Preparative Isolation of Bioactive Nitrile Glycoside “Niazirin” from the Fruits of Moringa oleifera Using Fast Centrifugal Partition Chromatography

Fast centrifugal partition chromatography was successfully applied in the separation of bioactive constituent niazirin directly from the chloroform extract of Moringa oleifera. The experiment was performed with a two-phase solvent system composed of ethyl acetate/BuOH/water (6:0.5:4 v/v/v) in which the upper organic layer was used as stationary phase and lower aqueous phase was used as mobile phase. From 1 g of crude extract, 70 mg of niazirin was obtained in 94.8% purity as determined by HPLC. The total yield recovery was >94%. The isolated nitrile glycoside (niazirin) was characterized on the basis of its ¹H, ¹³C-NMR and ESI-MS data.     

One Step Purification of Piperine Directly from Piper nigrum L. by High Performance Centrifugal Partition Chromatography

Abstract

In this study, a preparative high performance centrifugal partition chromatography (HPCPC) method for isolation and purification of the bioactive component piperine directly from the ethanol extract of Piper nigrum L. was successfully established by using n-hexane-ethyl acetate-methanol-water as the two-phase solvent system. The upper phase of n-hexane-ethyl acetate-methanol-water (6:5:6:5, v/v) was used as the stationary phase of CPC. Under the optimum conditions, 40 mg of piperine at 98.5% purity, as determined by HPLC, was yielded from 300 mg of the crude extract in a single CPC separation. The peak fraction of CPC was identified by ¹H NMR and ¹³C NMR.     

Preparative-Scale Separation of Anticancer Triterpenes from Eucalyptus hybrid by Centrifugal Partition Chromatography

Centrifugal partition chromatography was successfully applied in the separation of close R _f complex anticancer triterpenes directly from a fraction of Eucalyptus hybrid (Myrtaceae). The experiment was performed with a two-phase solvent system composed of hexane/ethyl acetate/methanol/water (1:2:1.5:1 v/v/v) where 2% ammonia solution was added in lower aqueous mobile phase to achieve pH 9.5. From 1.5 g of a fraction, 145 mg of ursolic acid and 72 mg of ursolic acid lactone were obtained in 95.4% and 94.8% purities. The total yield recovery was >94% and the isolated triterpenes were characterized on the basis of their ¹H, ¹³C-NMR, and ESI-MS data.     

Application of High-Speed Counter-Current Chromatography Preparative Separation of Flavone C-Glycosides From Lophatherum gracile

Preparative high-speed counter-current chromatography (HSCCC) was used to separate and purify bioactive constituents from the stems and leaves of Lophatherum gracile Brongn. Six flavone C-glycosides each at over 95% purity including two new compounds were obtained in one-step separation by HSCCC with an optimized two-phase solvent system composed of ethyl acetate-n-butanol-ethanol-water at volume ratio of 4:2:1.5:8.5 (v/v/v/v). The experiment yielded 19.9 mg of luteolin 6-C-β-D-galactopyranosiduronic acid (1→2)-β-D-glucopyranoside (1), 28.5 mg of luteolin 6-C-α-L-arabinopyranosyl-7-O-β-D-glucopyranoside (2), 31.5 mg of isoorientin (3), 44.8 mg of orientin (4), 25.3 mg of swertiajaponin (5) and 12.1 mg of apigenin 6-C-β-D-galactopyranosiduronic acid (1→2)-β-D-glucopyranoside (6) from 500 mg of crude extracts. The purity of these compounds was determined by high-performance liquid chromatography (HPLC). Their chemical structures were identified by electron spray ionization mass spectroscopy (ESI-MS), ¹H and ¹³C nuclear magnetic resonance spectroscopy (NMR).     

