Preparative separation of high-purity cordycepin from Cordyceps militaris(L.) Link by high-speed countercurrent chromatography

A high-speed counter-current chromatography (HSCCC) technique in a preparative scale has been applied to separate and purify cordycepin from the extract of Cordyceps militaris(L.) Link by a one-step separation. A high efficiency of HSCCC separation was achieved on a two-phase solvent system of n-hexane-n-butanol-methanol-water (23:80:30:155, v/v/v/v) by eluting the lower mobile phase at a flow rate of 2 ml/min under a revolution speed of 850 rpm. HSCCC separation of 216.2 mg crude sample (contained cordycepin at 44.7% purity after 732 cation-exchange resin clean-up) yielded 64.8 mg cordycepin with purity of 98.9% and 91.7% recovery. Identification of the target compound was performed by UV, IR, MS, (1)H NMR and (13)C NMR.     

Preparative isolation and purification of bergapten and imperatorin from the medicinal plant Cnidium monnieri using high-speed counter-current chromatography by stepwise increasing the flow-rate of the mobile phase

A high-speed counter-current chromatography (HSCCC) method was developed for the preparative separation and purification of bergapten and imperatorin from the Chinese medicinal plant Cnidium monnieri (L.) Cusson. The crude extract was obtained by extraction with ethanol from the dried fruits of Cnidium monnieri (L.) Cusson under sonication. Preparative HSCCC with a two-phase solvent system composed of n-hexane-ethyl acetate-ethanol-water (5:5:5:5, v/v/v/v) was successfully performed by increasing the flow-rate of the mobile phase stepwise from 1.0 to 2.0 ml min(-1) after 180 min. The components purified and collected were analyzed by high-performance liquid chromatography. The method yielded 45.8 mg of bergapten at 96.5% purity and 118.3 mg of imperatorin at 98.2% purity from 500 mg of the crude extract in a single run. The recoveries of bergapten and imperatorin were 92.1 and 93.7%, respectively.     

硅胶柱色谱结合高速逆流色谱法分离纯化荷花中黄酮类化合物

采用硅胶柱色谱结合高速逆流色谱法分离纯化了荷花中3种黄酮类化合物。荷花粗提物先经过硅胶柱色谱初步分离,得到黄酮含量高的组分,再经过高速逆流色谱分离,以乙酸乙酯-乙醇-水-乙酸(4:1:5:0.025, 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、流速2.0 mL/min、检测波长254 nm条件下,从150 mg样品中一次性分离制备得到6.1 mg槲皮素-3-O-β-D-葡萄糖醛酸苷(I), 14.8 mg杨梅素-3-O-β-D-葡萄糖苷(II)和20.2 mg紫云英苷(III),经高效液相色谱检测其纯度分别为97.0%、95.4%、96.3%,并通过质谱和核磁共振氢谱、碳谱鉴定各化合物的结构。该方法简便、快速、节省溶剂,可以对荷花中的黄酮类化合物进行快速有效的分离纯化,具有较好的实用价值,为荷花资源的进一步开发应用提供了参考依据。     

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     

正交设计试验优化高速逆流色谱分离制备玛咖中芥子油苷的条件

基于高速逆流色谱（HSCCC）技术从玛咖中分离制备出两种芥子油苷,苄基芥子油苷（glucotropaeolin, GTL）和甲氧基苄基芥子油苷（glucolimnanthin, GLI）。使用正交设计试验对分离条件进行优化,采用高分辨质谱对制备的组分进行鉴定,采用高效液相色谱法（HPLC）对组分进行定量分析。确定了两个组分GTL与GLI的HSCCC最佳分离条件：溶剂系统为正丁醇-乙腈-200 g/L硫酸铵溶液（1:0.5:2.4, v/v/v）,上相为固定相,下相为流动相,流动相流速2 mL/min,主机转速900 r/min,从玛咖根粗提物中一次性分离得到157.72 mg/kg纯度为97.9%的苄基芥子油苷和31.93 mg/kg的甲氧基苄基芥子油苷,固定相保留率达57.6%。该方法成本低,简便易行,样品损失量小,可大量循环进样制备。     

Isolation and purification of baicalein, wogonin and oroxylin A from the medicinal plant Scutellaria baicalensis by high-speed counter-current chromatography

The medicinal plant Scutellaria baicalensis Georgi has been used widely in traditional Chinese medicine for anti-inflammation, anticancer, antiviral and antibacterial infections, reducing the total cholesterol level and decreasing blood pressures. A high-speed counter-current chromatography (HSCCC) method was developed for the preparative separation and purification of three bioactive flavonoids, namely, baicalein, wogonin and oroxylin A, from S. baicalensis Georgi. Preparative HSCCC with a two-phase solvent system composed of n-hexane-ethyl acetate-n-butanol-water (1:1:8:10, v/v/v/v) was successfully performed by increasing the flow-rate of the mobile phase stepwise from 1.0 to 2.0 ml min(-1) after 4 h. The components purified and collected were analyzed by high-performance liquid chromatography. The method yielded 144.8 mg of baicalein at 95.7% purity, 50.2 mg of wogonin at 98.5% purity, and 12.4 mg of oroxylin A at 93.2% purity from 500 mg of the crude extract in a one-step separation. The recoveries of baicalein, wogonin and oroxylin A were 92.7%, 91.6% and 92.5%, respectively.     

