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.     

Isolation and characterization of flavonols from blackcurrant by high-performance counter-current chromatography and electrospray ionization tandem mass spectrometry

Blackcurrant is considered as a natural high-value food raw material and possesses a variety of therapeutic properties. The health benefits of blackcurrant have generally been credited to its high anthocyanin content; however, the therapeutic properties of other minor flavonoids constituents have not yet been investigated due the difficulties related to their isolation. Multiple steps of high-performance counter-current chromatography in combination with ESI tandem mass spectrometry (MS(n)) were successfully used for the preparative isolation of flavonols from blackcurrant extract, to study their electrospray ionization mass spectrometry fragmentation behavior. Seven flavonols, namely myricetin-3-O-rutinoside (145.5 mg), myricetin-3-O-hexoside (79.7 mg), myricetin-3-O-(6″-malonyl)-glucoside (17.4 mg), kaempferol-3-O-glucoside (20.5 mg), quercetin-3-O-rutinoside (55.1 mg), quercetin-3-O-hexoside (25.8 mg), and myricetin (129.1 mg) have been successfully isolated and their multistage MS(n) data were used for detailed structure characterization. The results of these experiments demonstrated that high-performance counter-current chromatography along with ESI-MS(n) is a sensitive, selective, and effective technology for isolation and characterization of minor constituents from a complex mixture.     

Separation of alkaloids from herbs using high-speed counter-current chromatography

Alkaloids represent a most widespread group of bioactive natural products. Because of their alkalinity and structural diversity, the fractionation and purification of the alkaloids from herbs can often present a number of practical difficulties using the conventional chromatographic techniques. High-speed counter-current chromatography (HSCCC) is a liquid-liquid partition chromatography with a support-free liquid stationary phase, and is gaining more and more popularity as a viable separation technique for bioactive compounds from natural resources. In the present review, focus is placed on the separation of alkaloids by both conventional HSCCC and pH-zone-refining counter-current chromatography (CCC) techniques from herbs. The review presents the separation of over 120 different alkaloid compounds from more than 30 plant species by the conventional HSCCC and pH-zone-refining CCC. Based on the data from the literature, the proper solvent systems for the separation of alkaloids by the conventional HSCCC and pH-zone-refining CCC are also summarized.     

Online extraction and isolation of highly polar chemical constituents from Brassica napus L. pollen by high shear technique coupled with high-performance counter-current chromatography

High shear technique coupled with high-performance counter-current chromatography was successfully used for the extraction and online isolation of seven highly polar chemical constituents from the Brassica napus L. The lower phase of ethyl acetate-n-butanol-water (1:4:5, v:v:v) was used as both the high shear technique solvent and high-performance counter-current chromatography mobile phase. Seven compounds of 14.2 mg of uridine, 4.6 mg of xanthosine, 7.8 mg of guanosine, 5.3 mg of adenosine, 19.5 mg of kaempferol-3,4'-di-O-β-D-glucopyranoside, 17.7 mg of kaempferol-3-O-(2-O-β-D-glucopyranosy1)-β-D-glucopyranoside, and 25.7 mg of an unknown compound, with a high-performance liquid chromatography (HPLC) purity over 95.0%, were obtained in a one-step extraction-separation process within 130 min from 20.0 g of raw material of pollen of Brassica napus L. Moreover, the mode of elution-extrusion was employed for the separation of the last one compound. The isolated compounds were analyzed by HPLC, and the chemical structures of the compounds mentioned above were identified by UV and NMR. It is the first time to combine the high shear technique and high-performance counter-current chromatography for the online isolation of the nature products.     

