Countercurrent Chromatography For Isolation Research Articles

Countercurrent chromatography isolation of 11′‐γ‐tocomonoenol from pumpkin seed oil with detection of novel minor tocochromanols

AbstractA non‐refined, organic pumpkin seed oil (PSO) was chosen for the isolation and structure verification of the rare vitamin E compound γ‐tocomonoenol (γ‐T1). Initial measurements indicated the presence of ~0.4 mg γ‐T1 per 100 g PSO. Saponification of ~2 L of PSO, followed by repeated countercurrent chromatography (CCC) with the solvent system n‐hexane/benzotrifluoride/acetonitrile (v/v/v) and silica gel column chromatography enabled the isolation of 6.8 mg γ‐T1 with a purity of 96.0% according to gas chromatography with mass spectrometry (GC/MS) analysis. Structural analysis by 1H nuclear magnetic resonance (NMR) spectroscopy and GC/MS of the γ‐T1 isolate confirmed the presence of a double bond in C‐11′‐position (11′‐γ‐tocomonoenol). In addition, CCC fractionation enabled the detection of 18 different tocochromanols, many of which were reported for the first time in PSO. This unmatched variety included among others α‐/γ‐tocopherol, α‐/γ‐tocomonoenol, two α‐ and two γ‐tocodienol isomers, α‐/γ‐tocotrienol as well as the rare 11′‐β‐tocomonoenol (β‐T1) and δ‐T1. Three uncommon tocochromanols were also detected whose origins and structure remained unclear.

High-speed countercurrent chromatography for isolation and enrichment of betacyanins from fresh and dried leaves of Atriplex hortensis L. var. "Rubra".

Betacyanins and their decarboxylated derivatives from fresh and dried edible leaves of Atriplex hortensis L. var. "Rubra" were fractionated for the first time by high-speed countercurrent chromatography. Pigments present in fresh leaf extract were separated in systems: ethanol - acetonitrile - n-propanol - ammonium sulphate - water (0.5:0.5:0.5:1.2:1.0, v/v/v/v/v) (tail-to-head mode) and tert-butyl methyl ether - n-butanol - acetonitrile - water with 0.7% heptafluorobutyric acid (2:2:1:5, v/v/v/v) (head-to-tail mode). The mobile phase flow rate was 2mL/min and the retention of the stationary phase was 79.8 and 75.2%, respectively. Pigments from dried leaves were separated in a similar ion-pair system with heptafluorobutyric acid in different volume proportions 1:3:1:5 (head-to-tail mode) and the flow rate of the mobile phase 3mL/min. The stationary phase retention was 64.0%. The application of the countercurrent chromatography for the fractionation of betacyanins from leaves of Atriplex hortensis enabled to isolate and pre-concentrate the pigments for further low- and high-resolution liquid chromatographic-tandem mass spectrometric detection. This study revealed the presence of 10 betacyanins in fresh and 16 in dried leaves of Atriplex hortensis. Two compounds were not previously identified in the whole Amaranthaceae family. Additionally, instead of (iso)amaranthin, celosianin and its epimer were dominant betacyanins in the Atriplex hortensis.

On-line coupling pressurised liquid extraction with two-dimensional counter current chromatography for isolation of natural acetylcholinesterase inhibitors from Astragalus membranaceus.

Radix Astragali, the dried root of Astragalus membranaceus (Fish.) Bge. (family Fabaceae), which is known as Huangqi in China, has been proven to be an immunostimulant, diuretic, antidiabetic, analgesic, and it has also been used as a health food supplement in some Asian populations and also serves as a lead herb in many traditional Chinese medicine formulations as well as in Chinese ethnic tonifying soups. Screening and purification of bioactive compounds from natural products is challenging work due to their complexity. We present the first report on the use of pressurised liquid extraction and on-line two-dimensional counter current chromatography as an efficient medium for scaled-up extraction and separation of six bioactive compounds from Astragalus membranaceus. We applied the established method with ultrafiltration-liquid chromatography to screen acetylcholinesterase inhibitors, which were then evaluated and confirmed for anti-Alzheimer activity using PC12 cell model. Six major compounds, namely, calycosin-7-O-β-d-glucoside, pratensein-7-O-β-d-glucoside, formononetin-7-O-β-d-glucoside, calycosin, genistein, and formononetin, with acetylcholinesterase binding affinities were identified and isolated from the raw plant materials via two sets of n-hexane/ethyl acetate/0.2% acetic acid (first-stage counter current chromatography) and n-hexane/ethyl acetate/methanol/water (second-stage counter current chromatography) solvent systems: 1.87:1.0:1.33 and 5.62:1.0:2.42:5.25, v/v/v/v, which were optimised by a mathematical model. Therefore, a useful platform for the large-scale production of bioactive and nutraceutical ingredients was developed herein. With the on-line system developed here, we present a feasible, selective, and effective strategy for rapid screening and identification of enzyme inhibitors from complex mixtures.