Enrichment and Separation of Sinomenine and Acutumine from Sinomenium acutum by pH-Zone-Refining Counter-Current Chromatography

Enrichment and separation of alkaloids from a chloroform extract of Sinomenium acutum has been successfully performed for the first time using pH-zone-refining counter-current chromatography. The two-phase solvent system used for enrichment was composed of Methyl tert-butyl ether (MtBE)–acetonitrile (CH₃CN)–water (4:1:5, v/v), where 10 mM triethylamine (TEA) was added to the upper organic stationary phase as a retainer and 10 mM hydrochloric acid (HCl) to the aqueous mobile phase as an eluter, which could enrich the alkaloids from the crude extract well. For the preparative separation, the solvent system consisted of MtBE–CH₃CN–water (4:0.5:5, v/v) with 10 mM TEA in organic stationary phase and 5 mM HCl in the aqueous mobile phase, which could separate and purify the enriched crude alkaloids successfully. 0.82 g of crude alkaloids was enriched from 1.60 g of chloroform extract in the first step separation. From the enriched crude alkaloids, 376 mg of sinomenine and 85 mg of acutumine were obtained in the second step separation with the purity of 98.1% and 98.7%, respectively. The chemical structures of the isolated compounds were identified by UV, ESI-MS and ¹H NMR.     

One Step Large-Scale Separation and Purification of Rutin from Boenninghausenia sessilicarpa by Countercurrent Chromatography

In this study, a preparative countercurrent chromatography (CCC) method for isolation and purification of the bioactive component rutin directly from the ethanol extract of Boenninghausenia sessilicarpa was successfully established by using n-butanol-ethyl acetate-water as the two-phase solvent system. The upper phase of n-butanol-ethyl acetate-water (4:1:5, v/v) was used as the stationary phase of CCC. Under the optimum conditions, 112 mg of rutin at 98.6% purity was obtained from 2.0 g of the crude extract in a single CCC separation. The peak fraction of CCC was identified by negative ESI, ¹H NMR, and ¹³C NMR.     

Effective and Preparative Separation of Bioactive Flavonoids From Gynostemma Pentaphyllum Tea Using Elution-Extrusion Counter-Current Chromatography

Elution-extrusion counter-current chromatography (EECCC) was successfully applied for screen and separation of four flavonoids from Gynostemma pentaphyllum tea, a popular herbal tea extract in China. With the hexane/ethyl acetate/methanol/water 5/6/5/6 (v/v) system, 300 mg of G. pentaphyllum tea extract were fractionated on a 180 mL-capacity preparative hydrodynamic CCC column. Satisfactory separation efficiency was achieved, producing milligram-amounts of quercetin, isorhamnetin, and cirsiliol over 90% pure in one EECCC process. Due to the hydrophilic property, the major flavonoid glycoside, rutin, was co-eluted with the solvent front as a mixture. Therefore, another carefully selected biphasic liquid system composed of ethyl acetate/n-butanol/water (4/1/5, v/v) was employed, yielding 35 mg of rutin with 97.1% purity. Structures of all separated compounds were identified by ESI-MS, ¹H NMR, and ¹³C NMR.     

Isolation and Purification of Two Constitutes from Dendrobium fimbriatum Hook by High-Speed Counter-current Chromatography Using Stepwise Elution

Abstract

Preparative high-speed counter-current chromatography (HSCCC) was successfully used for the isolation and purification of 2-hydroxyethyl caffeate and denhydroshizukanolide from Dendrobium fimbriatum Hook using stepwise elution with a pair of two-phase solvent systems composed of n-hexane-ethyl acetate-methanol-water at (1:1:1:1, v/v) and (3:1:3:1, v/v). Using a preparative unit of the HSCCC centrifuge, about a 100 mg amount of the sample was separated, yielding 13.3 mg of 2-hydroxyethyl caffeate and 18.0 mg of denhydroshizukanolide at a high purity of over 95%. The peak fraction of HSCCC was identified by ¹H NMR and ¹³C NMR.     

Isolation and Purification of Kaempferol-3,7-O-α-L-Dirhamnopyranoside from Siraitia grosvenori Leaves by High-Speed Counter-Current Chromatograph and Its Free Radical Scavenging Activity

Semi-preparative high-speed counter-current chromatography (HSCCC) was successfully used for the isolation and purification of flavonoid glycoside from the leaves of Siraitia grosvenori by using a two-phase-solvent system composed of ethyl acetate–n-butanol–water (4:1:5, v/v/v). kaempferol-3,7-O-α-L-dirhamnopyranoside was obtained in one-step separation and less than 5.5 h from 90 mg of crude extract from the S. grosvenori leaves. The chemical structure of this compound was identified by MS, ¹H NMR, and ¹³C NMR. Free radical scavenging activity of kaempferol-3,7-O-α-L-dirhamnopyranoside was also evaluated and the results showed that it had good free radical scavenging activity with its IC₅₀ value being 3.97 mg/ml.     