Isolation of bioactive components from Flaveria bidentis (L.) Kuntze using high-speed counter-current chromatography and time-controlled collection method

Semipreparative high-speed counter-current chromatography (HSCCC) by time-controlled collection method was successfully applied for isolation and purification of α-terthienyl, 5-(3-buten-1-ynyl)-2,2'-bithienyl, and 5-(3-penten-1-ynyl)-2,2'-bithienyl from Flaveria bidentis (L.) Kuntze for the first time. The two-phase solvent system composed of n-hexane and acetonitrile at the volume ratio of 1:1 (v/v) was used for the semipreparative HSCCC. The 5.2 mg α-terthienyl, 2.2 mg 5-(3-buten-1-ynyl)-2,2'-bithienyl, and 4.3 mg 5-(3-penten-1-ynyl)-2,2'-bithienyl with the purity of 99.9, 90.2, and 92.1% were produced from 265.6 mg crude extract, respectively, and 5-(3-penten-1-ynyl)-2,2'-bithienyl was first isolated from Flaveria bidentis (L.) Kuntze. The structures of the separated compounds were identified by electrospray-ionization mass spectrometry and proton and carbon nuclear magnetic resonance ((1)H- and (13)C-NMR).     

Isolation and purification of ginkgo flavonol glycosides from Ginkgo biloba leaves by high-speed counter-current chromatography

A high-speed counter-current chromatography method was developed for the separation and purification of bioactive flavonol glycosides from a crude ethanol extract of Ginkgo biloba leaves. The separation was performed with a two-phase solvent system composed of n-hexane-butanol-ethyl acetate-methanol-0.5% acetic acid (1:0.5:3.5:1:4, v/v) and three pure compounds were eluted in high purities in a one-step separation. Their purities were determined by HPLC and identified by MS,(1)H-NMR, and(13)C-NMR.     

Preparative separation of minor saponins from Platycodi Radix by high-speed counter-current chromatography

Platycosides (PSs), the saponins found in the root of Platycodon grandiflorum (Jacq.) A. DC. (Platycodi Radix), are typically composed of oleanene backbones with two side chains; one is a 3-O-glucose linked by a glycosidic bond, and the other is a 28-O-arabinose-rhamnose-xylose-apiose linked by an ester bond. Minor saponins, acetylated isomers of the major saponin on either the 2' or 3' position of rhamnose, were isolated from Platycodi Radix using a multi-step process including high-speed counter-current chromatography (HSCCC) and preparative reversed-phase high-performance liquid chromatography (RP-HPLC). After the separation of the major components, the enriched minor saponin fraction was used for this study. A two-phase solvent system consisting of chloroform-methanol-isopropanol-water (3:2:2:3, v/v) was used for HSCCC. HSCCC separation of the enriched minor saponin fraction yielded 2'-O-acetylplatycodin D, 3'-O-acetylpolygalacin D, 2'-O-acetylpolygalacin and a mixture of 3'-O-acetylplatycodin D and polygalacin D. The mixture fraction from HSCCC separation was further purified by preparative RP-HPLC, giving 3'-O-acetylplatycodin D and polygalacin D at a purity of over 98.9%. The developed method provides the preparative and rapid separation of minor saponins in the crude extract of Platycodi Radix. To the best of our knowledge, this is the first on the separation of acetylated PSs by HSCCC.     

Isolation of terpenoids from Pimpinella anisum essential oil by high‐performance counter‐current chromatography

High‐performance counter‐current chromatography was successfully used for the isolation and purification of terpenoid compounds from the essential oil of Pimpinella anisum L. A two‐phase solvent system composed of n‐heptane/methanol/ethyl acetate/water (5:2:5:2, v/v/v/v) was suitable for the purification of linalool, terpinen‐4‐ol, α‐terpineol, p‐anisaldehyde, while n‐heptane/methanol (1:1, v/v) was used for the isolation of anethole and foeniculin. A scale‐up process from analytical to preparative was developed. Additionally, a stepwise gradient elution was applied and instead of two different runs, 40 min each, one 80 min separation was performed; although the time of separation remains the same, it was possible to repeat the efficiency even if the water‐containing mobile phase was changed to a nonaqueous system. The obtained essential oil, as well as purified compounds, was analyzed by GC. A total of 0.64 mg of linalool, 0.52 mg of terpinen‐4‐ol, 0.10 mg of α‐terpineol, 0.62 mg of p‐anisaldehyde, 15 mg of anethole, and 2.12 mg of foeniculin were obtained from 210 mg of the essential oil of P. anisum L. in a short time with purities of 99, 98, 94, 93.54, 93, and 93.6%, respectively.     

