高速逆流色谱法分离制备母丁香和公丁香中的5种非挥发性化合物

应用高速逆流色谱法从母丁香和公丁香中快速分离了3种已知非挥发性化合物,并利用相同方法从公丁香中分离出2种色原酮类化合物。两相溶剂系统A为正己烷-乙酸乙酯-甲醇-水(5:8:6:13, v/v/v/v),系统B为正己烷-乙酸乙酯-甲醇-水(5:8:9:10, v/v/v/v),以系统A的上相为固定相,系统A和B的下相为流动相,利用梯度洗脱方式,在主机转速为880 r/min、流速1.2 mL/min条件下,成功地从70 mg母丁香粗提物中分离得到12.3 mg鞣花酸、9.6 mg鼠李素、17.2 mg槲皮素,从50 mg公丁香粗提物中分离得到5,7-二甲氧基-2-甲基色原酮10.2 mg、5,7-二甲氧基-2,6-二甲基色原酮8.6 mg,纯度均在96%以上。各化合物的结构均由质谱和核磁共振氢谱、碳谱鉴定。利用该方法可以对丁香不同药用部位中的非挥发性化合物进行有效的分离和纯化。     

生附子中C19型二萜生物碱的高速逆流色谱分离及其结构鉴定

应用高速逆流色谱法分离制备了生附子中的3个C19型二萜生物碱类化合物。以正己烷-乙酸乙酯-甲醇-水(3:5:4:5, v/v/v/v)为两相溶剂系统,上相为固定相,下相为流动相,在主机转速850 r/min、流动相流速2.0 mL/min、检测波长235 nm条件下进行分离制备;一次性从90 mg附子总碱粗提物中分离制备得到15.3 mg北草乌碱,35.1 mg中乌头碱和22.7 mg次乌头碱,经高效液相色谱分析,测得它们的纯度分别为97.9%、96.2%和99.2%。并应用波谱(电喷雾离子质谱、核磁共振氢谱和核磁共振13C谱)解析法确定了它们的结构。利用该方法可以对生附子中的二萜类生物碱成分进行快速的分离和纯化     

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.     

超高效液相色谱-串联质谱法快速同时测定生姜中10种营养成分

建立了超高效液相色谱-串联质谱（UHPLC-MS/MS）快速同时测定生姜中姜辣素类和姜黄素类营养成分的分析方法，具体包括6-姜酚、8-姜酚、10-姜酚、6-姜烯酚、8-姜烯酚、10-姜烯酚、四氢姜黄素、姜黄素、去甲氧基姜黄素、双去甲氧基姜黄素10种目标物。采用ZORBAX RRHD Eclipse Plus C18色谱柱（100 mm×2.1 mm，1.8 μm）分离，以0.1%（v/v）甲酸水溶液和0.1%（v/v）甲酸甲醇溶液为流动相进行梯度洗脱，采用电喷雾电离（ESI）源、正离子和多反应监测（MRM）模式对目标物进行定性确证和定量分析。10种营养成分的线性相关系数（r）均≥ 0.9995，方法的定量限为0.10~7.71 μg/L，样品基质在3个水平下的平均加标回收率为82.8%~115.3%，相对标准偏差（RSD）为0.58%~11.49%。分析结果显示，生姜中10种营养成分均有检出，其中6-姜酚的含量最高且集中分布于373.35~702.48 mg/kg。该法简便快速，准确可靠，适用于生姜中姜辣素类和姜黄素类营养成分的分析，可为生姜质量鉴定和控制提供技术手段。     

高速逆流色谱法从蝙蝠蛾拟青霉中快速分离制备麦角甾醇纯品

建立了用高速逆流色谱从蝙蝠蛾拟青霉中高效、快速分离制备高纯度麦角甾醇的方法。将蝙蝠蛾拟青霉的乙酸乙酯提取物直接进行高速逆流色谱分离,考察了不同溶剂系统的分离效果。结果表明,最佳的溶剂系统为正己烷-乙酸乙酯-甲醇-水(体积比为6:1.7:6:0.3),以上相为固定相,下相为流动相,转速为850 r/min,流速为2 mL/min,检测波长为280 nm。制备所得的麦角甾醇经紫外光谱(UV)和高分辨质谱(HRMS)鉴定及与标准品对照定性;纯度经高效液相色谱(HPLC)分析为99.2%(峰面积归一化法)。该方法制备麦角甾醇简便、快速,所得产物的纯度高,适合于麦角甾醇对照品的制备。     