Separation and Purification of Bioactive Flavonol Glycosides from Hedyotis diffusa Willd by High-Speed Counter-Current Chromatography

Three flavonoid glycosides including quercetin-3-O-[2″-O-(6″′-O-E-sinapoyl)-β-D-glucopyranosyl]-β-D-glucopyranoside(I), quercetin-3-O-[2″-O-(6″′-O-E-feruloyl)-β-D-glucopyranosyl]-β-D-glucopyranoside(II) and quercetin-3-O-[2″-O-(6″′-O-E-feruloyl)-β-D-glucopyranosyl]-β-D-galactopyranoside(III) were isolated and purified from Hedyotis diffusa Willd by high-speed counter-current chromatography (HSCCC). This run was carried out with a two-phase solvent system composed of n-hexane–ethyl acetate–n-butanol–methanol–1.0% acetic acid (1:1:3.5:1:4.5, v/v) by eluting the lower phase as the mobile phase with a flow-rate at 2.0 ml/min. Consequently, 29.6 mg of I, 35.1 mg of II, 41.3 mg of III with purities of over 95% were obtained from 200 mg of the crude extracts in a single run in less than 130 min. The structure of the isolated compounds was confirmed by MS, ¹H NMR, and ¹³C NMR analysis.     

Separation and Purification of Rosmarinic Acid and Rutin from Glechoma hederacea L. using High-Speed Counter-Current Chromatography

Rosmarinic acid and rutin were successfully separated from Glechoma hederaceaL. using high-speed counter-current chromatography for the first time. Eleven milligrams of rosmarinic acid (chromatographic purity 97.2 %) and 10 mg of rutin (chromatographic purity 98.1 %) were obtained from 100 mg ethyl acetate extract and 100 mg n -butanol extract of Glechoma hederacea L., respectively, with the separation procedure less than 2 h. Their structures were characterized by UV, MS, and NMR. The established methods were simple, fast, and convenient, which can be applied to the preparation of reference substances of rosmarinic acid and rutin.     

Separation of Radiogallium from Zinc Using Membrane-Based Liquid-Liquid Extraction in Flow: Experimental and COSMO-RS Studies

ABSTRACT

A switch from batch to continuous manufacturing of gallium-68 (⁶⁸Ga) and ⁶⁸Ga-labeled pharmaceuticals can be advantageous, as it recycles isotopically-enriched zinc-68 (⁶⁸Zn), removes pre- and post-irradiation target manipulations, and provides scalability via dose-on-demand production. Herein we report efficient extraction of radiogallium (^66,67,68Ga = *Ga) from ZnCl₂/HCl solutions in batch and in flow using a membrane-based liquid-liquid separator. From 5.6 M ZnCl₂/3 M HCl, a 1/2 (v/v) diisopropyl ether (ⁱPr₂O)/trifluorotoluene (TFT) solvent extracts 76.3 ± 1.9% of *Ga and 1.9 ± 1.6% of Zn in flow using a single pass through. From 1 M ZnCl₂/6 M HCl, a 1/2 (v/v) n-butyl methyl ether (n-BuOMe)/TFT solvent extracts 95.7 ± 2.0% of *Ga and 0.005 ± 0.003% of Zn in flow. TFT plays a key role in controlling the interfacial tension between the aqueous and the organic phases, ensuring clean membrane-based separation. The process did not extract Cu, Mn, and Co but did extract Fe. Using HGaCl₄ and ZnCl₂ as the extractable species, the COSMO-RS theory predicts the solvation-driven extraction of Ga and Zn with a mean unsigned error of prediction of 4.0% and 3.4% respectively.     

Ion interaction chromatography of anions in alkaline solutions: effect of temperature and the kind of stationary phase

Effect of temperature (5°–65°C) on the separation of 11 inorganic anions by ion interaction chromatography (IIC) was studied employing RP C₁₈ and C _PhenylHexyl columns and aqueous mobile phase: 2.8 mM NaHCO₃ + 0.7 mM TBAOH (tetra-n-butylammonium hydroxide). The apparent enthalpy changes, ΔH for hydrophobic ions like I^?, SCN^?, and ClO₄ ^? largely exceeded 3 kcal/mole suggesting that added to ion exchange they are retained by hydrophobic adsorption. Unlike conventional strongly basic anion exchangers, our system can be used at elevated temperatures with alkaline eluents without irreversible damaging the column.     

