pH-zone Refining Countercurrent Chromatography Research Articles

Aporphine Alkaloids from Triclisia dictyophylla Diels by pH-Zone Refining Countercurrent Chromatography

A new dimeric aporphine alkaloid, biscocsarmine, 2, together with lirioferine, 1, and cocsarmine, 3, were isolated from the ethanol extract of the stems of Triclisia dictyophylla Diels (Menispermaceae) using a preparative pH-zone refining CCC in a single step. The first solvent system family tested for this fractionation was hexane–ethyl acetate–methanol–water (HEMWat) at a series of solvent ratios, and hydrochloric acid and triethylamine at 5 mM as eluter/retainer. None of these HEMWat ratios, however, gave suitable K for the target alkaloids. Ethyl acetate–n-butanol–water (EBuWat) was tested instead and the selected solvent system was composed of ethyl acetate–n-butanol–water 5:5:10 (v/v), where triethylamine (60 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. The chemical structures of the isolated compounds were elucidated based on comprehensive spectroscopic and spectrometric techniques. This is the first report of aporphine alkaloids in this plant species.

Efficient extraction and purification of benzo[c]phenanthridine alkaloids from Macleaya cordata (Willd) R. Br. by combination of ultrahigh pressure extraction and pH-zone-refining counter-current chromatography with anti-breast cancer activity in vitro.

Macleaya cordata (Willd) R. Br. (Papaveraceae family) is a well-known traditional Chinese medicine used to treat muscle pain, inflamed wounds, and bee bites. Benzo[c]phenanthridine alkaloids are the main active ingredients in M. cordata. In this work, sanguinarine and chelerythrine were efficiently extracted and purified by ultrahigh-pressure extraction (UHPE) technique and pH-zone-refining counter-current chromatography (PZRCCC) from M. cordata. To develop an efficient UHPE method followed by an efficient separation technique using PZRCCC for benzo[c]phenanthridine alkaloids from the study plant species, and to evaluate the study samples for anti-breast cancer activity. The optimal extraction conditions were optimised as extraction pressure 200 MPa, extraction solvent 95% ethanol, solid-liquid ratio 1:30 (g/mL) and extraction time 2 min. A two-phase n-hexane/ethyl acetate/i-propanol/water (1:3:1.5:4.5, v/v) solvent system was optimised with 10 mmol triethylamine in the upper phase and 10 mmol trifluoroacetic acid in lower phase in PZRCCC. The sample loading was optimised as 2.50 g. Moreover, the samples were evaluated for anti-breast cancer activity later on. The 2.50 g sample loading yielded 0.45 g of sanguinarine and 0.59 g chelerythrine in one-step separation using PZRCCC. The anti-breast cancer activities of sanguinarine and chelerythrine were found stronger than positive control (vincristine 5.04 μg/mL) with half-maximal inhibitory concentration values of 0.96 and 3.00 μg/mL, respectively. This study showed that the established methods were efficient in extraction (UHPE) and separation (PZRCCC) of the sanguinarine and chelerythrine from M. cordata.

Isolation and purification of six aristolochic acids with similar structures from Aristolochia manshuriensis Kom stems by pH-zone-refining counter-current chromatography

Aristolochic acids (AAs), the major components in Aristolochia manshuriensis Kom stems (AMK), may cause Chinese herb nephropathy during clinical application. Therefore, it is necessary to distinguish AMK from other herbs and Chinese medicines using AAs with high purity as standards. So, an efficient method for separation and purification of AAs is required because of their similar structures. In this study, six AAs with purities of >98% were obtained by pH-zone-refining counter-current chromatography (PZRCCC) in a single run. The optimum two-phase solvent system was petroleum ether–ethyl acetate–methanol–water (3:7:3:7, v/v). Triethylamine (10 mM) was added to the aqueous mobile phase and trifluoroacetic acid (10 mM) to the organic stationary phase. As a result, 9.7 mg aristolochic acid IIIa, 12.0 mg aristolochic acid IVa, 32.2 mg aristolochic acid II, 103.7 mg aristolochic acid I, 24.6 mg aristolic acid II, and 26.1 mg aristolic acid I were obtained from 800 mg AAs crude extract. The elution order of AAs during PZRCCC separation corresponded with the pKa values and hydrophobicities of the target compounds. PZRCCC is an efficient method for isolation of AAs with similar structures.

Preparative and scaled-up separation of high-purity α-linolenic acid from perilla seed oil by conventional and pH-zone refining countercurrent chromatography.