半制备型高速逆流色谱分离制备铁棒锤根中的一种咪唑生物碱

采用高速逆流色谱（HSCCC）技术从铁棒锤根氯仿提取物中分离制备了一种高纯度咪唑类生物碱1H-imidazole-2-carboxylic acid,butyl ester （ICABE）。采用高效液相色谱（HPLC）测定目标化合物在两相溶剂中的分配系数,优化HSCCC分离ICABE的溶剂体系,确定了以正己烷-氯仿-乙醇-水（10：1：13：2,v/v/v/v）为HSCCC的两相溶剂系统,以上相为固定相,下相为流动相,流动相流速为1.8 mL/min,主机转速850 r/min,检测波长为230 nm条件下进行分离制备,在350 min内从100 mg粗样品中一步分离得到7.5 mg ICABE,经HPLC检测其纯度达98%以上（峰面积归一化法）,结构由UV、¹H-NMR和¹³C-NMR得以鉴定。该方法简便、快速,所得产物纯度高,适合于铁棒锤中ICABE的制备分离。     

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。     

Preparative high-speed counter-current chromatography for purification of shikonin from the Chinese medicinal plant Lithospermum erythrorhizon

The bioactive compound shikonin was successfully isolated and purified from the crude extract of the traditional Chinese medicinal plant Lithospermum erythrorhizon Sieb. et Zucc. by preparative high-speed counter-current chromatography (HSCCC). The preparative HSCCC was performed using a two-phase solvent system composed of n-hexane-ethylacetate-ethanol-water (16:14:14:5 (v/v)). A total amount of 19.6 mg of shikonin at 98.9% purity was obtained from 52 mg of the crude extract (containing 38.9% shikonin) with 96.9% recovery. The preparative isolation and purification of shikonin by HSCCC was completed in 200 min in a one-step separation.     

Allan J. Bard and Cynthia G. Zoski (Eds): Electroanalytical Chemistry

Coupled with evaporative light scattering detection, a high-speed counter-current chromatography (HSCCC) method was applied to the separation and purification of three tauro-conjugated cholic acids of taurochenodeoxycholic acid (TCDCA), taurohyodeoxycholic acid (THDCA) and taurohyocholic acid (THCA) from Pulvis Fellis Suis (Pig gallbladder bile) for the first time. The two-phase solvent system composed of chloroform-methanol-water-acetic acid (4:4:2:0.3, v/v/v/v) was selected for the one-step separation where the lower phase was used as the mobile phase in the head to tail elution mode. The revolution speed of the separation column, flow rate of the mobile phase and separation temperature were 800 rpm, 1.5 ml/min and 25°C respectively. From 100 mg of the crude extract, 10.2 mg of TCDCA, 11.8 mg of THDCA and 5.3 mg of THCA were obtained with the purity of 94.6%, 96.5% and 95.4%, respectively. in one step separation The HSCCC fractions were analyzed by high-performance liquid chromatography (HPLC) and the structures of the three tauro-conjugated cholic acids were identified by ESI-MS, (1)H NMR and (13)C NMR.     

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。     

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.     

高速逆流色谱法快速分离制备枸杞中莨菪亭

建立了用高速逆流色谱(HSCCC)从枸杞中快速分离莨菪亭的方法。将枸杞的乙醇提取物经D-101大孔树脂初步纯化后直接进行高速逆流色谱分离,用薄层色谱-荧光法考察了莨菪亭在不同溶剂体系中的分配情况。结果表明,最佳的溶剂体系为氯仿-甲醇-水(10:7:3, v/v/v),取上相为固定相,下相为流动相,在主机转速为850 r/min、流速为1.5 mL/min、检测波长为365 nm的条件下,从200 mg样品中一次性分离制备可得到10.2 mg纯度达到98.3%的莨菪亭。制备所得的莨菪亭与对照品的高效液相色谱(HPLC)保留时间一致,且经核磁共振氢谱、碳谱鉴定结构;纯度经HPLC法测定。研究发现,氯仿-甲醇-水(10:7:3, v/v/v)体系可连续二次进样而样品的峰形未受明显的影响。实验结果表明用薄层色谱-荧光法可快速选定HSCCC溶剂体系,进而可快速、简便地制备高纯度的莨菪亭。     

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.     

