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.     

Strategies of solvent system selection for the isolation of flavonoids by countercurrent chromatography

Flavonoids form a large class of important naturally occurring bioactive compounds. Their isolation and purification from natural sources can sometimes be very difficult and time‐consuming when traditional phytochemical techniques are used. Countercurrent chromatography (CCC), a support‐free liquid–liquid partition chromatography technique, is very useful for the isolation of polar compounds and its use is increasing in the natural products field. In this paper, we propose strategies of solvent system selection for the isolation of flavonoids by CCC, based on data from the literature, plus incorporation of own practical experiences. The selected references report the isolation of over 300 different flavonoid compounds from more than 100 plant species, using 40 different solvent systems, showing the versatility of this technique. The solvent system hexane‐ethylacetate‐methanol‐water is proposed as a starting point for the separation of samples containing free flavonoids, as it was cited in more than 60% of the papers. A “fine tuning” step is proposed at each level of this solvent family. Other modifications include exchanging the alcohol in the system as well as introducing a fifth solvent. The solvent system ethyl‐acetate‐butanol‐water is proposed as the starting point for glycosylated flavonoids. Other solvent systems are also discussed. The use of gradients is proposed for samples containing both free and glycosylated flavonoids, as the polarity window is larger in these cases. High‐speed countercurrent chromatography was used in 89% of the reviewed data.     

应用新型立式逆流色谱制备分离南蛇藤中的南蛇藤素

A versatile countercurrent chromatography with upright multilayer coil planet centrifuge, named upright countercurrent chromatography (UCCC), was applied to the isolation and purification of celastrol from the roots of Celastrus orbiculatus Thunb. The crude celastrol was obtained by elution with petroleum ether from ethanol extracts using a 15 cm length and 5 cm I.D. of silica gel flash chromatography. Preparative UCCC (Fig. 1) with a two-phase system composed of petroleum ether (b. p. 60 ～ 90 ℃ )-ethyl acetate-tetrachloromethanemethanol-water ( 1:1:8:6: 1, v/v) was successfully performed, yielding 705 mg celastrol at 99.5 % purity from 1020 rng of the crude extract in one step separation.     

The rôle of countercurrent chromatography in the fractionation of complex mixtures

The biologically active pricipals in nature are frequently present as only a few parts per million of complex mixtures of non-volatile components and often have limited stability. Their isolation often requires the application of all available techniques, such as adsorption chromatography, ion exchange procedures, size exclusion techniques, and solvent partition methods consistent with their physical properties and stability. The process of countercurret chromatography is essentially liquid-liquid chromatography in which the stationary liquid bed is retained in the column by a force field rather than by a solid supporting matrix. Adsorption effects are thereby eliminated. The technique is particularly advantageous in the preparative separation of milligram to gram quantities of polar and labile organic compounds and bio-particulate materials such as cells and cell fragments. Virtually any twophase solvent system, either aqueous or non-aqueous may be employed. Countercurrent chromatography (CCC) provides a convenlent alternative to adsorption chromatography for fractionation of natural products or other complex mixtures. In some cases, this high resolution method offers advantages with regard to the avoidance of contamination from solid adsorbents, versatility, and relatively inexpensive operation. The article covers some of the applications, selection of solvents, and advantages of CCC.     

Determination of chlorophenols in red wine using ionic liquid countercurrent chromatography as a new pretreatment method followed by high‐performance liquid chromatography

A countercurrent chromatography method for the enrichment and cleanup of chlorophenols from food samples was successfully established by using an ionic‐liquid‐modified two‐phase solvent system composed of dichloromethane containing 2% 1‐butyl‐3‐methylimidazolium bis(trifluoromethanesulfonyl)imide and water. The column was firstly filled with the organic stationary phase, and then a large volume of sample was pumped into the column after it was equilibrated with pure water at the rotation speed. Finally, the trace amounts of chlorophenols extracted and enriched in the stationary phase were eluted out by an alkaline mobile phase and determined by high‐performance liquid chromatography. Under optimized conditions, the enrichment and cleanup of the chlorophenols can be fulfilled online with enrichment factors (34–65) and high recoveries (84.69–95.23%). The method has been applied to the determination of chlorophenols in real red wine samples with the limits of detection in the range of 1.89–4.21 μg/L. The present method is highly suitable for the pretreatment of large volume of aqueous sample for the determination of trace amounts of contaminants in food and environmental samples.     

