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.     

工业制备高效液相色谱法制备淫羊藿中的黄酮系列对照品

淫羊藿甙和朝藿定A、B、C是淫羊藿中重要的活性成分,本研究应用工业制备高效液相色谱从淫羊藿粗提物中分离制备了这4个成分。淫羊藿粗提物经大孔吸附树脂粗分离获得相应的组分后,利用工业制备高效液相色谱完成精制纯化。采用自装填Chromatorex C18制备柱(220 mm×77 mm, 10 μm),乙腈-水(26:74或30:70, v/v)为流动相进行洗脱,在35 min内,实现了这4种成分的基线分离及规模制备。从300 g粗提物(总黄酮含量约20%)中获得淫羊藿甙33 g、朝藿定C 4.6 g、朝藿定B 3.7 g和朝藿定A 0.6 g,产品纯度均达到98%以上。此方法通过两步分离即可实现这4种成分的完全分离,具有快速高效、产品纯度高的特点,适于淫羊藿中淫羊藿甙、朝藿定A、B、C系列对照品的规模制备。     

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.     

Selective on-line extraction of trans-resveratrol and emodin from Polygonum cuspidatum using molecularly imprinted polymer

High-performance liquid chromatographic separation is performed to extract active components from the traditional Chinese medicine Polygonum cuspidatum using a trans-resveratrol imprinted polymer. Good separation and purification of trans-resveratrol and emodin from the Polygonum cuspidatum extract are achieved after condition optimization. The extraction recoveries are 83% and 99% for trans-resveratol and emodin, respectively. The results show that the molecularly imprinted polymer can be used as a selective extraction material for the extraction and purification of trans-resveratrol and emodin from Polygonum cuspidatum.     

Antiproliferative activity of Luehea candicans Mart. et Zucc. (Tiliaceae)

Luehea candicans Mart. et Zucc. (Tiliaceae) is known as 'a?oita-cavalo' and is one of the most important medicinal plants found in the Brazilian cerrado. The crude methanolic extracts of the branches and leaves and their fractions were evaluated using the following cancer cell lines: MCF-7 (breast), NCI-ADR (breast expressing the multidrug resistance phenotype), NCI-460 (lung), UACC-62 (melanoma), 786-0 (kidney), OVCAR (ovarian), PCO-3 (prostate), HT-29 (colon) and K-562 (leukaemia). The crude methanolic extracts from the branches (B) and leaves (L) were able to inhibit the growth of the K-562 and 786-0 cell lines in a dose-dependent manner, with GI(50) values of 8.1 and 5.4 μg mL(-1), respectively. The hexane (L1), chloroform (L2) and methanol (L4) fractions derived from extract L showed a high selectivity and pronounced cytostatic activity against 786-0 (GI(50) ~ 40 μg mL(-1)). A significant amount of lupeol was isolated from fraction L2. The chloroform (B2) and methanol (B3) fractions derived from extract (B) exhibited less selectivity, showing the highest cytostatic activity against K-562, NCI-ADR, OVCAR, MCF-7 and NCI-460 cells, with GI(50) values between 27 and 40 μg mL(-1). Lupeol, betulin, a mixture of steroids, (-)-epicatechin, vitexin and liriodendrin were isolated from these active fractions.     

Supercritical CO2 extraction of emodin and physcion from Polygonum cuspidatum and subsequent isolation by semipreparative chromatography

Emodin and physcion are abundant anthraquinone compounds found in the traditional Chinese medicinal herb Polygonum cuspidatum Sied. et Zucc. In this paper, emodin and physcion were successfully extracted with supercritical CO2 plus ethanol modifier after the extraction conditions were optimized with uniform design-sequential optimization. Results showed that the ethanol modifier concentration was the main factor for the effective extraction of the emodin. The optimal extraction condition was obtained: 20 MPa, 30 degrees C, and 95% ethanol, at which the yields of emodin and physcion were 0.616 and 0.178 g/100 g, respectively. The yield obtained by supercritical fluid extraction (SFE) was a little lower than that obtained by sonication extraction (SE). The crude extract obtained by SFE was further isolated and purified by semipreparative chromatography with the mobile phase composed of methanol-water (90:1, v/v). Emodin and physcion were obtained with purity 98.6 and 99.1%, respectively, when determined by HPLC, and identification was performed by retention time and UV spectra of the standards. The result suggested that SFE is an alternative and promising method for extraction of the two compounds from P. cuspidatum owing to its environment-friendly properties and fewer coextracts.     

