Combination of supercritical fluid extraction with counter‐current chromatography to isolate anthocyanidins from the petals of Chaenomeles sinensis based on mathematical calculations

Supercritical fluid extraction (SFE) coupled with high‐speed counter‐current chromatography (HSCCC) was successfully used for the extraction and on‐line isolation of the anthocyanidins from the petals of Chaenomeles sinensis in two stages. The SFE parameters were optimized by an orthogonal test, and the solvent systems of SFE and HSCCC were calculated and optimized with the help of a multiexponential function model. In the first stage, the lower phase of the solvent system of n‐butanol/tert‐butyl methyl ether/acetonitrile/0.1% aqueous TFA (0.715:1.0:0.134:1.592, v/v/v/v) was used as both the SFE modifier and the HSCCC stationary phase, after extraction, the extractants were pumped into HSCCC column, and then eluted with the corresponding upper phase to isolate the moderately hydrophobic compounds. In the second stage, the upper phase of the solvent system of n‐butanol/ethyl acetate/acetonitrile/0.1% aqueous TFA (1.348:1.0:0.605:2.156, v/v/v/v) was used as both the SFE modifier and the HSCCC stationary phase, followed by elution with the corresponding lower phase to separate the hydrophobic compounds. With the help of two‐stage SFE/HSCCC, six compounds including delphinidin‐3‐O‐glucoside (Dp3G), cyanidin‐3‐O‐glucoside (Cy3G), peonidin‐3‐O‐glucoside (Pn3G), delphinidin (Dp), peonidin (Pn), and malvidin (Mv) were successfully separated within 300 min. The targeted compounds were identified by UV spectrophotometry, MS, and NMR spectroscopy. This research has opened up great prospects for the industrial application of SFE–HSCCC for the automatic extraction and separation of unstable compounds.     

Application of supercritical fluid extraction coupled with counter–current chromatography for extraction and online isolation of unstable chemical components from Rosa damascena

Supercritical fluid extraction (SFE) coupled with high‐speed counter‐current chromatography (HSCCC) was successfully used for the extraction and online isolation of the unstable compounds from Rosa damascene in a single extraction and separation operation in two stages. The solvent systems of SFE/HSCCC were optimized with the help of multiexponential function model. At the first stage, the upper phase of the solvent system of n‐butanol–tert‐butyl methyl ether–acetonitrile–0.1% aqueous TFA (1.7:1.0:0.8:4.0, v/v/v/v) was used as both the SFE entrainer and the HSCCC stationary phase, and the target compounds were eluted with the corresponding lower phase to separate the hydrophobic compounds. At the second stage, the upper phase of the solvent system of n‐hexane–ethyl acetate–methanol–water (3.2:1.0:2.8:2.6, v/v/v/v) was used as both the SFE entrainer and the HSCCC stationary phase, followed by elution with the corresponding lower phase to separate the moderate hydrophobic compounds. Six compounds including formononetin, delphinidin, cyaniding, 5,6,4′‐trihydroxy‐7,8‐dimethoxy flavone, 5,3′‐dihydroxy‐7,8‐dimethoxy flavone, and 5‐hydroxy‐6,7,8,3′,4′‐pentamethoxy flavone were successfully separated in one extraction–separation operation within 300 min. The targeted compounds were identified by MS and NMR spectroscopy. This research has opened up great prospects for industrial application of SFE/HSCCC to the extraction and separation of unstable compounds.     