One-step separation of terpenoids from leaves extracts of Solanum cernuum by high-performance countercurrent chromatography

Solanum cernuum is a native shrub from Brazil that has been used as medicine since the nineteenth century. Although it has been utilized for a long time, there are few studies regarding its chemical composition and it motivated us to investigate this species. The dried leaves of S. cernuum were extracted with methanol and the obtained extract was further partitioned with different organic solvents. As part of our investigation, the n-hexane fraction was submitted to CCC with a nonaqueous solvent system composed of hexane, acetonitrile, and ethyl acetate (5:5:1, v/v/v). Three terpenoids were obtained by one-step separation and identified as 24-oxo-31-norcycloartanone (22.8 mg), lupeol (9.4 mg), and cycloeucalenone (13.4 mg). Complementarily, the solvents system used on the separation have had their compositions determined by gas chromotography–flame ionization detector (GC-FID).     

Application of high‐performance countercurrent chromatography for the isolation of steroidal saponins from Liriope plathyphylla

High‐performance countercurrent chromatography (HPCCC) with electrospray light‐scattering detection was applied for the first time to isolate a spirostanol and a novel furostanol saponin from Liriope platyphylla. Due to the large differences in K_D values between the two compounds, a two‐step HPCCC method was applied in this study. The primary HPCCC employed methylene chloride/methanol/isopropanol/water (9:6:1:4 v/v, 4 mL/min, normal‐phase mode) conditions to yield a spirostanol saponin ( 1 ). After the primary HPCCC run, the solute retained in the stationary phase (SP extract) in HPCCC column was recovered and subjected to the second HPCCC on the n‐hexane/n‐butanol/water system (1:9:10 v/v, 5 mL/min, reversed‐phase mode) to yield a novel furostanol saponin ( 2 ). The isolated spirostanol saponin was determined to be 25(S)‐ruscogenin 1‐O‐β‐d ‐glucopyranosyl (1→2)‐[β‐d ‐xylopyranosyl (1→3)]‐β‐d ‐fucopyranoside (spicatoside A), and the novel furostanol saponin was elucidated to be 26‐O‐β‐d ‐glucopyranosyl‐25(S)‐furost‐5(6)‐ene‐1β‐3β‐22α‐26‐tetraol‐1‐O‐β‐d ‐glucopyranosyl (1→2)‐[β‐d ‐xylopyranosyl‐(1→3)]‐β‐d ‐fucopyranoside (spicatoside D).     

Tubing modifications for countercurrent chromatography (CCC): Stationary phase retention and separation efficiency

Countercurrent chromatography (CCC) is a separation technique in which two immiscible liquid phases are used for the preparative purification of synthetic and natural products. In CCC the number of repetitive mixing and de-mixing processes, the retention of the stationary phase and the mass transfer between the liquid phases are significant parameters that influence the resolution and separation efficiency. Limited mass transfer is the main reason for peak broadening and a low number of theoretical plates along with impaired peak resolution in CCC. Hence, technical improvements with regard to column design and tubing modifications is an important aspect to enhance mixing and mass transfer.     

High‐speed countercurrent chromatography isolation of flavans from Ixeris chinensis and their identification by NMR spectroscopy

High‐speed countercurrent chromatography, combined with macroporous resin chromatography were applied to the separation and purification of flavans from Ixeris chinensis. Four flavans, namely, 5‐methoxy‐7,4′‐dihydroxyflavan‐3‐ol ( 1 ), 5,7‐dimethoxy‐4′‐hydroxyflavan‐3‐ol ( 2 ), 5,7‐dimethoxy‐4′‐hydroxyflavan ( 3 ), and 5,7‐dimethoxy‐8‐methyl‐4′‐hydroxyflavan ( 4 ), were obtained from I. chinensis for the first time. Their chemical structural identification was carried out by spectroscopic methods, including 1D and 2D NMR spectroscopy. Amounts of 13.2 mg of compound 1 , 6.4 mg of compound 2 , 5.8 mg of compound 3 , and 14.5 mg of compound 4 were separated from 120 mg 75% ethanol fraction. The purities of 1 – 4 were 99.1, 99.2, 97.3, and 98.6 %, respectively.     