Separation of three phenolic high‐molecular‐weight compounds from the crude extract of Terminalia Chebula Retz. by ultrasound‐assisted extraction and high‐speed counter‐current chromatography

This study presents an efficient strategy for separation of three phenolic compounds with high molecular weight from the crude extract of Terminalia chebula Retz. by ultrasound‐assisted extraction and high‐speed counter‐current chromatography. The ultrasound‐assisted extraction conditions were optimized by response surface methodology and the results showed the target compounds could be well enriched under the optimized extraction conditions. Then the crude extract was directly separated by high‐speed counter‐current chromatography without any pretreatment using n‐hexane/ethyl acetate/methanol/water (1:7:0.5:3, v/v/v/v) as the solvent system. In 180 min, 13 mg of A, 18 mg of B, and 9 mg of C were obtained from 200 mg of crude sample. Their structures were identified as Chebulagic acid (A, 954 Da), Chebulinic acid (B, 956 Da), and Ellagic acid (C) by ¹H NMR spectroscopy.     

Purification of salvianolic acid B from the crude extract of Salvia miltiorrhiza with hydrophilic organic/salt-containing aqueous two-phase system by counter-current chromatography

Establishment of hydrophilic organic/salt-containing aqueous two-phase system and purification of salvianolic acid B from crude extract of S. miltiorrhiza by counter-current chromatography with said system were studied. Ethanol and n-propanol were selected to constitute biphasic systems with ammonia sulphate, sodium chloride and phosphate separately, and related system characteristics including phase diagrams, phase ratio, separation time were tested. The partition coefficient of crude salvianolic acid B was also tested in above systems and further finely adjusted by altering the constitution of phosphate in n-propanol/phosphate system. Salvianolic acid B was purified to 95.5% purity by counter-current chromatography in 36% (w/w) n-propanol/8% (w/w) phosphate system with the ratio between dipotassium hydrogen phosphate and sodium dihydrogen phosphate of 94:6. One hundred and eight milligrams of salvianolic acid B was purified from 285 mg crude extract with the recovery of 89%.     

Isolation of the minor and rare constituents from fruits of Peucedanum alsaticum L. using high-performance counter-current chromatography

A high-performance counter-current chromatography (HPCCC) method was applied for the first time for the preparative separation and purification of three rare compounds which occur as minor constituents in the fruits of Peucedanum alsaticum L.: 5-substituted coumarin notoptol and two dihydropyranochromones: divaricatol and ledebouriellol. A scale-up process from analytical to preparative in a very short time was developed. In order to purify a range of rare and minor compounds with different polarity two separate experiments were performed, one in reverse phase, the other in normal phase, using the same crude extract. A two-phase solvent system composed of n-hexane/ethyl acetate/methanol/water (1:1:1:1) was developed. The components purified and collected were analyzed by high-performance liquid chromatography. The method yielded 0.7 mg of notoptol, 1.46 mg of ledebouriellol at purity of 99.5%, and 10 mg of mixtures of divaricatol, alsaticol and alsaticocoumarin, where divaricatol present 22% by peak area. These amounts were obtained from 1 g of the crude extract in a single run. This is the first time when minor notoptol, ledebouriellol, and divaricatol were isolated in a single run using HPCCC method and first time when these were identified in plant from Peucedanum genus.     