高速逆流色谱法分离纯化续随子种子中的七叶内酯

建立了高速逆流色谱(HSCCC)技术分离纯化续随子种子中七叶内酯的方法。将续随子种子的乙酸乙酯萃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,最佳的溶剂系统为氯仿-甲醇-水(体积比为4:3:2),以其上相为固定相,下相为流动相。从200 mg续随子种子乙酸乙酯萃取物中分离得到80 mg七叶内酯,纯度为99.04%。HSCCC技术可高效分离纯化续随子种子中的七叶内酯,为得到高纯度的七叶内酯提供了制备技术。     

A consecutive preparation method based upon accelerated solvent extraction and high‐speed counter‐current chromatography for isolation of aesculin from Cortex fraxinus

A consecutive preparation method based upon accelerated solvent extraction (ASE) coupled with high‐speed counter‐current chromatography (HSCCC) was presented and aesculin was obtained from Cortex fraxinus. The extraction condition of ASE was optimized with response surface methodology; some significant parameters such as the solvent system and its stability, the amount of loading sample in HSCCC were also investigated. The original sample was first extracted with methanol at 105°C and 104 bar for 7 min using ASE, then the extracts were consecutively introduced into the HSCCC system and separated and purified with the same ethyl acetate/n‐butanol/water (7:3:10, v/v/v) solvent system for five times without further exchange and equilibrium. About 3.1 ± 0.2 mg/g in each time and total of 15.4 mg/g aesculin with purity over 95% was isolated from Cortex fraxinus. The results demonstrated that the consecutive preparation method was time and solvent saving and high throughput, it was suitable for isolation of aesculin from Cortex fraxinus, and also has good potential on the separation and purification of effective compounds from natural product.     

Characterization,Large-Scale HSCCC Separation and Neuroprotective Effects of Polyphenols from Moringa oleifera Leaves

Moringa oleifera leaves have been widely used for the treatment of inflammation, diabetes, high blood pressure, and other diseases, due to being rich in polyphenols. The main objective of this work was to largely separate the main polyphenols from Moringa oleifera leaves using the technique of high-speed counter-current chromatography (HSCCC). The phenolic composition in Moringa oleifera leaves was first analyzed qualitatively and quantitatively by UPLC-Q-Exactive Orbitrap/MS and UPLC-QqQ/MS, respectively, indicating that quercetin and kaempferol derivatives, phenolic acid and apigenin are the main polyphenols in Moringa oleifera leaves, with quercetin and kaempferol derivatives predominating. Furthermore, the conditions of HSCCC for large-scale separation of polyphenols from Moringa oleifera leaves were optimized, which included the selection of the solvent system, flow rate and the sample load. Only by one-step HSCCC separation (within 120 min) under the optimized conditions, six quercetin and kaempferol derivatives, a phenolic acid and an apigenin could be individually isolated at a large scale (yield from 10% to 98%), each of which possessed high purity. Finally, the isolated polyphenols and phenolic extract from Moringa oleifera leaves (MLPE) were verified to have strong neuroprotective activities against H₂O₂-induced oxidative stress in PC-12 cells, suggesting that these compounds would contribute to the main beneficial effects of Moringa oleifera leaves.     

Preparative Isolation of Imperatorin, Oxypeucedanin and Isoimperatorin from a Traditional Chinese Herb Using a HSCCC Strategy Based on Optimization of Rapid Flow Rate

Preparative high-speed counter-current chromatography has been used successfully for the isolation and purification of imperatorin, oxypeucedanin and isoimperatorin from traditional Chinese herb “bai zhi”—Angelica dahurica (Fisch. ex Hoffm) Benth. et Hook using high-speed counter-current chromatography (HSCCC). This was achieved in two stages. The first stage used a high flow HSCCC protocol with a two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water (HEMW) with volume ratios of 5:5:5:5, v/v which isolated isoimperatorin but co-eluted imperatorin and oxypeucedanin. The second stage used HEMW 5:5:4:6, v/v at low flow rate to resolve the co-eluted components from the first stage. The flow rate was optimized by preparative HSCCC. 300 mg of the crude extract was separated, yielding 18.5 mg of imperatorin, 8.3 mg of oxypeucedanin and 9.8 mg of isoimperatorin all at a high purity of over 98%.     