高速逆流色谱分离制备椭圆叶花锚中的2种口山酮苷元

椭圆叶花锚的主要活性成分为口山酮类化合物,这类化合物具有利胆、抗炎、抗菌及抗病毒活性。应用高速逆流色谱法建立了2种高纯度口山酮苷元的分离制备方法。对椭圆叶花锚氯仿萃取部位运用高速逆流色谱分离纯化,以正己烷-乙酸乙酯-甲醇-水(5:5:7:5, v/v/v/v)为两相溶剂系统,上相为固定相,下相为流动相。在主机转速800 r/min,流动相流速1.5 mL/min,检测波长254 nm条件下进行分离制备。所得产物经高效液相色谱分析检测,其化学结构由核磁共振氢谱(1H NMR)和核磁共振碳谱(13C NMR)鉴定。在此条件下,从100 mg粗样品中一步分离得到18 mg 1-羟基-2,3,5-三甲氧基口山酮,14 mg 1-羟基-2,3,4,5-四甲氧基口山酮。经高效液相色谱分析,其纯度均达98%以上。该方法简便、快速,所得产物纯度高,适合于椭圆叶花锚口山酮苷元的制备分离。     

Effective two-dimensional counter-current chromatographic method for simultaneous isolation and purification of oridonin and ponicidin from the crude extract of Rabdosia rubescens

A preparative two-dimensional counter-current chromatographic system (2D-CCC) for simultaneous separation and purification of oridonin and ponicidin from the crude extract of Rabdosia rubescens was presented. It was based on the use of a high-speed CCC (HSCCC, total column volume V(c)=146ml) instrument in the first dimension (1st-D) and a preparative upright CCC (UCCC, V(c)=1500ml) column in the second dimension (2nd-D). The interface was a homemade column-switching system with a 50-ml sample loop. In this way, almost the whole interested region from the first dimension was transferred on-line to the second dimension for further separation. The use of a pair of two-phase solvent systems composed of n-hexane-ethyl acetate-methanol-water (1:5:1:5 and 3:5:3:5, v/v) in the two dimensions permitted the simultaneous separation of oridonin and ponicidin, making this 2D-CCC system with satisfactory resolution and peak capacity. During about 9h separation, two injections with about a 250mg amount of the crude extract for each, was accomplished using this 2D-CCC system, yielding 60mg of oridonin (1) and 10mg of ponicidin (2) all at a purity of over 95% as determined by HPLC analysis. Their chemical structures were identified by electrospray ionization (ESI) MS, (1)H NMR and (13)C NMR.     

Development and evaluation of a spiral tube column for counter-current chromatography

An improved type-J counter-current chromatography (CCC) planet centrifuge with two spiral tube columns (volume 2×15 mL, β value 0.3-0.7, tubing 0.8 mm id) was developed and evaluated for its retention ability of four typical different solvent systems including heptane-methanol (1:1, v/v) (A), hexane-ethyl acetate-methanol-water (1:1:1:1, v/v) (B), n-butanol-acetic acid-water (4:1:5, v/v) (C), PEG1000-K(2)HPO(4)-water (12.5:12.5:75, w/w) (D) under eight different operation modes. The results indicated that the spiral tube column could significantly increase the retention of four typical solvent systems compared with a traditional multilayer coil column with similar parameters (volume 35 mL, β value 0.3-0.7, tubing 0.8 mm id). The retention of stationary phase (S(f)) for the less polar system (A) and moderately polar solvent system (B) can be increased by about 10%, and for the polar system (C) and aqueous two-phase system (ATPS) (D) by 30-40%. The preliminary applications of this spiral tube column to the separation of small molecular compounds such as moderately polar theaflavins, polar anthocyanins and dipeptides were successful. Acceptable resolution can be obtained between cytochrome c and myoglobin, lysozyme and myoglobin when it was applied on protein separation; however, it still needs to be improved with regard to its column efficiency.     

Two-dimensional counter-current chromatography for the preparative separation of prenylflavonoids from Artocarpus altilis

A two-dimensional counter-current chromatographic system (2D-CCC) for preparative isolation and purification of three prenylflavonoids from Artocarpus altilis is presented. An upright CCC instrument (CCC1, total capacity: 1600 ml) was used as the first dimension. Effluent of interest from CCC1 was collected on-line into a 30 ml sample loop by a laboratory-prepared column-switching interface and introduced into a high-speed CCC instrument (CCC2, total capacity: 210 ml) for the second dimension separation. With this 2D-CCC system and a pair of two-phase solvent systems composed of n-hexane-ethyl acetate-methanol-water (5:5:7:3 and 5:5:6.5:3.5, v/v/v/v), which had been selected by high-speed CCC, about a 500 mg amount of the crude extract was separated, yielding 9 mg of compound 1, 28 mg of compound 2 and 78 mg of compound 3. The purities of the three prenylflavonoids were 98.7 (1), 98.3 (2) and 97.2% (3), respectively, as determined by HPLC analysis. Their chemical structures were identified by electrospray ionization MS, (1)H NMR and (13)C NMR.     

