Combination Of High-speed Counter-current Chromatography Research Articles

Characterization of Tyrosinase Inhibitors in Dryopteris crassirhizoma Rhizome Using a Combination of High-Speed Counter-Current Chromatography, Affinity-Based Ultrafiltration, and Liquid Chromatography-Tandem Mass Spectrometry.

Dryopteris crassirhizoma rhizome (DCR) inhibits melanin production in B16F10 melanoma cells and tyrosinase activity. The melanin content and tyrosinase activity of DCR-treated zebrafish embryos were determined to evaluate the in vivo inhibitory effect of DCR on melanogenesis. Moreover, an off-line hyphenated method combining the high-speed counter-current chromatography, affinity-based ultrafiltration, and liquid chromatography–tandem mass spectrometry was used to identify and characterize the DCR compounds with tyrosinase inhibitory activity. Our results indicated that DCR significantly decreased the melanin content and tyrosinase activity in zebrafish embryos in a dose-dependent manner; moreover, 22 compounds in DCR presented tyrosinase inhibitory activity. In silico molecular docking prediction data indicated that the 22 compounds in DCR can form stable conformations in the active site pocket of tyrosinase.

Systematic Separation and Purification of Alkaloids from Euchresta tubulosa Dunn. by Various Chromatographic Methods

High-speed countercurrent chromatography (HSCCC) and silica gel column chromatography were used to separate and purify alkaloids from Chinese herbal medicine Euchresta tubulosa Dunn. The purpose of this study is to provide a system mode for rapid separation of alkaloids from natural products. In the experiment, the eluent of silica gel column chromatography was screened by thin layer chromatography (TLC) to obtain four components with different polarity. Then, the two-phase solvent systems of different components were selected and purified by HSCCC. Four alkaloids with relatively high content were obtained by this mode successfully, including matrine (28 mg), oxymatrine (32 mg), N-formyl cytisine (24 mg), and cytisine (58 mg). The purity was higher than 91% by high performance liquid chromatography–ultraviolet (HPLC-UV) and their chemical structures were identified by nuclear magnetic resonance (NMR) and electron ionization mass spectrometry (EI-MS). The results showed that the combination of HSCCC and silica gel column chromatography could make alkaloids from natural products separate systematically.

Isolation and Purification of a Neuroprotective Phlorotannin from the Marine Algae Ecklonia maxima by Size Exclusion and High-Speed Counter-Current Chromatography.

Phlorotannins are polyphenolic metabolites of marine brown algae that have been shown to possess health-beneficial biological activities. An efficient approach using a combination of high-speed counter-current chromatography (HSCCC) and size exclusion chromatography with a Sephadex LH-20 has been successfully developed for the isolation and purification of a neuroprotective phlorotannin, eckmaxol, from leaves of the marine brown algae, Ecklonia maxima. The phlorotannin of interest, eckmaxol, was isolated with purity >95% by HSCCC using an optimized solvent system composed of n-hexane–ethyl acetate–methanol–water (2:8:3:7, v/v/v/v) after Sephadex LH-20 size exclusion chromatography. This compound was successfully purified in the quantity of 5.2 mg from 0.3 kg of the E. maxima crude organic extract. The structure of eckmaxol was identified and assigned by NMR spectroscopic and mass spectrometric analyses. The purification method developed for eckmaxol will facilitate the further investigation and development of this neuroprotective agent as a drug lead or pharmacological probe. Furthermore, it is suggested that the combination of HSCCC and size exclusion chromatography could be more widely applied for the isolation and purification of phlorotannins from marine algae.