Isolation and purification of inflacoumarin A and licochalcone A from licorice by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) technique in semi-preparative scale has been applied to isolate and purify bioactive flavone compounds from the ethanol extract of Glycyrrhiza inflata Bat., a particular plant species of licorice. HSCCC separation was performed with a two-phase solvent system composed of n-hexane-chloroform-methanol-water (5:6:3:2, v/v) by eluting the lower mobile phase at a flow rate of 1.8 ml/min and a revolution speed of 800 rpm. Purification was performed with a two-phase solvent system composed of n-hexane-chloroform-methanol-water (1.5:6:3:2, v/v) by eluting the lower mobile phase at a flow-rate of 1.5 ml/min and a revolution speed of 800 rpm. Two major flavone peaks: inflacoumarin A and licochalcone A were collected and the respective yields of the peaks amount to 6 mg (8.6%, w/w) and 8 mg (11.4%, w/w) from 70 mg of the crude extract sample. The purities of inflacoumarin A and licochalcone A reached 99.6% and 99.1%, respectively, after a sequential purification run. The structures of inflacoumarin A and licochalcone A were positively confirmed by 1H NMR and 13C NMR, 1H-13C-COSY, UV, FT-IR and electron ionization MS analyses.     

Isolation and purification of flavonoid glycosides from Trollius ledebouri using high-speed counter-current chromatography by stepwise increasing the flow-rate of the mobile phase

Three flavonoid glycosides including orientin, vitexin, quercetin-3-O-neohesperidoside and one unknown compound were isolated and purified by high-speed counter-current chromatography (HSCCC) and semi-preparative HPLC from Trollius ledebouri Reichb., a traditional Chinese medicine. Preparative HSCCC with a two-phase solvent system composed of ethyl acetate-n-butanol-water (2:1:3, v/v/v) was successfully performed by increasing the flow-rate of the mobile phase from 1.5 to 2.5 ml/min after 190 min. Consequently, 95.8 mg orientin, 11.6 mg vitexin, 9.3 mg unknown compound with purities of over 97% and one partially purified peak fraction (contained quercetin-3-O-neohesperidoside at 85.1% purity) were obtained from 500 mg of the crude extract. Then the partially purified fraction was further purified by reversed-phase semi-preparative high-performance liquid chromatography. The structure identification of all pure fractions was carried out by UV, MS, 1H NMR and 13C NMR.     

土豆叶中茄尼醇的高速逆流色谱法分离纯化及质谱解析

采用超微回流提取方法提取土豆叶中的茄尼醇,用高速逆流色谱(HSCCC)对粗提物中的茄尼醇进行分离纯化。以正己烷-甲醇(体积比为10∶7)作为两相溶剂系统,以其下相为流动相,上相为固定相,经过一步HSCCC从60 mg茄尼醇粗提物中分离得到了5 mg 纯度为98.7%的茄尼醇;对分离得到的茄尼醇进行大气压化学电离质谱解析,研究了茄尼醇的大气压化学电离质谱的一级电离规律和二级质谱裂解规律。     

Preparative isolation and purification of phillyrin from the medicinal plant Forsythia suspensa by high-speed counter-current chromatography

Forsythia suspensa (Thunb.) Vahl. has been used widely in traditional medicines to treat gonorrhea, erysipelas, inflammation, pyrexia and ulcer. It has also shown antioxidant activity, as well as antibacterial, antiviral, choleretic and antiemetic effects. A high-speed counter-current chromatography (HSCCC) method was developed for the preparative separation and purification of the bioactive molecule phillyrin from F. suspensa (Thunb.) Vahl. The crude phillyrin was obtained by extraction with 50% ethanol from the dried fruits of F. suspensa (Thunb.) Vahl. under sonication. Preparative HSCCC with a two-phase solvent system composed of n-hexane-ethyl acetate-ethanol-water (1:9:1:9, v/v/v/v) was successfully performed, and the components purified and collected were analyzed by high-performance liquid chromatography. The method yielded 5.6 mg phillyrin at 98.6% purity from 500 mg of the crude extract (1.2% phillyrin) with the recovery of 92% in a one-step separation.     

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.     

Separation and purification of baicalin and wogonoside from the Chinese medicinal plant Scutellaria baicalensis Georgi by high-speed counter-current chromatography

A preparative high-speed counter-current chromatography (HSCCC) method for isolation and purification of baicalin and wogonoside from the Chinese medicinal plant Scutellaria baicalensis Georgi (Huang-qin in Chinese) was successfully established by using ethyl acetate-methanol-1% acetic acid water (5:0.5:5, v/v) as the two-phase solvent system. The upper phase of ethyl acetate-methanol-1% acetic acid water (5:0.5:5, v/v) was used as the stationary phase of HSCCC. Baicalin (58.1 mg) and wogonoside (17.0mg) with the purity of 99.2 and 99.0%, respectively, were separated successfully in one-step separation from 120 mg of crude sample from S. baicalensi, Georgi. The structures of baicalin and wogonoside were identified by 1H NMR and 13C NMR.