苷和木犀草素-7'-O-β-D-葡萄糖苷为目标化合物,系统考察正己烷-乙酸乙酯-甲醇-水系统[(1∶19∶1∶19,v/v)、(1∶9∶1∶9,v/v)、(1∶9∶2∶8,v/v)、(1∶9∶3∶7,v/v)、(1∶6∶1∶6,v/v)、(1∶5∶1∶5,v/v)]不同比例组成的两相溶剂系统对目标化合物分离的影响。结果表明:橄榄苦苷、木犀草素-7'-O-β-D-葡萄糖苷随着溶剂系统的极性的减小,其分离效果呈现先增强后减弱的趋势,在最佳的分离条件下,分离制备所得橄榄苦苷、木犀草素-7'-O-β-D-葡萄糖苷的纯度按照HPLC归一化法检测,分别为87.8%和77.5%。本实验结果为HSCCC溶剂系统快速筛选及提高HSCCC在复杂体系中分离制备目标化合物的效率提供技术支持。     

高速逆流色谱制备玫瑰红景天中的三种酚性化合物

采用高速逆流色谱方法(HSCCC,High-speed Counter-current Chromatography)同时分离三种玫瑰红景天酚性化合物。玫瑰红景天提取物经聚酰胺吸附多酚后经硅胶柱分级得预分离样品,采用正己烷∶乙酸乙酯∶甲醇∶水(4∶5∶4∶5,v/v/v/v)组成的两相溶剂系统对预分离样品进行分离纯化,一次进样150 mg,一次色谱分离得到化合物1:68.5 mg、化合物2:8.5 mg、化合物3:45.5 mg,纯度都超过98%。通过ESI-MS、1H NMR对其结构进行鉴定化合物1为没食子酸(Gallic acid),化合物2为没食子酸甲酯(Methyl gallate),化合物3为山奈酚(Kaempferol)。结果表明利用HSCCC可以成功分离三种酚性化合物,分离效果好,产品纯度高。     

Combinative application of pH-zone-refining and conventional high-speed counter-current chromatography for preparative separation of alkaloids from Stephania kwangsiensis

A method which involves the combination of pH-zone-refining counter-current chromatography (pH-zone-refining CCC) and conventional high-speed counter-current chromatography (HSCCC) was established for the preparative separation of alkaloids from the crude extracts of Stephania kwangsiensis. pH-zone-refining CCC was first performed with the solvent system composed of n-hexane-ethyl acetate-methanol-water (3:7:1:9, v/v), where triethylamine (10 mM) was added to the upper organic stationary phase as a retainer and hydrochloric acid (5 mM) to the aqueous mobile phase as an eluter. From 2.0 g of crude extract, 370 mg of sinoacutine and 600 mg of a mixture of three other alkaloids were obtained. Then, the mixture was further separated by conventional HSCCC with the solvent system composed of n-hexane-ethyl acetate-methanol-water (7:3:6:4, v/v), yielding 42 mg of (-)-crebanine, 50 mg of (-)-stephanine and 30 mg of l-romerine from 150 mg mixture of three other alkaloids, respectively. The purities of the four compounds were all over 98% as determined by HPLC, and the chemical structures of the four compounds were confirmed by positive ESI-MS and (1)H NMR data. Results of the present study successfully indicated that this method was efficient for the preparative separation of alkaloids from natural plants.     

Preparative separation of alkaloids from Nelumbo nucifera leaves by conventional and pH-zone-refining counter-current chromatography

Two modes of high-speed counter-current chromatography (HSCCC) were successfully applied to the separation of alkaloids from crude extract of Nelumbo nucifera leaves. The conventional HSCCC separations were performed with a two-phase solvent system composed of tetrachloromethane–CHCl₃–methanol–0.1 M HCl at a volume ratio of 1:3:3:2 (v/v/v/v), and 120 mg crude extract could be successfully separated. pH-Zone-refining CCC was performed with a two-phase solvent system composed of petroleum ether (60–90 °C)–ethyl acetate–methanol–water (5:5:2:8, v/v/v/v) where triethylamine (10 mM) was added to the upper organic stationary phase as a retainer and hydrochloric acid (5 mM) to the aqueous mobile phase as an eluent. From 4.0 g of the crude extract, 120 mg N-nornuciferine, 1020 mg nuciferine and 96 mg roemerine were obtained in a single run each with a purity of over 98% as determined by HPLC. The structures of the isolated compounds were identified by ESI-MS, ¹H NMR and ¹³C NMR.     