Preparation from Lepidium meyenii Walpers using high-speed countercurrent chromatography and thermal stability of macamides in air at various temperatures

Originally cultivated in the Peruvian Andes, Maca (Lepidium meyenii) is now widely consumed as functional food which is known for its functional characteristic like relieving fatigue and enhancing stamina. Macamides are the unique fatty acids with long-chain and N-benzylamides among the various constituents extracted from Maca. N-benzyl-(9Z, 12Z, 15Z)-octadecatrienamide, N-benzyl-(9Z, 12Z)-octadecadienamide, N-benzylhexadecanamide and N-benzyl-9Z-octadecenamide were isolated from crude Maca extracts in two steps by using a combination of high-speed countercurrent chromatography (HSCCC) and semi-preparative HPLC. The four macamides were identified by NMR, UV, HRMS and IR spectra. The thermal stability and autoxidative kinetics for N-benzyl-(9Z, 12Z, 15Z)-octadecatrienamide and N-benzyl-(9Z, 12Z)-octadecadienamide in the air were also studied at various temperatures and the apparent activation energies were 28.4 kJ/mol and 29.9 kJ/mol, respectively. The purity of the isolated macamides was up to 98% and the amount was enough for application as a reference substance.

Separation and Purification of Five Flavone Glucosides and One Lignan from Caragana korshinskii Kom. by the Combination of HSCCC and Semi-preparative RPLC

A method of using high-speed counter-current chromatography (HSCCC) and semi-preparative reversed-phase liquid chromatography (semi-preparative RPLC) to preparatively separate flavone glucosides and lignan from the crude extracts of Caragana korshinskii has been established for the first time in this study. Five flavone glucosides, including 9 mg of kaempferol 3-O-{β-d-glucopyranosyl(1→2)-[α-l-rhamonopyranosyl(1 → 6)]-β-d-galactopyranoside}, 21 mg of kaempferol 3-O-α-l-rhamnopyranosyl(1→6)-β-d-galactopyranoside-7-O-α-l-rhamnopyranoside, 34 mg of kaempferol 3-O-β-d-galactopyranoside-7-O-α-l-rhamnopyranoside, 27 mg of kaempferol 3-O-β-d-glucopyranosyl-7-O-α-l-rhamnopyranoside, and 14 mg of calycosin 7-O-β-d-glucopyranoside, and one lignan, 16 mg of alangilignoside B, were successfully isolated from 1.8 g of the crude sample through the combination of HSCCC with a two-phase solvent system composed of ethyl acetate–n-butanol–0.5 % formic acid (4:1:5, v/v/v) and semi-preparative RPLC with a mobile phase of methanol–water (20:80, v/v). The purities of the six compounds are all over 95 % as determined by HPLC and the structures are confirmed by the analysis of UV, 1H-NMR, and 13C-NMR and compared with published data.

Fast isolation of cytotoxic compounds from the native Chilean species Gypothamnium pinifolium Phil. collected in the Atacama Desert, northern Chile

Gyphothamnium pinifolium Phil. is an evergreen Chilean native bush with white–violet flowers growing in Paposo valley in the cost of the Atacama Desert. The combination of high-speed counter-current chromatography (HSCCC) and medium pressure column chromatography allowed the fast isolation and purification of one rare phenolic derivative: 2-Nor-1,2-secolycoserone, one 5-methyl-coumarin: Aphyllodenticulide, one ent-labdane: ent-labda-8,13-E-diene-15-ol (compounds 1–3) and a inseparable mixture of common sterols (mainly composed of lupeol and taraxerol) from this Chilean species. In order to prepare the main individual compounds, a rapid enrichment step (only 100min) was achieved by HSCCC using the solvent system n-hexane: ethyl acetate: acetonitrile 5:2:5 v:v:v, followed by a fast final purification step of the mentioned compounds by normal phase medium pressure column chromatography (MPC). The isolated compounds were evaluated against breast cancer MCF-7 cells, showing good cytotoxic activity (<50% cell viability at 100μM). Besides, compound 1 showed the best cytotoxicity, (IC50=15.12μM), but was also toxic against its normal counterpart MCF-10F cells (IC50=13.79μM). Based on these results, it is concluded that HSCCC is a viable separation method for the purification of novel compounds from Gyphotamnium pinifolium with cytotoxic activity in cancer cells. Furthermore, the X-ray crystal-structure for compound 2 is reported in this paper for the first time.