Preparative isolation of alkaloids from Corydalis bungeana Turcz. by high-speed counter-current chromatography using stepwise elution

High‐speed counter‐current chromatography (HSCCC) was successfully applied for the preparative separation and purification of alkaloids from Corydalis bungeana Turcz. (Kudiding in Chinese) for the first time. After the measurement of partition coefficient of seven target alkaloids in the nine two‐phase solvent systems composed of CHCl₃–MeOH–(0.1 M; 0.2 M; 0.3 M) HCl (4:1.5:2; 4:2:2; 4:3:2, v/v), CHCl₃–MeOH–0.2 M HCl (4:2:2, v/v) and CHCl₃–MeOH–0.3 M HCl (4:3:2, v/v) were finally selected for the HSCCC separation using the first upper phase as the stationary phase and the stepwise elution of the two lower mobile phases. Consequently, sanguinarine (10 mg), corynoline (25 mg), protopine (20 mg), corynoloxine (18 mg), and 12‐hydroxycorynoline (8 mg) were obtained from 200 mg of crude alkaloid extracts with purities of 94–99% as determined by HPLC. Their chemical structures were characterized on the basis of ¹H‐NMR, ¹³C‐NMR, and LC‐ESI‐Q‐TOF‐MS/MS analyses.     

High‐performance countercurrent chromatography separation of Peucedanum cervaria fruit extract for the isolation of rare coumarin derivatives

For the first time, rare major and minor compounds from fruits of Peucedanum cervaria were isolated. High‐performance countercurrent chromatography with two different solvent systems, heptane/ethyl acetate/methanol/water (3:2:3:2 and 2:1:2:1, v/v), was successfully used in the reversed‐phase mode. A scale‐up process from analytical to semipreparative in a very short time was developed. The structures of isolated compounds were evaluated by high‐performance liquid chromatography with diode array detection and electrospray ionization mass spectrometry, gas chromatography with mass spectrometry, and one‐ and two‐dimensional NMR spectroscopy. (8S,9R)‐9‐(3‐Methylbutenoyloxy)‐O‐acetyl‐8,9‐dihydrooroselol (compound B), (8S,9R)‐9‐(2‐methyl‐Z‐butenoyloxy)‐O‐acetyl‐8,9‐dihydrooroselol (edultin, compound C), and (8S,9R)‐9‐acetoxy‐O‐(2α‐methylbutyryl)‐8,9‐dihydrooroselol (compound D) were obtained using heptane/ethyl acetate/methanol/water (2:1:2:1, v/v) in <40 min. The method yielded 4.6 mg of a mixture of compounds B and C (11:89) and 3.7 mg of compound D. These amounts were obtained from the crude extract (0.5 g) in a single run. Although the compounds are known, their isolation by countercurrent chromatography and the analysis of their relative stereochemistry by two‐dimensional NMR spectroscopy have been performed for the first time. Additionally, heptane/ethyl acetate/methanol/water (3:2:3:2, v/v) led to the isolation of oxypeucedanin (1.2 mg; compound A). This is the first time that angular dihydrofuranocoumarin was isolated from plant extract by countercurrent chromatography.     