Modeling gradient elution in countercurrent chromatography: efficient separation of tanshinones from Salvia miltiorrhiza Bunge

Countercurrent chromatography (CCC) is a support-free liquid-liquid chromatography using centrifugal fields to hold the liquid stationary phase. CCC has been widely applied in the separation of various natural and synthetic components using a variety of biphasic liquid systems. The related hexane or heptane/ethyl acetate/methanol or ethanol/water biphasic liquid systems demonstrated their significance in CCC. Gradient is difficult in CCC since any composition change in one phase induces a composition change of the other phase to maintain phase equilibrium. This work provides a new insight into linear gradient elution in CCC that is feasible with some biphasic liquid systems such as selected compositions of the hexane/ethyl acetate/ethanol/water systems. The equations modeling solute motion inside the CCC column are proposed. Particular compositions of the liquid system, namely the hexane/ethyl acetate/ethanol/water 8:2:E:W compositions with E + W = 10, were studied from W = 1 to 9. They showed moderate changes in the upper organic phase compositions. The model is tested with the separation of tanshinones from the rhizome of Salvia miltiorrhiza Bunge. Different linear solvent gradient profiles were experimentally performed between 8:2:5:5 and 8:2:3:7 compositions and the results were evaluated using the proposed model. Five tanshinones including dihydrotanshinone I, cryptotanshinone, tanshinone I, 1,2-dihydrotanshinquinone, and tanshinone IIA have been successfully separated (>95% purities) using a gradient profile optimized by the developed model. The gradient model can be used only with biphasic liquid systems in which one phase shows minimum composition changes when the other phase composition changes notably. This case is not the general case for biphasic liquid systems but can be applied with specific compositions of the quaternary hexane or heptane/ethyl acetate/methanol or ethanol/water most useful CCC liquid systems.     

Microwave‐assisted extraction coupled with countercurrent chromatography for the rapid preparation of flavonoids from Scutellaria barbata D. Don

As a famous Chinese herb having good inhibitory effects on numerous human cancers both in vitro and in vivo, Scutellaria barbata D. Don attracts extensive attention worldwide. In this work, four flavonoids named scutellarin, baicalin, luteolin, and apigenin were simply and rapidly prepared from S. barbata by microwave‐assisted extraction coupled to countercurrent chromatography. Extraction conditions including irradiation time, extraction temperature, liquid/solid ratio, and microwave power were optimized using an orthogonal array design method. The extract of S. barbata was separated and purified with a two‐phase solvent system composed of hexane/ethyl acetate/methanol/acetic acid/water (1:5:1.5:1:4, v/v/v/v/v) and 4.5 mg of scutellarin, 4.6 mg of baicalin, 1.1 mg of luteolin, 2.1 mg of apigenin were obtained from 2.0 g original sample in a single run. The purities of scutellarin, baicalin, luteolin, and apigenin determined by HPLC were 93.6, 97.3, 97.6, and 98.4%, respectively. The targeted compounds were identified by LC with MS and ¹H NMR spectroscopy. The total time including extraction, separation, and purification was <300 min. Compared to traditional methods, microwave‐assisted extraction coupled to countercurrent chromatography method is more simple and rapid for the extraction, separation, and purification of flavonoid compounds from natural products.     

Automated solvent system screening for the preparative countercurrent chromatography of pharmaceutical discovery compounds

A fully automated countercurrent chromatography system has been constructed to rapidly screen the commonly used heptane/ethyl acetate/methanol/water solvent system series and translate the results to preparative scale separations. The system utilizes “on‐demand” preparation of the heptane/ethyl acetate/methanol/water solvent system upper and lower phases. Elution‐extrusion countercurrent chromatography was combined with non‐dynamic equilibrium injection reducing the screening time for each heptane/ethyl acetate/methanol/water system to 17 min. The result enabled solvent system development to be reduced to under 2 h. The countercurrent chromatography system was interfaced with a mass spectrometer to allow selective detection of target components in crude medicinal chemistry reaction mixtures. Mass‐directed preparative countercurrent chromatography purification was demonstrated for the first time using a synthetic tetrazole epoxide derived from a routine medicinal chemistry support workflow.     

Application of off‐line two‐dimensional high‐performance countercurrent chromatography on the chloroform‐soluble extract of Cuscuta auralis seeds

In this study, the chloroform‐soluble extract of Cuscuta auralis was separated successfully using off‐line two‐dimensional high‐performance countercurrent chromatography, yielding a γ‐pyrone, two alkaloids, a flavonoid, and four lignans. The first‐dimensional countercurrent separation using a methylene chloride/methanol/water (11:6:5, v/v/v) system yielded three subfractions (fractions I–III). The second‐dimensional countercurrent separations, conducted on fractions I–III using n‐hexane/ethyl acetate/methanol/water/acetic acid (5:5:5:5:0, 3:7:3:7:0, and 1:9:1:9:0.01, v/v/v/v/v) systems, gave maltol ( 1 ), (−)‐(13S)‐cuscutamine ( 2 ), (+)‐(13R)‐cuscutamine ( 3 ), (+)‐pinoresinol ( 4 ), (+)‐epipinoresinol ( 5 ), kaempferol ( 6 ), piperitol ( 7 ), and (9R)‐hydroxy‐d ‐sesamin ( 8 ). To the best of our knowledge, maltol was identified for the first time in Cuscuta species. Furthermore, this report details the first full assignment of spectroscopic data of two cuscutamine epimers, (−)‐(13S)‐cuscutamine and (+)‐(13R)‐cuscutamine.     