A simple method to optimize the HSCCC two-phase solvent system by predicting the partition coefficient for target compound

A simple method was developed to optimize the solvent ratio of the two-phase solvent system used in the high-speed counter-current chromatography (HSCCC) separation. Some mathematic equations, such as the exponential and the power equations, were established to describe the relationship between the solvent ratio and the partition coefficient. Using this new method, the two-phase solvent system was easily optimized to obtain a proper partition coefficient for the CCC separation of the target compound. Furthermore, this method was satisfactorily applied in determining the two-phase solvent system for the HSCCC preparation of pseudolaric acid B from the Chinese herb Pseudolarix kaempferi Gordon (Pinaceae). The two-phase solvent system of n-hexane/EtOAc/MeOH/H(2)O (5:5:5:5 by volume) was used with a good partition coefficient K = 1.08. As a result, 232.05 mg of pseudolaric acid B was yielded from 0.5 g of the crude extract with a purity of 97.26% by HPLC analysis.     

Thiophene derivatives with antileishmanial activity isolated from aerial parts of Porophyllum ruderale (Jacq.) Cass

Porophyllum ruderale (Jacq.) Cass. is a plant native to Brazil and in the northwest region of the state of Paraná, Brazil, aerial parts of P. ruderale have been used popularly in the treatment of lesions caused by Leishmania sp.. In this study the antileishmanial and cytotoxic activities of the crude extract, fractions, and isolated compounds from aerial parts of P. ruderale was evaluated. The dichloromethane extract was submitted to chromatography to yield compounds active against Leishmania amazonensis. Their structures were established by comparison of their spectroscopic data with literature values. The activities of crude extract against promastigote and axenic amastigote forms of L. amazonensis (IC(50)) were 60.3 and 77.7 μg/mL, respectively. Its cytotoxic activity against macrophage cells (CC(50)) was 500 μg/mL. The thiophene derivatives isolated were: 5-methyl-2,2':5',2"-terthiophene (compound A) and 5'-methyl-[5-(4-acetoxy-1-butynyl)]-2,2'-bithiophene (compound B). The activity of compound A against promastigote and axenic amastigote forms were 7.7 and 19.0 μg/mL and of compound B were 21.3 and 28.7 μg/mL, respectively. The activity of the isolated compounds against promastigote and axenic amastigote forms was better than that of the crude extract and more selective against protozoa than for macrophage cells.     

Rapid separation of three glucosylated resveratrol analogues from the invasive plant Polygonum cuspidatum by high‐speed countercurrent chromatography

Three glucosylated resveratrol analogues (piceid, piceatannol glucoside, resveratroloside) were successfully isolated from the crude MeOH extract of the invasive plant species Polygonum cuspidatum by semi‐preparative high‐speed countercurrent chromatography with a two‐phase solvent system composed of cyclohexane‐ethyl acetate‐methanol‐water (1:5:1:5, v/v/v/v). Piceid (23 mg), resveratroloside (17 mg), piceatannol glucoside (15 mg) of purities over 80% were isolated from 500 mg crude MeOH extract in one step. Subsequent passage over a SPE column was used to quickly bring their purities to over 90%. The purities were determined by HPLC analysis and their structures were elucidated by proton nuclear magnetic resonance (¹H‐NMR), HMBC, ESI‐MS and HR‐MS.     

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。     

Preparative isolation and purification of six diterpenoids from the Chinese medicinal plant Salvia miltiorrhiza by high-speed counter-current chromatography

A high-speed counter-current chromatography (HSCCC) method was developed for the preparative separation and purification of six diterpenoids. dihydrotanshinone I, cryptotanshinone, methylenetanshiquinone, tanshinone I, tanshinone IIA and danshenxinkun B from the Chinese medicinal plant Salvia miltiorrhiza Bunge. The crude diterpenoids were obtained by extraction with ethanol-n-hexane (1:1, v/v) from S. miltiorrhiza Bunge. Preparative HSCCC with the two-phase solvent systems A composed of n-hexane-ethanol-water (10:5.5:4.5, v/v) and B composed of n-hexane-ethanol-water (10:7:3, v/v) was successfully performed in a stepwise elution yielding six relatively pure diterpenoids from 300 mg of the crude extract in a single run. The purities of dihydrotanshinone I, cryptotanshinone, methylenetanshiquinone, tanshinone I, tanshinone IIA and danshenxinkun B were 88.1, 98.8, 97.6, 93.5, 96.8 and 94.3%, respectively.     