High-performance counter-current chromatography isolation and initial neuroactivity characterization of furanocoumarin derivatives from Peucedanum alsaticum L (Apiaceae)

BackgroundMedicinal plants are a proven source of drug-like small molecules with activity towards targets relevant for diseases of the central nervous system (CNS). Plant species of the Apiaceae family have to date yielded a number of neuroactive metabolites, such as coumarin derivatives with acetylcholinesterase inhibitory activity or anti-seizure activity. PurposeTo accelerate the discovery of neuroactive phytochemicals with potential as CNS drug leads, we sought to rapidly isolate furanocoumarins, primary constituents of the dichloromethane (DCM) extract of the fruits of Peucedanum alsaticum L. (Apiaceae), using high-performance counter-current chromatography (HPCCC) and to evaluate their neuroactivity using both in vitro and in vivo microscale bioassays based on cholinesterase ELISAs and zebrafish epilepsy models. Research methods and procedureIn this study the DCM extract was subjected to HPCCC for the efficient separation (60 min) and isolation of furanocoumarins. Isolated compounds were identified with TOF-ESI-MS and NMR techniques and examined as inhibitors of AChE and BChE using ELISA microtiter assays. Anti-seizure properties of the extract and of the isolated compounds were evaluated using a zebrafish epilepsy model based on the GABAA antagonist pentylenetetrazol (PTZ), which induces increased locomotor activity and seizure-like behavior. ResultsThe solvent system, composed of n-heptane, ethyl acetate, methanol and water (3:1:3:1, v/v/v/v), enabled the isolation of 2.63 mg lucidafuranocoumarin A (purity 98%) and 8.82 mg bergamottin (purity 96%) from 1.6 g crude DCM extract. The crude extract, at a concentration of 100 µg/ml, exhibited a weak inhibitory activity against acetylcholinesterase (AChE) (9.63 ± 1.59%) and a moderate inhibitory activity against butyrylcholinestrase (BChE) (49.41 ± 2.19%). Lucidafuranocoumarin A (100 µg/ml) was inactive against AChE but showed moderate inhibition towards BChE (40.66 ± 1.25%). The DCM extract of P. alsaticum fruits (0.62–1.75 µg/ml) and bergamottin (2–10 µm) exhibited weak anti-seizure activity, while lucidafuranocoumarin A (10–16 µm) was found to significantly inhibit PTZ-induced seizures. The percentage of seizure inhibition for the isolated compounds, at their most bioactive concentration, was 26% for bergamottin and 69% for lucidafuranocoumarin A. ConclusionOur findings underscore the utility of HPCCC for the rapid isolation of rare coumarin derivatives, and the potential of microscale in vivo bioassays based on zebrafish disease models for the rapid assessment of neuroactivity of these drug-like natural products.

K-targeted strategy for isolation of phenolic alkaloids of Nelumbo nucifera Gaertn by counter-current chromatography using lysine as a pH regulator