Enhancement of Diosgenin Production in Plantlet and Cell Cultures of &lt;i&gt;Dioscorea zingiberensis&lt;/i&gt; by Palmarumycin C&lt;sub&gt;13&lt;/sub&gt; from the Endophytic fungus, &lt;i&gt;Berkleasmium &lt;/i&gt;sp. Dzf12

Purpose: To study the effect of palmarumycin C13, an elicitor from the endophytic fungus Berkleasmium sp. Dzf12, on growth and diosgenin production in plantlet or cell cultures of its host plant, Dioscorea zingiberensis.Methods: Palmarumycin C13 was isolated from the ethyl acetate extract of the endophytic fungus Berkleasmium sp. Dzf12 using a combination of high-speed counter-current chromatography (HSCCC), Sephadex LH-20 chromatography and preparative high performance liquid chromatography (HPLC). The biomass of the plantlet and cell cultures of D. zingiberensis as well as their diosgenin content and yield were analyzed after treatment with palmarumycin C13.Results: Optimal elicitation of diosgenin production by palmarumycin C13 in D. zingiberensis plantlet and cell cultures was achieved when palmarumycin C13 was added to the medium at a concentration of 60 mg/L (for plantlet culture) at the beginning of culturing or 10 mg/L (for cell culture) on day 25 after inoculation. By using these optimal concentrations, the diosgenin yield of the cultured plantlets reached its maximum of 6.44 mg/L, that is, &gt; 1.4-fold increase, while diosgenin yield of the cultured cells reached a maximum of 10.73 mg/L, which is an &gt; 8.0-fold increase.Conclusion: Addition of palmarumycin C13 from the endophytic fungus, Berkleasmium sp. Dzf12, is a potentially effective strategy for enhancing diosgenin production in D. zingiberensis plantlet and cell cultures.Keywords: Endophytic fungus, Berkleasmium sp. Dzf12, Spirobisnaphthalene, Palmarumycin C13, Dioscorea zingiberensis, Diosgenin, Elicitation, Cell culture, Plantlet culture

Rapid Screening, Identification, Separation, and Purification of Four Bioactive Compounds from Swertia mussotii Franch

Gentiopicroside, mangiferin, sweroside, and isoorientin, the bioactive constituents of Swertia mussotii Franch, have various pharmacological effects, and are used in particular for treating liver disorders. However, efficient methods for their separation are not currently available. In this study, these bioactive compounds were detected in S. mussotii extracts using high-performance liquid chromatography coupled with mass spectrometry, and separated using a combination of high-speed counter-current chromatography and Sephadex LH-20 column chromatography. Their structures were determined by using 1H and 13C nuclear magnetic resonance spectroscopies. The results show that this method is effective for the separation and purification of bioactive compounds from S. mussotii.

Purification of six lignans from the stems of Schisandra chinensis by using high-speed counter-current chromatography combined with preparative high-performance liquid chromatography

A method for the preparative purification of lignans from Schisandra chinensis was established using a combination of high-speed counter-current chromatography (HSCCC) and preparative high-performance liquid chromatography (HPLC). The crude extracts obtained from S. chinensis by using 70% ethanol were separated on a macroporous resin column and then eluted with a graded ethanol series. A two-phase solvent system consisting of n-hexane–ethyl acetate–methanol–water (1:1:1:1, v/v) was used for HSCCC, and a mobile phase of acetonitrile–water (50:50, v/v) was used for preparative HPLC. The results obtained using HSCCC were compared with those obtained using preparative HPLC, and their advantages were further integrated to improve the separation efficiency. Six known lignans were identified by electrospray ionisation mass spectrometry and 1H nuclear magnetic resonance (NMR) and 13C NMR analyses; the purities of all the compounds were more than 91%.

Separation and purification of bioactive botrallin and TMC-264 by a combination of HSCCC and semi-preparative HPLC from endophytic fungus Hyalodendriella sp. Ponipodef12

Two dibenzo-α-pyrones, botrallin (1) and TMC-264 (2) were preparatively separated from crude ethyl acetate extract of the endophytic fungus Hyalodendriella sp. Ponipodef12, which was isolated from the hybrid 'Neva' of Populus deltoides Marsh × P. nigra L. using a combination of high-speed counter-current chromatography (HSCCC) and semi-preparative HPLC. Botrallin (1) with 74.73% of purity and TMC-264 (2) with 82.29% of purity were obtained through HSCCC by employing a solvent system containing n-hexane-ethyl acetate-methanol-water at a volume ratio of 1.2:1.0:0.9:1.0. It was the first time for TMC-264 (2) to be isolated from this fungus. TMC-264 (2) showed strong antimicrobial and antinematodal activity, and botrallin (1) exhibited moderate inhibitory activity on acetylcholinesterase.