Purification of (+)-dihydromyricetin from leaves extract of Ampelopsis grossedentata using high-speed countercurrent chromatograph with scale-up triple columns

Purification of (+)-dihydromyricetin from an extract (16 g) of leaves of Ampelopsis grossedentata was performed using a preparative triple-column countercurrent chromatograph. With a solvent system composed of n-hexane-ethyl acetate-methanol-water (1:3:2:4, v/v) 11.3 g of (+)-dihydromyricetin was obtained at a high purity of over 99% by HPLC at 254 nm in 9 h.     

Preparative isolation and purification of five compounds from the Chinese medicinal herb Polygonum cuspidatum Sieb. et Zucc by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) was applied to the separation and purification of five compounds from the Chinese medicinal herb Polygonum cuspidatum Sieb. et Zucc. The crude extracts from P. cuspidatum Sieb. et Zucc were treated with light petroleum-ethyl acetate-methanol-water (2:5:4:6, v/v). Sample 1 was obtained from the lower phase and sample 2 from the upper phase. The sample 1 was separated with light petroleum-ethyl acetate-water (1:5:5, v/v) and yielded 19.3mg of piceid, 17.6 mg of anthraglycoside B from 200mg of sample 1. The sample 2 was separated with light petroleum-ethyl acetate-methanol-water (3:5:4:6, v/v) and light petroleum-ethyl acetate-methanol-water (3:5:7:3, v/v) in a gradient elution and yielded 18.5mg of resveratrol, 35.3mg of emodin and 8.2mg of physcion from 220 mg of sample 2. The purity of each compound is over 95% as determined by HPLC. The chemical structures of these components were identified by (1)H NMR and (13)C NMR.     

Semi-industrial isolation of salicin and amygdalin from plant extracts using slow rotary counter-current chromatography

Salicin in the bark extract of Salix alba and amygdalin in the fruit extract of Semen armeniacae were each separated by slow rotary counter-current chromatography (SRCCC). The apparatus was equipped with a 40-L column made of 17 mm i.d. convoluted Teflon tubing. A 500g amount of crude extract containing salicin at 13.5% was separated yielding 63.5 g of salicin at 95.3% purity in 20h using methyl tert-butyl ether-l-butanol (1:3) saturated by methanol-water (1:5) as a stationary phase and methanol-water (1:5) saturated by methyl tert-butyl ether-1-butanol (1:3) as a mobile phase. A 400g amount of crude extract containing amygdalin at 55.3% was isolated to yield 221.2g of amygdalin at 94.1% purity in 19h using ethyl acetate-1-butanol (1:2) saturated by water as a stationary phase and water saturated by ethyl acetate-1-butanol (1:2) as a mobile phase. The flow rate of the mobile phase was 50 ml/min. The results show that industrial SRCCC separation of salicin and amygdalin is feasible using a larger column at a higher flow rate of the mobile phase.     

SEPARATION OF THE MINOR FLAVONOLS FROM FLOS GOSSYPII BY HIGH-SPEED COUNTERCURRENT CHROMATOGRAPHY

An effective high-speed countercurrent chromatography (HSCCC) method was established for further separation and purification of four minor flavonols in addition to five major flavonols which were reported by our previous study from extracts of Flos Gossypii. HSCCC was performed with three two-phase solvent systems composed of n-hexane-ethyl acetate-methanol-water (7.5:15:6:7, v/v), (2.5:15:2:7, v/v) and (0:1:0:1, v/v). The separation was repeated 3 times, and 3.8 mg of 8-methoxyl-kaempferol-7-O-β-D-rhamnoside (HPLC purity 98.27%), 6.7 mg of astragalin (HPLC purity 94.18%), 3.3 mg of 4'-methoxyl-quercetin-7-O-β-D-glucoside (HPLC purity 94.30%) and 8.2 mg of hyperoside (HPLC purity 93.48%) were separated from 150 mg of the crude sample. The chemical structures of the flavonols were confirmed by MS, (1)H NMR and (13)C NMR. Meanwhile, the results indicated that the target compound with smaller K value (<0.5) can be separated by increasing column length of HSCCC. And four separation rules of flavonols according to the present study and references were summarized, which can be used as a useful guide for separation of flavonols by HSCCC.     

A preparative isolation and purification of arctigenin and matairesinol from Forsythia koreana by centrifugal partition chromatography

Centrifugal partition chromatography was applied to separate arctigenin and matairesinol from Forsythia koreana extract with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (5:5:5:5 v/v). Using this method, arctigenin and matairesinol were successfully separated from partially purified F. koreana extracts in only one step. The purities of isolated compounds were determined to be over 90% by HPLC analysis.     