Orthogonal test design for optimization of suitable conditions to separate C-phycocyanin from Spirulina platensis by high-speed counter-current chromatography using reverse micelle solvent system

High-speed counter-current chromatography (HSCCC) was applied to separate C-phycocyanin (C-PC) from Spirulina platensis in the article. The suitable conditions were optimized by an orthogonal test design (L(9)(3)(3)), including the stationary phase of reverse micelle solvent system (0.10 g/mL cetyltrimethylammonium bromide [CTAB]/isooctane-hexylalcohol), mobile phase A (0.05 mol/L sodium phosphate buffer, pH 4.0, containing 0.2 mol/L KCl) and mobile phase B (0.05 mol/L sodium phosphate buffer, pH 8.0, containing 0.4 mol/L KCl). Under the selected conditions, 78.7 mg protein was purified from 200 mg crude extract of S. platensis, and the purity of the product was 4.25 based on the absorbance ratio of A(620)/A(280) , which was increased 6.85 times compared with the crude extract. Then, the protein was identified to be C-PC by MALDI-TOF/TOF-MS and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis compared with the standard. The application of HSCCC used in the separation of C-PC from S. platensis was first reported in the article. Furthermore, three kinds of tumor cell lines including human hepatoma cell line SMMC-7721, human ovarian carcinoma cell line ES-2, and human lung adenocarcinoma cell line SPCA-1 were used to evaluate the anticancer activities of the separated product, and the results showed that the separated C-PC had excellent anti-tumor actions with the IC(50) values at 2.998, 4.854, and 8.423 μg/mL, respectively, for 48 h treatment. The outcome indicates that an effective method for C-PC purification by HSCCC has been established.     

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.     

Preparative isolation and purification of salvianolic acid B from the Chinese medicinal plant Salvia miltiorrhiza by high-speed counter-current chromatography

High-speed counter-current chromatography was applied to the isolation and purification of salvianolic acid B from the Chinese medicinal plant Salvia miltiorrhiza Bunge. The crude salvianolic acid B was obtained by extraction with ethanol-water from S. miltiorrhiza Bunge. Preparative high-speed counter-current chromatography with a two-phase solvent system composed of n-hexane-ethyl acetate-ethanol-water (3:7:1:9, v/v) was successfully performed yielding 342 mg salvianolic acid B at 98% purity from 500 mg of the crude extract in a one-step separation.     

Rapid and high-throughput purification of salvianolic acid B from Salvia miltiorrhiza Bunge by high-performance counter-current chromatography

A large-scale purification of salvianolic acid B from Salvia miltiorrhiza Bunge is presented. The method development began with selection of the solvent system, then optimization of the operating parameters and ended up with linear scale-up from an analytical to a preparative instrument. Three factors were used for method optimization and scale-up estimation: purity, process throughput and process efficiency. Preparation was achieved using a two-phase solvent system comprising hexane–ethyl acetate–methanol–acetic acid–water (1:5:1.5:0.00596:5, v/v). This preparation yielded 475 mg of salvianolic acid B with a purity of 96.1% from 1.5 g of crude extract. The process throughput of crude was 2.23 g/h while process efficiency per gram of target compound was 0.769 g/h. Two factors—process environmental risk factor and process evaluation factor were used for evaluation of the separation process.     

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。     

Preparative isolation and purification of salidroside from the Chinese medicinal plant Rhodiola sachalinensis by high-speed counter-current chromatography.

High-speed counter-current chromatography was applied to the isolation and purification of salidroside from the Chinese medicinal plant Rhodiola sachalinensis A. Bor. The crude salidroside was obtained by extraction with methanol from Rhodiola sachalinensis A. Bor. Preparative high-speed counter-current chromatography with a two-phase solvent system composed of n-butanol-ethyl acetate-water (2:3:5, v/v) was successfully performed yielding salidroside (32 mg) at 98% purity from 250 mg of the crude extract in a one-step separation.