Counter-current chromatography (CCC) is an efficient liquid-liquid partition chromatography technique without support matrix. Despite there are many significant advancements in the CCC separation of natural products especially for non-ionic neutral compounds, CCC isolation of ionic compounds including alkaloids is still a challenging process guide by classical partition coefficients (K) or distribution ratio (KC) because their partition coefficient could not be equal to distribution ratio in common ionic conditions. Here, taking the extract of embryo of the seed of Nelumbo nucifera Gaertn as sample, we introduced a modified K-targeted strategy for isolation of phenolic alkaloids by use of lysine as a pH regulator. The results indicated that if the mass of basic regulators such as aqueous ammonia and lysine added into the solvent system were high enough to inhibit the ionization of the targeted alkaloids, the distribution ratio of targets with ionic and non-ionic molecular forms got stable and might not been changed as the concentration of the pH regulator. In this case, the distribution ratio of target was almost equal to the partition coefficient. Thus, the targets could be isolated by K-targeted CCC separation through adding a certain amount pH regulators into the solvent system. Further experiments also showed that the sample concentration was an important factor on the distribution ratio of targets. Meanwhile, CCC experiments indicated that lysine was more suitable than aqueous ammonia for the separation of phenolic alkaloids because the chemical property of lysine-target complex in the CCC fractions was more stable. Therefore, the preparative CCC separation was performed using 20mM lysine as a pH regulator with more than 800mg injection mass. After simple back-extraction with dichloromethane, the lysine in the CCC fraction was removed completely and pure isoliensinine and neferine were obtained. In summary, the whole results indicated that the modified K-targeted CCC strategy using lysine as the pH regulator was efficient for isolation of phenolic alkaloids from crude plant extracts. It not only provided a practical strategy for the isolation of neferine and its analogues, but also introduced a powerful method to resolve the peak skewing (leading or tailing) in CCC separation of ionic compounds.

Sugaring-out strategy for counter-current chromatography isolation: podophyllotoxins and flavones from Dysosma versipellis as examples

In this work, we introduced a sugaring-out strategy for optimizing two-phase solvent systems for counter-current chromatography by adding some sugars into the selected two-phase solvent system.

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.

Preparative isolation of a cytotoxic principle of a forest mushroom Suillus luteus by sodium dodecyl sulfate based "salting-in" countercurrent chromatography.

In the course of screening new anticancer natural products, an edible forest mushroom Suillus luteus (L. Ex Franch). Gray was found to have potent cytotoxicity against several human cancer cells. However, the lipophilic sample made some countercurrent chromatography solvent systems emulsify, which caused difficulties in the separation of its cytotoxic components. Here, we found that the addition of an organic salt sodium dodecyl sulfate could efficiently shorten the settling time of the mushroom sample solutions by eliminating the emulsification of two-phase solvent systems. Moreover, we found that sodium dodecyl sulfate could play a new "salting-in" role and made the partition coefficients of the solutes decrease with the increased concentrations. Thus, a sodium dodecyl sulfate based salting-in countercurrent chromatography method has been successfully established for the first time for preparative isolation of a cytotoxic principle of the mushroom. The active component was identified as isosuillin. Whole results indicated that sodium dodecyl sulfate could be used as an efficient salting-in reagent for two-phase solvent system selection and targeted countercurrent chromatography isolation. It is very useful for current natural products isolation and drug discovery.

A novel 9 × 9 map-based solvent selection strategy for targeted counter-current chromatography isolation of natural products

Counter-current chromatography (CCC) is an efficient liquid–liquid chromatography technique for separation and purification of complex mixtures like natural products extracts and synthetic chemicals. However, CCC is still a challenging process requiring some special technical knowledge especially in the selection of appropriated solvent systems. In this work, we introduced a new 9×9 map-based solvent selection strategy for CCC isolation of targets, which permit more than 60 hexane–ethyl acetate–methanol–water (HEMWat) solvent systems as the start candidates for the selection of solvent systems. Among these solvent systems, there are clear linear correlations between partition coefficient (K) and the system numbers. Thus, an appropriate CCC solvent system (i.e., sweet spot for K=1) may be hit by measurement of k values of the target only in two random solvent systems. Besides this, surprisingly, we found that through two sweet spots, we could get a line (“Sweet line”) where there are infinite sweet solvent systems being suitable for CCC separation. In these sweet solvent systems, the target has the same partition coefficient (K) but different solubilities. Thus, the better sweet solvent system with higher sample solubility can be obtained for high capacity CCC preparation. Furthermore, we found that there is a zone (“Sweet zone”) where all solvent systems have their own sweet partition coefficients values for the target in range of 0.4<K<2.5 or extended range of 0.25<K<16. All results were validated by using 14 pure GUESSmix mimic natural products as standards and further confirmed by isolation of several targets including honokiol and magnolol from the extracts of Magnolia officinalis Rehd. Et Wils and tanshinone IIA from Salvia miltiorrhiza Bunge. In practice, it is much easier to get a suitable solvent system only by making a simple screening two to four HEMWat two-phase solvent systems to obtain the sweet line or sweet zone without special knowledge or comprehensive standards as references. This is an important advancement for solvent system selection and also will be very useful for isolation of current natural products including Traditional Chinese Medicines.