α-Linolenic acid is an essential omega-3 fatty acid needed for human health. However, the isolation of high-purity α-linolenic acid from plant resources is challenging. The preparative separation methods of α-linolenic acid by both conventional and pH-zone refining countercurrent chromatography were firstly established in this work. The successful separation of α-linolenic acid by conventional countercurrent chromatography was achieved by the optimized solvent system n-heptane/methanol/ water/acetic acid (10:9:1:0.04, v/v), producing 466mg of 98.98% α-linolenic acid from 900mg free fatty acid sample prepared from perilla seed oil with linoleic acid and oleic acid as by-products. The scaled-up separation in 45× is efficient without loss of resolution and extension of separation time. The separation of α-linolenic acid by pH-zone refining countercurrent chromatography was also satisfactory by the solvent system n-hexane/methanol/water (10:5:5, v/v) and the optimized concentration of trifluoroacetic acid 30mM and NH4 OH 10mM. The separation can be scaled up in 180× producing 9676.7mg of 92.79% α-linolenic acid from 18000mg free fatty acid sample. pH-zone refining countercurrent chromatography exhibits a great advantage over conventional countercurrent chromatography with 20× sample loading capacity on the same column.

An efficient method for the preparative separation and isolation of ginkgolic acids from the sarcotesta of Ginkgo biloba L by pH-zone-refining counter-current chromatography coupled with inner-recycling mode

An efficient method for the preparative separation and isolation of ginkgolic acids (GAs) from the sarcotesta of Ginkgo biloba L was developed by combining pH-zone-refining counter-current chromatography (PZRCCC) with inner-recycling mode. Two grams of GAs extract were separated by PZRCCC using a two-phase solvent system of n-heptane–ethyl acetate–methanol–water (2:1:3:1, v/v) with 10 mM HCl added to the upper phase as the retention acid and 10 mM triethylamine (TEA) in the lower phase as the elution base. In a one-step PZRCCC separation, two GA compounds, C15:1 and C17:2, were obtained with purities over 98% along with two mixtures. The mixtures were further separated by inner-recycling CCC mode with n-heptane–ethyl acetate–methanol–acetic acid (5:4:1:1, v/v) as the solvent system. Five main GAs were successfully separated with purities over 98%. The structures of the separated GAs were determined based on standard samples coupled with Electrospray Ionization Mass Spectrometry (ESI-MS) results. This separation method of coupling PZRCCC with inner-recycling CCC is effective for the preparation of GAs from the sarcotesta of G. biloba and could be further applied to separate organic acids of low polarity in other natural products.

Preparative separation of two alkaloids from devil pepper radix (Rauvolfia verticillata [Lour.] Baill.) by ph-zone-refining counter-current chromatography

A method was developed for the preparative separation of two alkaloids from the crude extract of the radix of Rauvolfia verticillata (Lour.) Baill. in a single run. The two-phase solvent system composed of petroleum ether–ethyl acetate–methanol–water (5:5:2:8, v/v), where triethylamine (40 mmol/L) was added to the upper organic phase as the stationary phase and hydrochloric acid (10 mmol/L) was added to the lower aqueous phase as the mobile phase, was selected for this separation by pH-zone-refining counter-current chromatography (PZRCCC). For the preparative separation, the apparatus was rotated at a speed 850 rpm, while the mobile phase was pumped into the column at 2 mL/min. As a result, 112 mg of reserpine and 21 mg of yohimbine were obtained from 3 g of crude extract in a single run. The analysis of the isolated compounds was determined by high-performance liquid chromatography (HPLC) at 230 nm with purities of over 91.0%, and the chemical identification was carried out by the data of electrospray ionization–mass spectrometry (ESI–MS) and nuclear magnetic resonance (NMR) spectroscopy. The technique introduced in this paper is an efficient method for preparative separation of reserpine and yohimbine from devil pepper radix. It will be beneficial to utilize medicinal materials and also useful for the separation, purification, and pharmacological study of Chinese herbal ingredients.

PREPARATIVE SEPARATION OF ISOQUINOLINE ALKALOIDS FROM PHELLODENDRI CORTEX BY PH‑ZONE-REFINING COUNTER-CURRENT CHROMATOGRAPHY

pH-Zone-refining counter-current chromatography (PZRCCC) was successfully used for preparative separation isoquinoline alkaloids from Phellodendri Cortex. Two target compounds of berberine and palmatine were completely resolved by a two-phase solvent system chloroform/methanol/water (4:2:2, v/v), with 10 mmol L-1 HCl in upper stationary phase and 10 mmol L-1 TEA in lower organic phase. From 2.0 g of the crude alkaloids, palmatine (448 mg, 95.1%) and berberine (159 mg, 95.4%) were obtained in one-step PZRCCC separation. The chemical structures were identified by ESI-MS and NMR data. The results indicated that PZRCCC separation is an excellent preparative separation method for isoquinoline alkaloids from Phellodendri Cortex.