An Economical Method for Isolation of Dioscin from Dioscorea nipponica Makino by HSCCC Coupled with ELSD, and a Computer-Aided UNIFAC Mathematical Model

An economical method for isolation of dioscin from Dioscorea nipponica Makino by high-speed counter-current chromatography (HSCCC) was successfully developed by using a UNIFAC mathematical model coupled with computer-aided counter-current chromatography solvent-selection software (CCC-SSS) for separate preparation of the components of the solvent system (i.e., the stationary and mobile phases). The solvent system n-hexane–ethyl acetate–ethanol–water 2:5:2:5 (v/v) was selected to demonstrate the feasibility of the approach. A comparative study was also carried out on different methods for preparation of the solvent system, namely conventional preparation of the mobile and stationary phases together in the same vessel and the method developed for separate preparation of the phases. The results indicated that purity and recovery of dioscin were no different when solvent systems prepared by the different methods were used for HSCCC separation. Much less n-hexane, ethyl acetate, and ethanol was used when the mobile and stationary phases were prepared separately, however. This was not only environmentally sensible, but also enabled conservation of resources. Use of the UNIFAC mathematical model combined with the CCC-SSS technique for separate preparation of the components of the solvent system in HSCCC is reported and explained. It is a simple and economical means of isolating pure dioscin from Dioscorea nipponica Makino.     

Enrichment and isolation of barbigerone from Millettia pachycarpa Benth. using high‐speed counter‐current chromatography and preparative HPLC

Enrichment of the anti‐tumor compound barbigerone along with a rotenoid derivative from Millettia pachycarpa Benth. was performed by a two‐step high‐speed counter‐current chromatography (HSCCC) separation process. In the first step, 155.8 mg of target fraction (Fra6) was obtained from 400 mg ethyl acetate extract of M. pachycarpa Benth. with an increase in barbigerone from 5.1 to 13% via HSCCC using a solvent system of n‐hexane–ethyl acetate–methanol–water (5:4:5:3, v/v) under normal phase head to tail elution. HSCCC was repeated to eliminate the major contaminant in this initial fraction 6. After a separation time of 65 min, 22.1 mg barbigerone of 87.7% purity was obtained from Fra6 with the ternary solvent system of n‐hexane–methanol–water (2:2:1, v/v) under normal phase elution. Finally, preparative HPLC was employed for the further isolation of barbigerone and the rotenoid derivative. The structures were confirmed by ESI‐MS, ¹H NMR and ¹³C NMR.     

Separation of phenolic acids from sugarcane rind by online solid‐phase extraction with high‐speed counter‐current chromatography

Sugarcane rind contains some functional phenolic acids. The separation of these compounds from sugarcane rind is able to realize the integrated utilization of the crop and reduce environment pollution. In this paper, a novel protocol based on interfacing online solid‐phase extraction with high‐speed counter‐current chromatography (HSCCC) was established, aiming at improving and simplifying the process of phenolic acids separation from sugarcane rind. The conditions of online solid‐phase extraction with HSCCC involving solvent system, flow rate of mobile phase as well as saturated extent of absorption of solid‐phase extraction were optimized to improve extraction efficiency and reduce separation time. The separation of phenolic acids was performed with a two‐phase solvent system composed of butanol/acetic acid/water at a volume ratio of 4:1:5, and the developed online solid‐phase extraction with HSCCC method was validated and successfully applied for sugarcane rind, and three phenolic acids including 6.73 mg of gallic acid, 10.85 mg of p‐coumaric acid, and 2.78 mg of ferulic acid with purities of 60.2, 95.4, and 84%, respectively, were obtained from 150 mg sugarcane rind crude extracts. In addition, the three different elution methods of phenolic acids purification including HSCCC, elution–extrusion counter‐current chromatography and back‐extrusion counter‐current chromatography were compared.     