Separation and purification of 9,10-dihydrophenanthrenes and bibenzyls from Pholidota chinensis by high-speed countercurrent chromatography.

Stilbenoids are the main components of leaves and stems of Pholidota chinensis. In the present investigation, high-speed counter-current chromatography was used for the separation and purification of two classes of stilbenoids, namely, bibenzyls and 9,10-dihydrophenanthrenes, on a preparative scale from whole plants of P. chinensis with different solvent systems after silica gel column chromatography fractionation. n-Hexane/ethyl acetate/methanol/water (1.2:1:1:0.8, v/v/v/v) was selected as the optimum solvent system to purify 1-(3,4,5-trimethoxyphenyl)-1',2'-ethanediol (1), coelonin (2), 3,4'-dihydroxy-5,5'-dimethoxybibenzyl (3), and 2,​7-​dihydroxy-​3,​4,​6-​trimethoxy-​9,​10-​dihydrophenanthrene (4). While 2,7-dihydroxy-3,4,6-trimethoxy-​9,​10-​dihydrophenanthrene (5), batatasin III (6), orchinol (7), and 3'-O-methylbatatasin III (8) were purified by n-hexane/ethyl acetate/methanol/water (1.6:0.8:1.2:0.4, v/v/v/v). After the high-speed counter-current chromatography isolation procedure, the purity of all compounds was over 94% assayed by ultra high performance liquid chromatography. The chemical structure identification of all compounds was carried out by mass spectrometry and (1)H and (13)C NMR spectroscopy. To the best of our knowledge, the current investigation is the first study for the separation and purification of bibenzyls and 9,10-dihydrophenanthrenes by high-speed counter-current chromatography from natural resources.

Application of High-Speed Counter-Current Chromatography for Isolation and Purification of Paclitaxel and Related Taxanes from Taxus chinensis Cell Culture

Six taxanes were isolated and purified from Taxus chinensis cell culture extract using high-speed counter-current chromatography. The crude cell culture extract was first treated with Al2O3 column chromatography and then divided into two parts: fraction 1 and fraction 2. 52 mg of taxuyunnanine C and 11 mg of sinenxane C were yielded from 150 mg of fraction 1. 5 mg of 9-dihydrobaccatin III, 12 mg of baccatin III, 12 mg of paclitaxel, and 17 mg baccatin VI were yielded from 150 mg of fraction 2. The purities of the six compounds were 93.51%, 98.02%, 98.61%, 97.55%, 98.93%, and 98.76%.

Application of High-Speed Counter-Current Chromatography for Isolation of Triterpenes from Schisandra Chinensis (Turcz.) Baill and Induction Apoptosis Mechanism of HSC-T6

Triterpenes have shown many beneficial activities in researches, but their separation and preparation usually require multiple methods. Following an initial cleaning-up step on the AB-8 macroporous resin, a preparative high-speed counter-current chromatography (HSCCC) with a two-phase solvent system comprising chloroform-n-butyl alcohol-methanol-water (10:1:7:4, v/v/v/v) was used to isolate and separate triterpenes from caculis of Schisandra Chinensis (Turcz.) baill. A total of 89 mg corosolic acid with purities of 98.5% were obtained from 400 mg crude extract in one-step elution and less than 4 h, and the structure identification was performed by UV, IR, MS, 1H NMR and 13C NMR. The inhibition on liver fibrosis activities of the triterpenes against HSC-T6 in vitro were studied by cell culture methods. The results showed that the corosolic acid have better inhibitory effects on HSC-T6 cells with the IC50 value of 5~25 μg/mL and the study also indicated that corosolic acid might be a potential Chinese medical component to inhibit liver fibrosis.

Antioxidant Properties and Principal Phenolic Compositions of Cynomorium Songaricum Rupr.