Determination of Gingerols in Ginger by Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry

A rapid and sensitive ultra-high performance liquid chromatography–tandem mass spectrometry method was developed and validated for the determination of 6-, 8-, and 10-gingerol in the rhizomes of Zingiber officinale Rosc. The chromatographic separation was achieved isocratically on a C₁₈ (2.1 mm × 100 mm; 1.7 µm) column using acetonitrile:water (90:10 v/v) at a flow rate of 0.25 mL/min. Mass spectrometric transitions for 6-gingerol, 8-gingerol, and 10-gingerol occurred at m/z 277.2467/177.1470, 305.3196/177.1617, and 333.3614/177.1666, respectively. The analysis time was 3.0 min and the elution of 6-, 8-, and 10- gingerol occurred at 1.27, 1.51, and 1.80 min. The linear dynamic range was established over the concentration range of 2 and 1000 ng/mL for the analytes. The limits of detection and quantitation for 6-gingerol, 8-gingerol, and 10-gingerol were 0.921, 0.856, and 1.069 ng/mL, and 2.951, 2.727, and 3.013 ng/mL, respectively. The relative standard deviation and accuracy were found to be satisfactory. The results showed significant variation (0.176%–0.290% w/w) in gingerols among twelve accessions and that the Patna and Lucknow varieties contained the highest concentrations and were concluded to be superior. These results may be used to devise strategies for cultivating this plant for large scale production.     

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.     

Preparative separation of flavonoid glycosides in leaves extract of Ampelopsis grossedentata using high-speed counter-current chromatography

Preparative separation of flavonoid glycosides in leaves extract of Ampelopsis grossedentata was conducted using high-speed counter-current chromatograph (HSCCC) with a solvent system composed of n-hexane-ethyl acetate-methanol-water (1:6:1.5:7.5, v/v). In a single operation, 28 mg of 5,7-dihydroxy-3',4'-trihydroxyflavone-3-O-6'-rhamnose and 18 mg of 5,7-dihydroxy-3',4'-dihydroxyflavone-3-O-6'-rhamnose was obtained from 150 mg of the extract. The chemical structure of the two compounds was elucidated by electrospray ionization (EIS) MS and NMR.     

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.     

Isolation of three sesquiterpene lactones from the roots of Cichorium glandulosum Boiss. et Huet. by high-speed counter-current chromatography

Because of the skeletal complexity and similarity of the polarity, little research was reported on the isolation of sesquiterpene lactones by high-speed counter-current chromatography (HSCCC). Herein, three sesquiterpene lactones were successfully purified from the ethyl acetate extract of the roots of the traditional Uyghur medicinal plant Cichorium glandulosum Boiss. et Huet. by HSCCC. The separation was performed in two steps with two solvent systems: n-hexane-ethyl acetate-methanol-water (1.5:5:2.75:5, v/v/v/v) and ethyl acetate-methanol-water (20:1:20, v/v/v). From 166 mg of the ethyl acetate extract, 19 mg of lactucopicrin was isolated with the first solvent system and 10 mg of 11beta,13-dihydrolactucin and 16 mg of lactucin were obtained with the second solvent system. All purified compounds were over 94% purity as determined by HPLC analysis, and these chemical structures were confirmed by (1)H NMR and (13)C NMR.     

Liquid chromatographic determination of 6-, 8-, 10-gingerol, and 6-shogaol in ginger (Zingiber officinale) as the raw herb and dried aqueous extract

The determination of 6-, 8-, 10-gingerol, and 6-shogaol in dried ginger (Zingiber officinale) and in the dried aqueous extract of ginger is reported. This is the first study to report a validated method for the determination of these 4 analytes. Several extraction solvents and methods were examined, and the optimum combination was determined. The samples were extracted at room temperature by sonication with methanol, and the extract was analyzed by liquid chromatography with photodiode array detection. A C18 column was used with a water-acetonitrile gradient mobile phase. Quantification was at 200 nm. The levels of 6-, 8-, 10-gingerol, and 6-shogaol in the raw herb were 9.3, 1.6, 2.3, and 2.3 mglg, respectively. The levels of gingerols found in the dried aqueous extract were between 5 and 16 times lower than those in the raw herb, but the level of 6-shogaol was higher. Analyte identity was confirmed by negative-ion electrospray ionization tandem mass spectrometry, in which 2 daughter ions were obtained for each analyte. The average recovery was 97% with a relative standard deviation of <8%. The limits of detection for 6-, 8-, 10-gingerol, and 6-shogaol in the raw herb were 0.22, 0.04, 0.09, and 0.07 mglg, respectively, and in the dried aqueous extract, 0.11, 0.02, 0.02, and 0.14 mg/g, respectively.