Isolation and purification of seven lignans from Magnolia sprengeri by high-speed counter-current chromatography

Seven lignans including (-)-maglifloenone, futoenone, magnoline, cylohexadienone, fargesone C, fargesone A and fargesone B were isolated and purified from Magnolia sprengeri Pamp. using high-speed counter-current chromatography (HSCCC) with two-step separation. In the first step, a stepwise elution mode with the two-phase solvent system composed of petroleum ether-ethyl acetate-methanol-water (1:0.8:0.6:1.2, 1:0.8:0.8:1, v/v) was used and 15.6 mg of (-)-maglifloenone, 19.2 mg of futoenone, 10.8 mg of magnoline, 14.7 mg of cylohexadienone and 217 mg residues were obtained from 370 mg crude extract. In the second step, the residues were successfully separated by HSCCC with the solvent system composed of petroleum ether-ethyl acetate-methanol-water (1:0.8:1.2:0.6, v/v), yielding 33.2 mg of fargesone C, 47.5 mg of fargesone A and 17.7 mg of fargesone B. The purities of the separated compounds were all over 95% determined by HPLC. The chemical structures of these compounds were confirmed by (1)H NMR, (13)C NMR and ESI-MS.     

高速逆流色谱法分离制备丹酚酸B

采用高速逆流色谱法分离纯化丹参水溶性成分丹酚酸类物质,制备丹酚酸B化学对照品。分离采用的溶剂系统为正己烷-乙酸乙酯-水-甲醇(1.5:5:5:1.5),上相做固定相,下相做流动相,流速为1.7 mL/min,仪器转速850 rpm,进样量80 mg,纯度用HPLC方法测定。结果表明:一次分离可制备63.4 mg丹酚酸B,其纯度为98.6%。该方法操作简单,可作为高纯度丹酚酸B化学对照品的制备分离方法。     

高速逆流色谱法分离凤尾草中的芹菜素和木犀草素

建立了高速逆流色谱分离纯化芹菜素和木犀草素的方法。两相溶剂系统为氯仿-甲醇-水（4：3：2）,上相为固定相,下相为流动相进行洗脱。从100mg粗提物中一步分离得到17．0mg芹菜素和22．5mg木犀草素,经高效液相色谱分析,纯度分别为92％和96％。其化学结构由1H和13CNMR鉴定。     

Separation and purification of four chromones from radix saposhnikoviae by high-speed counter-current chromatography

A preparative high-speed counter-current chromatography (HSCCC) method for the isolation and purification of 1'-O-glucosylcimifugin (1), 4'-O-beta-d-glucosyl-5-O-methylvisamminol (2), cimifugin (3) and 3'-O-glucosylhamaudol (4) from the Chinese medicinal herb radix saposhnikoviae has been successfully developed. A sample of 300 mg of crude extract was separated using ethyl acetate:n-butanol:1% aqueous acetic acid (1:4:5, v/v) as the two-phase solvent system and yielded 102.4 mg of 1 and 81.6 mg of 2. During this separation 3 and 4 remained in the stationary phase, which was collected, evaporated to dryness and separated with another two-phase solvent system involving ethyl acetate:n-butanol:1% aqueous acetic acid (5:0.5:5, v/v) to yield 31.4 mg of 3 and 12.7 mg of 4. The purities of compounds 1-4 were 98.4, 98.7, 99.3 and 98.2%, respectively, as determined by HPLC. The chemical structures of these components were established by (1)H-NMR and (13)C-NMR.     