The antioxidant properties of crude acetone extract and its fractions separated using n-hexane, chloroform, ethyl acetate, and butanol of Cynomorium songaricum Rupr. were examined by 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) and 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity assay. The activity of the crude acetone extract was significantly higher than that of vitamin E and lower than that of ascorbic acid. The ethyl acetate fraction had the highest activity among the four fractions. Catechins and proanthocyanidins were identified as the predominant components in ethyl acetate fraction by high performance liquid chromatography–mass spectrometry. Catechin was demonstrated as the major phenolic compound in ethyl acetate fraction of C. songaricum after high-speed counter-current chromatography isolation. In summary, C. songaricum exhibiting high antioxidant activity and abundant procyanidins might be considered as a potential source of antioxidants.

PH-gradient counter-current chromatography isolation of natural antioxidant chlorogenic acid from Lonicera japonica Thumb. using an upright coil planet centrifuge with three multi-layer coils connected in series

A new pH-gradient counter-current chromatography method for the isolation of chlorogenic acid from flowers and buds of Lonicera japonica Thumb. has been successfully established using a novel upright coil planet centrifuge with three multi-layer coils connected in series with 600 mL capacity. The crude extracts were first prepared by direct extraction with hot water and following concentration to remove the solution. Then the two-phase solvent system composed of ethyl acetate– n-butanol–water (2:1:3, v/v) was applied to the separation. Its neutral upper phase was used as stationary phase, whereas both its neutral lower phase and base lower phase with 10 mM NH 3 were employed as mobile phase with gradient elution in the head to tail mode. As a result, 330 mg quantity of crude extract was purified in one-step separation for 180 min, yielding 20.5 mg chlorogenic acid with over 98% purity. Structure of the compound is further identified by electrospray ionization tandem mass spectrometry (ESI-MS/MS) and nuclear magnetic resonance (NMR).

Preparative counter-current chromatography isolation of liensinine and its analogues from embryo of the seed of Nelumbo nucifera GAERTN. using upright coil planet centrifuge with four multilayer coils connected in series

Preparative counter-current chromatography (CCC) isolation of liensinine and its analogues, isoliensinine and neferine from embryo of the seed of Nelumbo nucifera GAERTN. has been successfully performed for the first time using upright coil planet centrifuge with four multilayer coils connected in series with 1600 mL capacity. Two kinds of two-phase solvent systems were applied to preparative CCC isolation. The first was the system composed of light petroleum (b.p. 60–90 °C)–ethyl acetate–tetrachloromethane–chloroform–methanol–water (1:1:4:4:6:2, v/v) which was very suitable for fast and small-scale CCC isolation. The second was the system composed of ethyl acetate–tetrachloromethane–methanol–water (1:6:4:1, v/v), which was the optimum for large-scale CCC isolation. Using the first system, 1102 mg of the crude alkaloid was purified in one-step separation of 150 min, yielding 350 mg neferine, 100 mg isoliensinine and 95 mg liensinine with over 95% purity. While using the second solvent system, 5850 mg of the crude alkaloid was purified in one-step separation of 9 h, yielding 2545 mg neferine, 698 mg isoliensinine and 650 mg liensinine with over 97% purity. Structures of the compounds were identified by electrospray ionization multiple mass spectrometry, one- and two-dimensional NMR.

3-Butyl-5,6-dihydro-4H-isobenzofuran-1-one, a sensorial active phthalide in parsley roots

The analysis of parsley root revealed the presence of a novel phthalide derivative with low odor threshold. The structure 3-butyl-5,6-dihydro-4H-isobenzofuran-1-one followed from interpretation of MS, GC-FTIR, and NMR spectroscopic data. It can be derived from sedanenolide. Due to its restricted stability, a high-speed countercurrent chromatography isolation and purification method, which allows separation from other phthalides, had to be elaborated

Preparative countercurrent chromatography for isolation of charge density-fractionated heparin.

A preparative method is described for continuous flow fractionation of heparin according to charge density in two-phase systems of butanol-hexadecylpyridinium chloride/aquaous NaCl. The separation is carried out on a "Slowly-Rotating Coil" device for countercurrent chromatography with 6 coil units and a sample capacity of 80 mg heparin. A turbidometric method is described which can be used to rapidly quantify the distribution of heparin. Characterization of fractions showed that linear charge density, binding constant for acridine orange, and specific activities in a "global" plasma anticoagulant assay and in a specific activities in a specific biochemical thrombin-inhibition assay varied with the NaCl concentration in the eluting phase. Some partial resolution of the two functional activities was observed. The fractions are much more homogeneous in composition than are the usual heparin preparations and may be useful as standards or in structure-function studies.