Separation of the potential G-quadruplex ligands from the butanol extract of Zanthoxylum ailanthoides Sieb. & Zucc. by countercurrent chromatography and preparative high performance liquid chromatography

G-quadruplex DNA structure is considered to be a very attractive target for antitumor drug design due to its unique role in maintaining telomerase activities. Therefore, discovering ligands with high stability of G-quadruplex structure is of great interest. In this paper, pH-zone refining counter current chromatography (CCC) and preparative high performance liquid chromatography (HPLC) were employed for the separation of potent G-quadruplex ligands from the n-butanol fraction of the crude extract of Zanthoxylum ailanthoides, which is a traditional Chinese medicine recently found to display high inhibitory activity against several human cancer cells. The 75% aqueous ethanol extract of the stem bark of Z. ailanthoides and its fractions with petroleum ether, ethyl acetate and n-butanol displayed almost the same G-quadruplex stabilization ability. Here, pH-zone refining CCC was used for the separation of the alkaloids from the n-butanol fraction by a seldom used solvent system composed of dichloromethane-methanol-water (4:1:2.5) with 10mM TEA in the organic stationary phase as retainer and 10mM HCl in the aqueous mobile phase as eluter. Compounds I, II and III were obtained, with purity greater than 95%, in the quantities of 31.2, 94.0, and 26.4mg respectively from 300mg of lipophilic fraction within 80min, which were identified as three tetrahydroprotoberberines isolated for the first time in this plant. In addition, a phenylpropanoid glycoside compound IV (Syringin), an isoquinoline (Magnoflorine, V), and two lignin isomers (+)-lyoniresiol-3α-O-β-d-glucopyranoside (VI) and (−)-lyoniresinol −3α-O-β-D −glucopyranoside (VII) were isolated by traditional CCC together with preparative HPLC. Compounds IV, V, VI and VII were obtained, with purity greater than 95%, in the quantities of 4.0, 13.2, 6.7, and 6.5mg respectively from 960mg of hydrophilic fraction. Among the seven isolated compounds, tetrahydroprotoberberine I, II and III were found to display remarkable stabilization effects on G-quadruplex by increasing G-quadruplex's Tm approximately 10°C, which may be the most potent G-quadruplex ligands in Z. ailanthoides.

Optimisation and establishment of separation conditions of organic acids from Usnea longissima Ach. by pH-zone-refining counter-current chromatography: Discussion of the eluotropic sequence

The major bioactive constituents of Usnea longissima Ach. are organic acids. However, few recent literatures involve the preparative separation of these organic acids. In the present study, pH zone-refining counter-current chromatography is used to separate organic acids from crude sample of U. longissima Ach. The crude extract was separated with the two-phase solvent system Pet–EtAc–MeOH–H2O (5:5:3:7, v/v) with 10mM TFA in organic stationary phase and different concentration of the eluter in aqueous mobile phase for the screening of the most suitable separation conditions. From the crude extract (1.2g), 74.0mg of orsellinic acid at 92.7% purity, 55.5mg of 4-O-methylorsellinic acid at 97.7% purity, 353.5mg of evernic acid at 93.8% purity, 102.0mg of barbatic acid at 94.8% purity, 19.4mg of diffractaic acid at 92.2% purity, and 44.9mg of usnic acid at 95.7% purity were obtained using the selected conditions in which the concentration of TFA in stationary phase was 10mM and the concentration of NaOH in mobile phase was 10–20mM. The purities of the separated organic acids were measured by HPLC. And the data of electrospray ionization-liquid chromatography/mass spectrometry (ESI–LC/MS), 1H NMR, and 13C NMR were used for confirming chemical structures.

Modeling pH-zone refining countercurrent chromatography: A dynamic approach

A model based on mass transfer resistances and acid–base equilibriums at the liquid–liquid interface was developed for the pH-zone refining mode when it is used in countercurrent chromatography (CCC). The binary separation of catharanthine and vindoline, two alkaloids used as starting material for the semi-synthesis of chemotherapy drugs, was chosen for the model validation. Toluene/CH3CN/water (4/1/5, v/v/v) was selected as biphasic solvent system. First, hydrodynamics and mass transfer were studied by using chemical tracers. Trypan blue only present in the aqueous phase allowed the determination of the parameters τextra and Pe for hydrodynamic characterization whereas acetone, which partitioned between the two phases, allowed the determination of the transfer parameter k0a. It was shown that mass transfer was improved by increasing both flow rate and rotational speed, which is consistent with the observed mobile phase dispersion. Then, the different transfer parameters of the model (i.e. the local transfer coefficient for the different species involved in the process) were determined by fitting experimental concentration profiles. The model accurately predicted both equilibrium and dynamics factors (i.e. local mass transfer coefficients and acid–base equilibrium constant) variation with the CCC operating conditions (cell number, flow rate, rotational speed and thus stationary phase retention). The initial hypotheses (the acid–base reactions occurs instantaneously at the interface and the process is mainly governed by mass transfer) are thus validated. Finally, the model was used as a tool for catharanthine and vindoline separation prediction in the whole experimental domain that corresponded to a flow rate between 20 and 60mL/min and rotational speeds from 900 and 2100 rotation per minutes.