Separation and purification of furanocoumarins from Toddalia asiatica (L.) Lam. using microwave-assisted extraction coupled with high-speed counter-current chromatography

A method of microwave-assisted extraction coupled with high-speed counter-current chromatography was established for separation and purification of isopimpinellin, pimpinellin and phellopterin from Toddalia asiatica (L.) Lam. The conditions of MAE including the extraction solvent, size of sample, solid/liquid ratio, extraction temperature and extraction time were optimized by a mono-factor test. That is, 2.0 g dried powder of T. asiatica (L.) Lam of 0.30-0.15 mm size was extracted with 20 mL (solid/liquid ratio of 1:10, g/mL) methanol under 50 °C for 1 min. The crude extract was separated and purified by high-speed counter-current chromatography with hexane-ethyl acetate-methanol-water (5:5:5.5:4.5, v/v/v/v) solvent system. 0.85 mg/g of isopimpinellin, 2.55 mg/g of pimpinellin and 0.95 mg/g of phellopterin were obtained from original sample in one-step within 240 min, the purity determined by high performance liquid chromatography was 95.0%, 99.1% and 96.4%, respectively. Their chemical structures were further identified by mass spectroscopy and nuclear magnetic resonance spectroscopy. The results demonstrated that microwave-assisted extraction coupled with high-speed counter-current chromatography was a feasible, economical and efficient technique for rapid extraction, separation and purification of effective compounds from natural products.     

Rapid preparative isolation of a new phenylpropanoid glycoside and four minor compounds from Sparganium stoloniferum using high-speed counter-current chromatography as a fractionation tool

A rapid high-speed counter-current chromatography (HSCCC) method was used to isolate five minor compounds from rhizome of Sparganium stoloniferum namely San Leng in Chinese, including two phenylpropanoid glycosides, sparganiaside A (1) and 1-O-feruloyl-3-p-coumaroylglycerol (2), and three aromatic acids, vanillic acid (3), p-hydroxylcinnamic acid (4), and p-hydroxybenzoic acid (5), of which, compound 1 was a new one. Five compounds were preparatively enriched at top efficiency by one-step HSCCC operation in the isolation procedure. A suitable solvent system composed of chloroform-methanol-water (4:3.5:1.8, v/v/v) was used. And the operation time was less than 4 h. The purities of compounds (1-5) in the enriched fractions were determined to be 75.8%, 66.3%, 90.6%, 79.9%, and 98.2%, respectively. The mean recoveries of the five compounds were 84.8%, 87.3%, 81.8%, 90.3%, and 92.7%, respectively. Compounds 1-4 were further purified by semi-preparative high-performance liquid chromatography (HPLC). This is the first report on the use of HSCCC as a fractionation tool for preparative isolation of minor compounds from S. stoloniferum. The method was proved to be rapid, convenient, high yield, and low cost. HSCCC was shown to be a quick and effective tool in isolation of natural products even though the compounds were not abundant.     

Preparative isolation and purification of two amides from Mallotus lianus Croiz by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) was applied to the preparative isolation and purification of two amides from Mallotus lianus Croiz. In a single HSCCC separation, using the two-phase solvent system composed of n-hexane/ethyl acetate/methanol/water (5:1:5:1 v/v), 247.5 mg of the enriched crude sample was separated to afford 10.3 mg of N-isobutyl-2E,4E,12Z-octadecatrienamide and 15.7 mg of (7Z,10Z,18Z)tricosa-7,10,18-trienamide, a novel compound, with the purities of 98.0 and 94.6%, respectively. The HSCCC fractions were analyzed by HPLC and chemical structures of the compounds were identified by 1D- and 2D-NMR, ESI-, and GC-MS.     