Separation and identification of polyphenols in apple pomace by high-speed counter-current chromatography and high-performance liquid chromatography coupled with mass spectrometry

Apple pomace, a by-product in the processing of apple juice, was investigated as a potential source of polyphenols. Two methods of separation and purification of polyphenols from apple pomace extract were established by combination of gel chromatography with high-speed counter-current chromatography (HSCCC) and solvent extraction with HSCCC, respectively. The optimal separation was performed on a Sephadex LH-20 column using gradient aqueous ethanol as eluting solvent from 0% to 100% in increments of 10%. HPLC analysis indicated that main polyphenols existed in fractions eluted between 40% and 50% aqueous ethanol. The fractions of interest from column were separated by HSCCC with the solvent system hexane–ethyl acetate–1% aqueous acetic acid (0.5:9.5:10, v/v/v). Ethyl acetate fractionation of the apple pomace extract followed by direct HSCCC separation by the same solvent system in the volume ratio of 1:9:10 also produced a good separation of the main polyphenols of interest. Six high-purity polyphenols were achieved tentatively and identified by HPLC/MS: chlorogenic acid (1, m/z 354), quercetin-3-glucoside/quercetin-3-glacaside (2, m/z 464), quercetin-3-xyloside (3, m/z 434), phloridzin (4, m/z 436), quercetin-3-arabinoside (5, m/z 434), and quercetin-3-rhamnoside (6, m/z 448). These results provided a preliminary foundation for further development and exploration of apple pomace.     

Isolation of the new minor constituents dihydropyranochromone and furanocoumarin from fruits of Peucedanum alsaticum L. by high-speed counter-current chromatography

A preparative high-speed counter-current chromatography (HSCCC) method was successfully used for isolation of two new minor compounds – alsaticol and alsaticocoumarin A. A two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water (1:1:1:1) was developed. Compounds were obtained from the dichloromethane extract of Peucedanum alsaticum fruits and their identification was performed with NMR and MS methods. Optimized HSCCC offers a rapid method of obtaining new natural compounds.     

Preparative Isolation and Purification of Three Sesquiterpenoid Lactones from <em>Eupatorium lindleyanum</em> DC. by High-Speed Counter-Current Chromatography

A high-speed counter-current chromatography (HSCCC) method was established for the preparative separation of three sesquiterpenoid lactones from Eupatorium lindleyanum DC. The two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (1:4:2:3, v/v/v/v) was selected. From 540 mg of the n-butanol fraction of Eupatorium lindleyanum DC., 10.8 mg of 3β-hydroxy-8β-[4'-hydroxy-tigloyloxy]-costunolide, 17.9 mg of eupalinolide A and 19.3 mg of eupalinolide B were obtained in a one-step HSCCC separation, with purities of 91.8%, 97.9% and 97.1%, respectively, as determined by HPLC. Their structures were further identified by ESI-MS and ¹H-NMR.     

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.     

Separation of tanshinones from Salvia miltiorrhiza Bunge by high-speed counter-current chromatography using stepwise elution

High-speed counter-current chromatography (HSCCC) was successfully used for isolation and purification of tanshinones from the roots of Salvia miltiorrhiza Bunge by stepwise elution. A set of three solvent systems and other experimental conditions were determined by analytical HSCCC. Using the optimized conditions, the preparative HSCCC separation was performed on 50 mg of crude light petroleum extract yielding pure tanshinones of tanshinone HA (7 mg), tanshinone I (3 mg) and cryptotanshinone (4 mg) all at purities of over 95% in a single run.     

Preparative separation of capsaicin and dihydrocapsaicin from Capsicum frutescens by high‐speed counter‐current chromatography

Capsaicin and dihydrocapsaicin are two main bioactive components of Capsicum frutescens and are widely used as food additives and drugs in China and India. Due to their similarity in structures, isolation of capsaicin and dihydrocapsaicin with traditional methods such as silica gel column chromatography, normal‐phase thin‐layer chromatography (TLC) becomes difficult. This study involves separating capsaicin and dihydrocapsaicin with sufficient purity and recovery using high‐speed counter‐current chromatography (HSCCC) with a solvent system composed of n‐hexane–ethyl acetate–methanol–water–acetic acid (20:20:20:20:2, v/v/v/v/v). Separation parameters such as sample volume, and sample concentration were first optimized on analytical HSCCC, and then scaled up to preparative HSCCC. 0.65 g capsaicin and 0.28 g dihydrocapsaicin were obtained from 1.2 g crude extract and their purities were 98.5 and 97.8%, respectively. The recoveries of the two compounds were 86.3 and 85.4%, respectively. The purity of the isolated compounds was analyzed by high‐performance liquid chromatography (HPLC) and their structures were identified by ¹H nuclear magnetic resonance (NMR) and ¹³C NMR analysis.     