Preparative isolation of alkaloids from Dactylicapnos scandens using pH-zone-refining counter-current chromatography by changing the length of the separation column

pH-Zone-refining counter-current chromatography was successfully applied for the preparative separation of alkaloids from Dactylicapnos scandens. The two-phase solvent system was composed of petroleum ether-ethyl acetate-methanol-water (3:7:1:9, v/v), where 20 mM of triethylamine (TEA) was added to the upper phase as a retainer and 5 mM of hydrochloric acid (HCl) to the aqueous phase as an eluter. In this experiment, the apparatus with an adjustable length of the separation column was used for the separation of alkaloids from D. scandens and the resolution of the compounds can be remarkably improved by increasing the length of the separation column. As a result, 70 mg protopin, 30 mg (+) corydine, 120 mg (+) isocorydine and 40 mg (+) glaucine were obtained from 1.0 g of the crude extracts and each with 99.2%, 96.5%, 99.3%, 99.5% purity as determined by HPLC. The chemical structures of these compounds were confirmed by positive ESI-MS and (1)H NMR.     

Accelerated solvent extraction of monacolin K from red yeast rice and purification by high-speed counter-current chromatography

Monacolin K from red yeast rice was extracted by accelerated solvent extraction (ASE). The effects of various extraction parameters including extraction temperature, static extraction time and cycle index on yield were investigated using a DIONEX ASE 300 system to select the optimal conditions by an orthogonal test design L₉ (3)³. The optimum extraction conditions were determined as follows: extraction temperature 120 °C, static extraction time 7 min, and cycle index 3. Under the optimal conditions, the yield of ASE extract and monacolin K was 5.35% and 9.26 mg/g of dry red yeast rice, respectively. A separation and purification method of monacolin K was then established using high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water (8:2:5:5, v/v/v/v). From 300 mg of crude extract, 51.2 mg of monacolin K was obtained with the purity of 98.7%. The chemical structure of isolated compound was identified by UV, ESI-MS and ¹H NMR.     

Sensitive quantification of ranolazine in human plasma by liquid chromatography--tandem mass spectrometry with positive electrospray ionization

A rapid, selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method with positive electrospray ionization (ESI) was developed for the quantification of ranolazine in human plasma. After liquid-liquid extraction of ranolazine and internal standard (ISTD) phenoprolamine from a 100 microl specimen of plasma, HPLC separation was achieved on a Nova-Pak C(18) column, using acetonitrile-water-formic acid-10% n-butylamine (70:30:0.5:0.08, v/v/v/v) as the mobile phase. The mass spectrometer was operated in multiple reaction monitoring (MRM) mode using the transition m/z 428.5-->m/z 279.1 for ranolazine and m/z 344.3-->m/z 165.1 for the internal standard, respectively. Linear calibration curves were obtained in the concentration range of 5-4000 ng/ml, with a lower limit of quantitation (LLOQ) of 5 ng/ml. The intra- and inter-day precision values were below 3.7% and accuracy was within +/-3.2% at all three quality control (QC) levels. This method was found suitable for the analysis of plasma samples collected during the phase I pharmacokinetic studies of ranolazine performed in 28 healthy volunteers after single oral doses from 200 mg to 800 mg.     

Screening of iron- and zinc-enriched yeast strain and optimization of cultivation conditions

Saccharomyces cerevisiae LN-17 was selected from 26 kinds of primary yeast strains that belong to different genera and species. The iron- and zinc-enriched capability of strain LN-17 was higher than the others. The highest iron and zinc contents of the strain were obtained when the strain grew up under the following conditions: The strain was incubated (5%, v/v) in 50 mL wort medium (pH 6.0) with 100 mg/L Fe ion and 120 mg/L Zn ion. The medium was loaded into a 250-mL Erlenmeyer flask and shaken in a rotary shaker (200 rpm) at 30°C for 60 h. Ferrous sulfate and zinc sulfate were chosen as the source of Fe and Zn. The Fe and Zn contents of the dry cells were determined by atomic absorption spectrum analysis. Under the optimized cultivation conditions, the Fe and Zn contents reached 7.854 mg/g dry cells and 4.976 mg/g dry cells.