Analysis and determination of oestrogen-active compounds in fructus amomi by the combination of high-speed counter-current chromatography and high performance liquid chromatography

Amomum longiligulare or Amomum villosum showed oestrogenic activity. In the present study, oestrogen-active components in fructus amomi, the seeds of A. longiligulare were separated by high-speed countercurrent chromatography (HSCCC) using stepwise elution of eight mobile phases with gradient polarity and advanced separation by high performance liquid chromatography (HPLC). The results yielded 17 compounds with the amount of 8-138mg and a purity of 94.3-99.8% from a 3g ethanolic extract of fructus amomi. The chemical structures of the compounds were identified by ESI-MS and NMR spectra, in which eight diarylheptanoids were demonstrated as the main oestrogen-active compounds in the fructus amomi. Determination of the diarylheptanoids in fructus amomi from various origins showed that fructus amomi contains more than 0.5% total diarylheptanoids. The results showed that fructus amomi is a diarylheptanoids-rich food resource possessing oestrogen-activity. The combination method of HSCCC and HPLC can be applied for the analysis of bioactive compounds by detecting the corresponding bioactivity in the HSCCC fractions and separating the target compounds with HPLC.

Efficient preparative isolation and identification of walnut bioactive components using high-speed counter-current chromatography and LC-ESI-IT-TOF-MS

Preparative isolation of complex mixtures of compounds from walnut polar extracts was established by a combination of high-speed counter-current chromatography (HSCCC) and electrospray ionization-ion trap-time of flight mass spectrometry (ESI-IT-TOF-MS). Compounds were isolated after a solvent optimisation selection based on solute distribution in a biphasic solvent system. Isolation was achieved through one or two successive HSCCC runs, and final purification on Sephadex LH-20. Isolated compounds included ellagitannins (1–11), gallic acid (12), dicarboxylic acid glucosides (13–15), hydrojuglone glucoside (16), catechin (17), procyanidin B2 (18), and megasterone glucosides (19–20). Praecoxin D (4) was isolated for the first time from walnut, while praecoxin A methyl ester (5) and glansreginin A n-butyl ester (14) are newly identified compounds. The purity and identity of isolated compounds were confirmed by NMR and HPLC-ESI-MS/MS. These results provided a foundation for in depth characterisation of walnut compounds and offered an efficient strategy for isolation of potentially health-relevant phytochemicals from walnuts.

Isolation and Identification of Two Novel Attractant Compounds from Chinese Cockroach (Eupolyphaga sinensis Walker) by Combination of HSCCC, NMR and CD Techniques

High-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hextane-ethyl acetate-methanol-water (1.5:1:1.5:1, v/v/v/v) was applied to the isolation and purification of attractants from Chinese cockroach, Eupolyphaga sinensis Walker. Two new attractants with attractant activity towards the male insects were obtained from the extract sample in a one-step separation. Their purities were determined by HPLC. Subsequent MS, NMR and CD analyses have led to the characterization of (R)-3-ethyl-6,8-dihydroxy-7-methyl-3,4-dihydroisochromen-1-one (1) and (R)-6,8-dihydroxy-3,7-dimethyl-3,4-dihydroisochromen-1-one (2), two novel isocumarin type attractants. Based on these results, it is concluded that HSCCC is a viable separation method option for purifying insect attractants, while effectively maintaining the attracting activity of the isolates. This is the first attempt to apply counter-current chromatography technique to separate attractants from Chinese cockroach.