Preparative purification of gentamicin components using high-speed counter-current chromatography coupled with electrospray mass spectrometry

We developed a useful and preparative method based on high-speed counter-current chromatography with mass spectrometry (HSCCC/MS) to purify gentamicin C1a, C2/2a and C1 from standard powder. The analytes were purified on the HSCCC model CCC-1000 (multi-layer coil planet centrifuge) with a volatile two-phase solvent system composed of n-butanol/10% aqueous ammonia solution (50:50, v/v) and detected on an LCMS-2020EV quadrupole mass spectrometer fitted with an electrospray ionization (ESI) source system in positive ionization following scan mode (m/z 100-500). The HSCCC/ESI-MS peaks indicated that gentamicin C1a (m/z 450: [M+H](+)), C2/2a (m/z 464: [M+H](+)) and C1 (m/z 478: [M+H](+)) have the peak resolution values of 1.3 and 1.7 from 30 mg of loaded gentamicin powder. The HSCCC yielded 3.9 mg of gentamicin C1a, 12.6 mg of gentamicin C2/2a and 12.0 mg of gentamicin C1. These purified substances were analyzed by LC/MS with scan positive-mode. Based on the LC/MS chromatograms and spectra of the fractions, analytes were estimated to be over 95% pure. These gentamicin isomers of C1a, C2/2a and C1 were evaluated for their antibacterial activities. The overall results indicate that this approach of HSCCC/MS is a powerful technique for the purification of gentamicin components.     

Preparative isolation and purification of flavone compounds from sophora japonica L. by high-speed counter-current chromatography combined with macroporous resin column separation

High-speed counter-current chromatography combined with macroporous resin column separation was applied to the isolation and purification of genistein-7,4'-di-O-beta-D-glucoside (I), genistein-7-O-beta-D-glucopyranoside-4'-O-[(alpha-L-rhamnopyransoyl)-(1-2)-beta-D-glucopyranoside] (II), kaempferol-3-O-beta-D-sophoroside(III), quercetin-3-O-beta-L-ramnopyranosyl-(1 - 6)-beta-D-glucopyranoside (IV), genistein-4'-beta-L-rhamnopyransoyl-(1 - 2)-alpha-D-glucopyranoside (V), and kaempferol-3-O-beta-L-ramnopyranosyl-(1 - 6)-beta-D-glucopyranoside (VI) from the Chinese medicinal herb Sophora japonica L. The crude extracts from the pericarps of Sophora japonica L. were pre-separated on a D-101 macroporous resin column and divided into two parts as sample 1 and sample 2. An 80-mg portion of sample 1 was separated by using n-butanol-acetic acid (1%) (5:5, v/v) as the two-phase solvent system and yielded 30.1 mg of compound I, 23.3 mg of compound II. A 120 mg portion of sample 2 was separated by using ethyl acetate-n-butanol-acetic acid (1%) (5:0.8:5, v/v) as the two-phase solvent system and yielded 5.5 mg of compound III, 31.7 mg of compound IV, 37.4 mg of compound V, and 6.2 mg of compound VI. The purities of compounds I, II, III, IV, V, and VI were 98.7, 98.2, 97.8, 98.5, 99.3, and 98.9%, respectively, as determined by HPLC. The chemical structures of these components were identified by 1H-NMR and 13C-NMR.     

Separation and purification of harmine and harmaline from Peganum harmala using pH-zone-refining counter-current chromatography

pH-zone-refining counter-current chromatography was successfully applied to the separation of alkaloids from a crude extract of Peganum harmala L. using a multilayer coil planet centrifuge. The experiment was performed with a two-phase solvent system composed of methyl tert-butyl ether/THF/water (2:2:3 by volume) where triethylamine (10 mM) was added to the upper organic stationary phase as a retainer and hydrochloric acid (5 mM) to the aqueous mobile phase as an eluter. From 1.2 g of the crude extract, 554 mg harmine and 325 mg harmaline were obtained each with a purity of over 96% as determined by HPLC. The structures of the isolated compounds were identified by electron ionization MS (EI-MS), (1)H NMR, and (13)C NMR.     