Preparative Separation of Six Rhynchophylla Alkaloids from Uncaria macrophylla Wall by pH-Zone Refining Counter-Current Chromatography

pH-Zone refining counter-current chromatography was successfully applied to the preparative isolation and purification of six alkaloids from the ethanol extracts of Uncaria macrophylla Wall. Because of the low content of alkaloids (about 0.2%, w/w) in U. macrophylla Wall, the target compounds were enriched by pH-zone refining counter-current chromatography using a two-phase solvent system composed of petroleum ether–ethyl acetate–isopropanol–water (2:6:3:9, v/v), adding 10 mM triethylamine in organic stationary phase and 5 mM hydrochloric acid in aqueous mobile phase. Then pH-zone refining counter-current chromatography using the other two-phase solvent system was used for final purification. Six target compounds were finally isolated and purified by following two-phase solvent system composed of methyl tert-butyl ether (MTBE)–acetonitrile–water (4:0.5:5, v/v), adding triethylamine (TEA) (10 mM) to the organic phase and HCl (5 mM) to aqueous mobile phase. The separation of 2.8 g enriched total alkaloids yielded 36 mg hirsutine, 48 mg hirsuteine, 82 mg uncarine C, 73 mg uncarine E, 163 mg rhynchophylline, and 149 mg corynoxeine, all with purities above 96% as verified by HPLC Their structures were identified by electrospray ionization-mass spectrometry (ESI-MS) and 1H-NMR spectroscopy.

Application of pH-zone-refining countercurrent chromatography for the separation of indole alkaloids from Aspidosperma rigidum Rusby

Species of Aspidosperma (Apocynaceae) are characterized by the occurrence of indole alkaloids, but few recent reports on Aspidosperma rigidum Rusby chemical constituents were found. The present work shows the application of pH-zone refining countercurrent chromatography on the separation of alkaloids from the barks of A. rigidum. In this study, the dichloromethane extract was fractionated with the solvent system composed of methyl-tert-butyl ether and water with different concentrations of the retainer triethylamine in the organic stationary phase and formic or hydrochloric acids as eluters in the aqueous mobile phase, in order to evaluate the most suitable condition. In each experiment, from circa 200mg of the dichloromethane extract of A. rigidum, three major alkaloids were isolated and identified as 3α-aricine (circa 17mg), isoreserpiline (ca. 22mg) and 3β-reserpiline (ca. 40mg), with relative purity of 79%, 89% and 82% respectively, in a one-step separation of 2h. Two of them – 3α-aricine and isoreserpiline – were isolated and identified for the first time in this species.

Use of pH-Zone Refining Countercurrent Chromatography for Preparative Separation of Fangchinoline and Tetrandrine from Stephania tetrandra

pH-Zone-refining countercurrent chromatography with a multilayer coil planet centrifuge has been successfully used for separation of fangchinoline and tetrandrine from crude extracts of Stephania tetrandra. The two target compounds were completely resolved by use of the two-phase solvent system petroleum ether (60–90 °C)–ethyl acetate–methanol–water 5:5:1:9 (v/v), with 10 mm triethylamine in the organic stationary phase and 5 mm hydrochloric acid in the aqueous mobile phase. Separation of 3.5 g sample yielded 126 mg fangchinoline (LC purity >93%) and 249 mg tetrandrine (LC purity >95%). The structures of the compounds were confirmed by use of electrospray ionization mass spectrometry and 1H NMR.

Tropane aromatic ester alkaloids from a large-scale re-collection of Erythroxylum pervillei stem bark obtained in Madagascar.

Fractionation by pH zone-refining countercurrent chromatography of an extract of the stem bark of Erythroxylum pervillei, obtained on a kilogram scale in southern Madagascar, led to the isolation and characterization of four tropane aromatic ester alkaloids as minor constituents, namely, pervilleines G (5) and H (6) and cis-pervilleines B (7) and F (8). Their structures were determined by spectroscopic data interpretation.