Fishing and knockout of bioactive compounds using a combination of high-speed counter-current chromatography (HSCCC) and preparative HPLC for evaluating the holistic efficacy and interaction of the components of Herba Epimedii

Due to the complex chemical compositions and pharmacological effects of traditional Chinese medicines, we developed a strategy based on fishing and knockout of bioactive compounds using a combination of high-speed counter-current chromatography (HSCCC) and preparative HPLC for evaluating the holistic activity and interaction of the components of Herba Epimedii. First, osteoblast target cell extraction was used for preliminary screening of the potential bioactive compounds of Herba Epimedii. Second, the bioactive compounds identified (epimedin A, epimedin B, epimedin C and icariin) were fished and knocked out using high-speed counter-current chromatography and preparative HPLC. Third, the bioactivity of resulting fractions was assessed by determining their influence on cell proliferation and differentiation, thereby allowing for an evaluation of their interaction.The pharmacodynamic contribution ratio of each bioactive compound to the efficacy of the herbal medicine could then be comprehensively and intuitively determined based on the spectra-activity correlations (VIP values) of the tested compositions using partial least-squares regression (PLS-R), through which the reliability of the screening and isolation of bioactive compounds by the target cell extraction technique were verified. The proposed strategy is a useful approach with potential application in other traditional Chinese medicines.

(6R,9S)-6″-(4″-Hydroxybenzoyl)-Roseoside, a New Megastigmane Derivative fromOuratea polyanthaand its Effect on Hepatic Glucose-6-phosphatase

A new megastigmane derivative, (6R,9S)-6'-(4"-hydroxybenzoyl)-roseoside (1) and two known compounds, the biflavoneagathisflavone (2) and 4-hydroxybenzoic acid (3) were isolated and purified from leaves and stems of Ouratea polyantha Engl. Agathisflavone was isolated in a single high-speed countercurrent chromatography run, while the megastigmane was purified in two steps, by using a combination of high-speed countercurrent chromatography and analytical column chromatography. All structures were elucidated on the basis of spectral evidence and comparison with literature data. Compound 1 was characterized by [alpha]D20, UV-Vis, IR, MS, 1H NMR, 13C NMR, HMQC, HMBC, COSY and NOESY. Compounds 1 and 2 showed an inhibitory effect of 63.6 and 13.7% on the G-6-Pase intact microsomes, respectively.

Combination of high-speed countercurrent chromatography and reversed phase C18 chromatography for large-scale isolation of cyanidin-3-O-β-d-glucoside from black rice bran extract

Cyanidin-3-O-β-d-glucoside (C3G), the best known and most investigated anthocyanin, has many pharmacological properties. Black rice bran is an outstanding source for the separation of C3G due to its high C3G content and a simple anthocyanin composition. The present study described a large-scale isolation of C3G from the crude black rice bran extract by combining high-speed countercurrent chromatography (HSCCC) and reversed phase C18 column chromatography. After HSCCC enrichment with the solvent system water–n-butanol–tert-butyl-methyl-ether (TBME)–acetonitrile–trifluoroacetic acid (TFA) (5:2:2:1:0.1%), the C3G content was concentrated about 16-fold and reached to 563.23mg/g with a recovery 96.89%. Further purification was carried out using reversed phase C18 column chromatography and 1.43g of pure C3G (purity 94.38%) was obtained from 3.0g of anthocyanin-enriched extract. The method is time-saving, low risk of sample denaturation, high sample recovery, and so is suitable for large-scale preparation of C3G for further studies of bioactivities.

Preparative isolation and purification of four flavonoids from the petals of nelumbo nucifera by high‐speed counter‐current chromatography