Separation of abietane-type diterpenoids from Clerodendrum kaichianum Hsu by high-speed counter-current chromatography using stepwise elution

High-speed counter-current chromatography (HSCCC) was successfully used for the separation of abietane-type diterpenoids from the medicinal plant C. kaichianum, which were not separated in our previous study using preparative HPLC. The HSCCC separation employed the lower phases of n-hexane–ethyl acetate–methanol–water (HEMW) 4:5:4:5 and HEMW 4:5:5:4 as the mobile phase for stepwise elution while the upper phase of HEMW 4:5:4:5 was used as the stationary phase. HSCCC separation yielded 90.5 mg of compound 1(kaichianone A), 137.7 mg of compound 2 (kaichianone B), 125.0 mg of compound 3 (teuvincenone E), and 227.6 mg of compound 4 (taxusabietane A) with purities of 95.3%, 97.2%, 97.8%, and 98.6%, respectively, as determined by HPLC. Compounds 1–2 are two new abietane-type diterpenoids while Compounds 3–4 are known abietane-type diterpenoids, analyzed by ESIMS and NMR data. The results demonstrated that HSCCC can be an excellent alternative for other separation methods. The two new compounds showed significant cytotoxicity against ileocecal carcinoma HCT-8 and breast adenocarcinoma MCF-7 cells.     

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。     

Comparison of high-speed counter-current chromatography instruments for the separation of the extracts of the seeds of Oroxylum indicum

Analytical Milli high-speed counter-current chromatography (HSCCC) was used for the selection and optimization of the two-phase solvent system to separate flavonoids from the extracts of the seeds of Oroxylum indicum. The optimum solvent system obtained from Milli-CCC was also the best solvent system for preparative HSCCC and led to the successful separation of two crude flavonoids from the seeds of O. indicum by Lab/Prep (laboratory preparative) HSCCC using different sized coils. Four flavonoids were isolated by preparative HSCCC: baicalein-7-O-diglucoside (25.0 mg, 92% purity), baicalein-7-o-glucoside (50.4 mg; 95% purity), baicalein (75 mg; purity 98%) and chrysin (100 mg; purity 98%).     

High-Speed Counter-Current Chromatography Combined with Column Chromatography for Isolation of Methyllycaconitine from Delphinium pseudocyanthum

Preparative high-speed counter-current chromatography (HSCCC) combined with conventional column chromatography (CC) has been used for isolation and purification of methyllycaconitine from Delphinium pseudocyanthum. n-Hexane-ethyl acetate-methanol-water, 1:1:1:2 (v/v), was used as the solvent system for HSCCC. Separation of methyllycaconitine from an HSCCC fraction was successfully achieved by CC on silica gel using chloroform-methanol, 7:1 (v/v), as mobile phase. A total of 113.45 mg methyllycaconitine of purity >95% was obtained from 1.044 g extract of D. pseudocyanthum. Its structure was identified by MS and NMR.

     

High-Speed Counter-Current Chromatography Combined with Column Chromatography for Isolation of Methyllycaconitine from Delphinium pseudocyanthum

Preparative high-speed counter-current chromatography (HSCCC) combined with conventional column chromatography (CC) has been used for isolation and purification of methyllycaconitine from Delphinium pseudocyanthum. n-Hexane-ethyl acetate-methanol-water, 1:1:1:2 (v/v), was used as the solvent system for HSCCC. Separation of methyllycaconitine from an HSCCC fraction was successfully achieved by CC on silica gel using chloroform-methanol, 7:1 (v/v), as mobile phase. A total of 113.45 mg methyllycaconitine of purity >95% was obtained from 1.044 g extract of D. pseudocyanthum. Its structure was identified by MS and NMR.