Off‐line comprehensive two‐dimensional reversed‐phase countercurrent chromatography with high‐performance liquid chromatography: Orthogonality in separation of Polygonum cuspidatum Sieb. et Zucc

Off‐line comprehensive two‐dimensional reversed‐phase countercurrent chromatography with high‐performance liquid chromatography was investigated in separation of crude ethanol extract from traditional Chinese medicinal herb Polygonum cuspidatum Sieb. et Zucc. Two‐dimensional contour plots for countercurrent chromatography with high‐performance liquid chromatography was obtained after comprehensive separation was completed. Total peak capacity was evaluated and approximately 810 peaks were obtained through a comprehensive two‐dimensional separation. A highly orthogonality of 52.23% and a large separation space occupancy of 88.86% were achieved. Meanwhile, it was found that several components could be well separated by countercurrent chromatography while they could not be separated by high‐performance liquid chromatography, and vice versa, which further indicated the orthogonality of the two separation methods. The off‐line comprehensive two‐dimensional countercurrent chromatography with high‐performance liquid chromatography provided a promising and powerful method for separation of complex natural products.     

Tubing modifications for countercurrent chromatography: Investigation of geometrical parameters

Countercurrent chromatography (CCC) is a separation technique which may be described as a combination of a great number of liquid–liquid distributions of analytes in a two-phase solvent system with liquid chromatography (LC) features. Even optimized CCC separations currently provide a lower number of theoretical plates when compared to LC. For this reason, instrumental advancements are indispensable to, at least partly, overcome this drawback. Recently, we found that improvement of the classic CCC coil, that is using a long hollow tubing, may be achieved by the introduction of tubing crimpings which increase the stationary phase retention. In this study, we systematically investigated the effects of three geometrical parameters (crimping depth, distance between two crimpings as well as partial or complete crimping of the tubing) on the stationary phase retention by a factorial design of experiments approach. Separation efficiency tests were performed with two groups of analytes: fatty acid methyl esters (FAME) in the n-hexane/acetonitrile (HAcn) and alkyl p-hydroxybenzoates in the n-hexane/tert-butylmethylether/methanol/water solvent system. The most narrow crimping distance and the deepest crimping of the tubing were the best configurations in the examined flow rate range.     

Enantioseparation of 2‐(substituted phenyl)propanoic acids by high‐speed countercurrent chromatography and investigation of the influence of substituents in enantiorecognition

This paper concentrates on the enantioseparation of racemic 2‐(substituted phenyl)propanoic acids by high‐speed countercurrent chromatography with substituted β‐cyclodextrin as the chiral selector, and an investigation of the influence of the substituent on the benzene ring in enantiorecognition between the chiral selector and enantiomer of each racemate is presented. This is an extension research of our previous work on the enantioseparation of 2‐phenyl propanoic acid derivatives, to investigate the relationship between the value of enantioseparation factor and the different substituent on the benzene ring. In total, ten racemic 2‐(substituted phenyl)propanoic acids were investigated, of which four including 2‐(4‐nitrophenyl)propanoic acid, 2‐(4‐methylphenyl)propanoic acid, 2‐(4‐hydroxyphenyl)propanoic acid, and 2‐(4‐chlorophenyl)propanoic acid, were studied by countercurrent chromatography for the first time, and two racemates were successfully enantioseparated. The distribution ratio and enantioseparation factor for all the ten racemates were determined by enantioselective liquid–liquid extraction. The results showed that an electron‐donating group on the benzene ring presents a higher enantiorecognition induced by chiral selector than that of racemates with an electron‐withdrawing group on the benzene ring.     