Flavonoids, the primary constituents of the petals of Nelumbo nucifera, are known to have antioxidant properties and antibacterial bioactivities. However, efficient methods for the preparative isolation and purification of flavonoids from this plant are not currently available. To develop an efficient method for the preparative isolation and purification of flavonoids from the petals of N. nucifera by high-speed counter-current chromatography (HSCCC). Following an initial clean-up step on a polyamide column, HSCCC was utilised to separate and purify flavonoids. Purities and identities of the isolated compounds were established by HPLC-PAD, ESI-MS, (1)H-NMR and (13)C-NMR. The separation was performed using a two-phase solvent system composed of ethyl acetate-methanol-water-acetic acid (4 : 1 : 5 : 0.1, by volume), in which the upper phase was used as the stationary phase and the lower phase was used as the mobile phase at a flow-rate of 1.0 mL/min in the head-to-tail elution mode. Ultimately, 5.0 mg syringetin-3-O-beta-d-glucoside, 6.5 mg quercetin-3-O-beta-d-glucoside, 12.8 mg isorhamnetin-3-O-beta-d-glucoside and 32.5 mg kaempferol-3-O-beta-d-glucoside were obtained from 125 mg crude sample. The combination of HSCCC with a polyamide column is an efficient method for the preparative separation and purification of flavonoids from the petals of N. nucifera.

Excellent combination of counter-current chromatography and preparative high-performance liquid chromatography to separate galactolipids from pumpkin

Galactolipids in the fruits of Cucurbita moschata (pumpkin) could not be completely separated by high-performance liquid chromatography (HPLC). Preparative HPLC was not available for preparing major galactolipid monomers in pumpkin. In the present paper, a combination of high-speed counter-current chromatography (HSCCC) and preparative HPLC was used for preparing the galactolipids. A fraction containing galactolipids (Fr60) from the purification of the n-butanol extract of pumpkin by macro-porous resin column chromatography was first separated by HSCCC to result in three sub-fractions of each containing two galactolipid monomers. The three sub-fractions were further separated by preparative HPLC respectively to yield six galactolipid monomers with purity more than 96%. The method is a good one for preparing galactolipid monomers from plant materials for the studies of bioactivities.

Combination of HSCCC and Sephadex LH-20 methods: An approach to isolation and purification of the main individual theaflavins from black tea

In order to separate the main individual theaflavin monomers from black tea, high-speed countercurrent chromatography (HSCCC) and Sephadex LH-20 column chromatography were applied. The results showed that theaflavin (TF1), theaflavin-3-gallate (TF2A), theaflavin-3′-gallate (TF2B) and theaflavin-3,3′-digallate (TF3) can be obtained by HSCCC using a solvent system composed of n-hexane–ethyl acetate–methanol–water (1:3:1:6, v/v/v/v), but the TF1 was containing epicatechin-3-gallate (ECG). Similarly, Sephadex LH-20 can also effectively separate TF2A(B) and TF3, but epigallocatechin-3-gallate (EGCG) contaminated TF1, too. Combination of HSCCC and Sephadex LH-20, the preferably purified TF1, TF2A(B) and TF3 were obtained than single separation technique. In addition, ECG and EGCG were also suggested to be able to be comprehensively separated by combination of the two techniques.

Isolation and identification of 5-carboxy-pyranoanthocyanins in beverages from cherry (Prunus cerasus L.)

Abstract Novel pyranoanthocyanins were identified by HPLC–ESI-MSn in fermented and unfermented fruit juices from cherry (Prunus cerasus L.). The two major compounds, namely 5-carboxy-pyranocyanidin 3-O-(2G-glucosylrutinoside) and 5-carboxy-pyranocyanidin 3-O-rutinoside were isolated by a combination of high-speed countercurrent chromatography with semi-preparative HPLC. Their structures were fully elucidated by means of NMR spectroscopy and mass spectrometry. As minor compounds 5-carboxy-pyranocyanidin 3-glucoside, 5-carboxy-pyranopeonidin-3-glucoside, and 5-carboxy-pyranopeonidin 3-rutinoside could be identified. These compounds are formed during storage of the juice through the reaction of pyruvic acid with the respective genuine anthocyanins. Industrial relevance For the consumers the color of fruit juices is one of the most important quality markers. However due to a fast degradation of anthocyanins, changes in color from purple/red hues to orange/brown hues can be observed during storage. The main objective of this study was the isolation, structural characterization and evaluation of pigments that are formed during aging of fruit juices. Pyranoanthocyanins were reported to possess a color stabilizing effect. In the present study it could be demonstrated that the color contribution of these newly formed pigments to the overall color of fruit juice was overestimated. Instead, the formation of polymeric pigments yields the highest impact (> 80%) on the color of aged fruit juices.