Isolation and purification of oridonin from Rabdosia rubescens using upright counter-current chromatography

A preparative counter-current chromatography (CCC) method for isolation and purification of oridonin, a new cancer chemoprevention agent, from the Chinese medicinal plant Rabdosia rubescens was successfully established. The crude oridonin was obtained by elution with a light petroleum/acetone solvent mixture from ethanol extracts of R. rubescens using column chromatography on silica gel. With a two-phase solvent system composed of n-hexane/ethyl acetate/methanol/water (1:2:1:2, v/v), 120 mg of oridonin at the purity of 97.8% was obtained from 200 mg of the crude sample in a single-step CCC separation. The structure of oridonin was identified by ESI-MS, 1H NMR, and 13C NMR.     

Preparative purification of five bioactive components from Agrimonia pilosa Ledeb by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) coupled with ultraviolet (UV) detection or evaporative light-scattering detection was successfully applied for preparative separation of five bioactive compounds from Agrimonia pilosa Ledeb. In preliminary process, D101 macroporous resin was used to separate the crude extract of the plant and four fractions (20, 40, 50, and 60% aqueous ethanol elutions) were produced. Then, these fractions were directly subjected to HSCCC purification. Five chemicals including taxifolin-3-glucoside (6.4 mg), quercetin-3-rhamnoside (13.0 mg), tiliroside (14.7 mg), agrimonolide (21.4 mg), and tormentic acid (29.8 mg) with the purities of 94.24, 95.37, 97.42, 95.29, and 96.34% were separated from each 200 mg prepared fraction. The purities were analyzed by high-performance liquid chromatography, and the chemical structures of the products were identified by UV detection, mass spectrometry, nuclear magnetic resonance, and the standards. This paper used a simple method to separate five bioactive compounds from A. pilosa Ledeb, and it could provide a new idea for the purification of bioactive compounds from other medicinal plants.     

Separation and purification of four compounds from Desmodium styracifolium using off‐line two‐dimensional high‐speed counter‐current chromatography

An off‐line 2D high‐speed counter‐current chromatography technique in preparative scale has been successfully applied to separate and purify the main compounds from the ethyl acetate extract of Desmodium styracifolium. A two‐phase solvent system composed of n‐hexane/ethyl acetate/methanol/water at an optimized volume ratio of 1:2:1:2 v/v/v/v was used. Conventional high‐speed counter‐current chromatography was used as the first dimension, and the upper phase of the solvent system was used as the stationary phase in the head‐to‐tail elution mode at a flow rate of 2.0 mL/min and a rotation speed of 900 rpm. Recycling high‐speed counter‐current chromatography served as the second dimension to separate an impure fraction of the first dimension. A total of four well‐separated substances including vanillic acid ( 1 ), β‐sitosterol ( 2 ), formononetin ( 3 ), and aromadendrin ( 4 ) were obtained, and their purities and structures were identified by HPLC–MS and ¹H NMR spectroscopy. The results illustrated that off‐line 2D high‐speed counter‐current chromatography is an effective way to isolate compounds in complex samples.     

Preparative isolation and purification of atractylon and atractylenolide III from the Chinese medicinal plant atractylodes macrocephala by high-speed counter-current chromatography

The medicinal plant Atractylodes macrocephala (Baizhu in Chinese) has been widely used in traditional Chinese medicine for energy and stomach complaints, treatment of dyspepsia and anorexia, anti-inflammation, anticancer and for increasing assimilation. A high-speed counter-current chromatography (HSCCC) method was developed for the preparative separation and purification of two main bioactive components, namely, atractylon and atractylenolide III from A. macrocephala by using light petroleum (60-90 degrees C)-ethyl acetate-ethanol-water (4:1:4:1 v/v) as the two-phase solvent system in dual-mode elution. Compared with the separation using the normal-mode elution, the dual-mode HSCCC can be achieved with shorter elution time. Atractylenolide III (32.1 mg) at 99.0% purity and 319.6 mg atractylon at 97.8% purity could be obtained from 1000 mg crude sample in a single run. The recoveries of atractylenolide III and atractylon were 95.4 and 92.6%, respectively.