Performance of an aqueous two‐phase‐based countercurrent chromatographic system for horseradish peroxidase purification

Countercurrent chromatography (CCC) purification of horseradish peroxidase (HRP) from Armoracia rusticana root extracts was achieved by employing polymer‐phosphate aqueous two‐phase systems (ATPS). By using preparative columns at 1000 rpm, a 25–30% retention of the top phase of an ATPS composed of 10% w/w PEG 1540 and 14.8% w/w phosphate – with added 2 mol/kg sodium chloride – was obtained. The retention level was stable during the standard separation running time (4 h). Horseradish root extract samples were injected into the system (10–25 mL; 200–250 U/mL peroxidase; 2.0–4.0 mg/mL total protein). Retention of HRP in the CCC “column” during the chromatographic run was attained in the selected ATPS, where the partition coefficient K for the enzyme was ≥ 8. Replacement of the mobile phase with a fresh one but in the absence of added salt brought about product elution. Recovery of HRP in this fraction accounts for ≥ 45% of the total activity loaded, with a purification factor of 6. Enzyme activity was also found in the pass‐through fraction and in the remaining liquid (stationary) phase, a fact that should be ascribed to the existence of multiple peroxidase isoforms. SDS‐PAGE of the active fraction showed a protein band at 44 kDa, compatible with the presence of HRP. Thus, the optimised CCC system allowed the separation of HRP directly from a complex biological material. These results open up the possibility of achieving protein separation with CCC/ATPS and of scaling‐up processes in industrial separators.     

Isolation of a furan fatty acid from Hevea brasiliensis latex employing the combined use of pH‐zone‐refining and conventional countercurrent chromatography

Furan fatty acids are valuable and bioactive minor fatty acids that usually contribute <0.1% to the fatty acid content of food samples. Their biological role still remains unclear as authentic furan fatty acid standards are not readily available and thorough experimental studies verifying the relevance of furan fatty acids are thus virtually impossible. An efficient protocol for the isolation of the furan fatty acid 9‐(3‐methyl‐5‐pentylfuran‐2‐yl)‐nonanoic acid from hydrolyzed and centrifuged latex of Hevea brasiliensis was developed using countercurrent chromatography. A first run using pH‐zone‐refining countercurrent chromatography provided 48.4 mg of 9‐(3‐methyl‐5‐pentylfuran‐2‐yl)‐nonanoic acid from 210 mg latex extract in a purity of 95%. The purity was increased to 99% by means of one second run in conventional countercurrent chromatography mode. The Structure and purity of 9‐(3‐methyl‐5‐pentylfuran‐2‐yl)‐nonanoic acid were determined by gas chromatography coupled to mass spectrometry and ¹H and ¹³C NMR spectroscopy.     

Separation of isorhamnetin 3‐sulphate and astragalin from Flaveria bidentis (L.) Kuntze using macroporous resin and followed by high‐speed countercurrent chromatography

D4020 resin offered the best dynamic adsorption and desorption capacity for total flavonoids based on the research results from ten kinds of macroporous resin. A column packed with D4020 resin was used to optimize the separation of total flavonoids from Flaveria bidentis (L.) Kuntze extracts. The content of flavonoids in the product was increased from 4.3 to 30.1% with a recovery yield of 90%. After the treatment with gradient elution on D4020 resin, the contents of isorhamnetin 3‐sulfate and astragalin were increased from 0.49 to 8.70% with a recovery yield of 74.1% and 1.16 to 30.8%, with a recovery yield of 92.2%, respectively. Further purification was carried out by one‐run high‐speed countercurrent chromatography yielding 4.5 mg of isorhamnetin 3‐sulfate at a high purity of 96.48% and yielding 24.4 mg of astragalin at a high purity of over 98.46%.     

Single‐step isolation of embelin using high‐performance countercurrent chromatography and determination of the fatty acid composition of seeds of Embelia schimperi

Embelin (2,5‐dihydroxy‐3‐undecyl‐p ‐benzoquinone) is known for its potent anthelmintic activity, but also for wound‐healing, antidiabetic, anticonvulsant, antitumour, anti‐inflammatory, analgesic, hepatoprotective, antioxidant, antibacterial and antispermatogenic activities. A high‐performance countercurrent chromatography method was developed for the purification of embelin from an extract of the seeds of Embelia schimperi fruit. The optimized solvent system (n ‐hexane–ethylacetate–ethanol–water, 7:3:7:3) resulted in the isolation of 13.9 mg of embelin, directly from 100 mg of crude extract, in a single step within a short time (40 min). Although the compound appeared to be completely pure when analysed by ultra‐performance liquid chromatography (UPLC) with photo diode array detection, the purity was established as ~90% by UPLC–mass spectrometry. Furthermore, we report the fatty acid composition of the seeds of E. schimperi fruit. Nine fatty acids were quantified from the fruit seed extract by gas chromatography–mass spectrometry, with linoleic (46.4%), palmitic (21.5%) and oleic (19.6%) acids making up the largest proportions.