Molecularly imprinted monolithic material for the extraction of three organic acids from Salicornia herbacea L

A molecularly imprinted monolithic material was designed and prepared by in situ thermally initiated copolymerization for the extraction of protocatechuic acid, caffeic acid, and ferulic acid from Salicornia herbacea L. Field emission scanning electron microscopy and offline solid‐phase extraction (SPE) were investigated for the characterization of this material. The extract samples were loaded onto and passed through the monolithic material; the target compounds were selectively retained on the material, whereas other interfering substances were quickly washed out. Chromatographic analysis was conducted on a C₁₈ column with ultraviolet detection at 270 nm, and an eluting solution consisting of acetonitrile, water, and acetic acid (14/86/0.5 v/v/v, pH 5.0) was used as the mobile phase at a flow rate of 0.8 mL/min. The linearity was confirmed in the concentration ranges of 0.10–200.00, 0.20–400.00, and 0.30–600.00 μg/mL for protocatechuic acid, caffeic acid, and ferulic acid, respectively, with r² greater than 0.9997. The SPE recoveries of the three organic acids ranged from 71.08 to 81.02%, and the coefficient of variation (precision) was 3.08–5.70%. This method is simple, economical, and specific and has been used successfully in the extraction of three organic acids from S. herbacea L. This cartridge can be used as a potential tool for the extraction of drugs from natural plants. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Antioxidant and Antibacterial Activity of Nutraceutical Compounds from Chlorella vulgaris Extracted in Hydrothermal Condition

Abstract

Water in hydrothermal condition has been used for extraction of nutraceutical compounds from Chlorella vulgaris. Hydrothermal extraction was carried out in a semi-batch and a batch extractor at various temperatures (120–200°C), pressures (2–10 MPa), and extraction times (30–300 min) to extract antioxidant and antibacterial compounds. The effect of extraction condition on the yield of extract was investigated. The antioxidant and antibacterial activity of extracts obtained by hydrothermal extraction were examined. The increasing extraction temperature resulted in higher antioxidant activity, but lower antimicrobial activity. As comparison with hot water extraction, the antioxidant activity of extract obtained by hydrothermal extraction was higher than that obtained by hot water extraction, but the antibacterial activity of the extract obtained by hydrothermal extraction was lower.     

Isolation and Purification of Isoquercitrin and Quercitrin from sf Hypericum Japonicum Thunb.ex Murray by Counter-Current Chromatography

Isoquercitrin and quercitrin were successfully isolated and purified from Hypericum japonicum Thunb.ex Murray by counter-current chromatography with a solvent system of n-hexane-ethyl acetate-methanol-water (1:7:1:7, v/v/v/v) in one step. From 100 mg of the extract of Hypericum japonicum Thunb.ex Murray, 9.8 mg of isoquercitrin and 12 mg of quercitrin were obtained with the purities of 95.9% and 99.1%, respectively, as determined by HPLC. Their structures were identified by UV, MS, and NMR analysis. In this study, a rapid method for isolation and purification of the two major compounds from Hypericum japonicum Thunb.ex Murray crude extract was established.     

Using High-Performance Counter-Current Chromatography Combined with Preparative High Performance Liquid Chromatogramphy for the Separation of Bioactive Compounds from the Water Extract of Gentiana macrophylla Pall

In this paper, a combined high performance counter-current chromatography (HPCCC) and preparative high-performance liquid chromatography (HPLC) method was employed for rapid separation and enrichment of bioactive constituents from a water extract of Gentiana macrophylla Pall. With a two phase solvent system composed of ethyl acetate-n-butanol-water-acetic acid (2: 3: 5: 0.6, v/v), the water extract of G. macrophylla Pall was fractionated into six fractions with three targets isolated and four others highly concentrated, which were then further purified by preparative-HPLC. As a result, 37 mg deglucoserrulatoside, 22.4 mg loganic acid, 3.9 mg isoorientin, 22.4 mg swertiamarin, 52.3 mg gentiopicroside, 27.5 mg sweroside, and 7.9 mg macrophylloside D with the purity of 95.3%, 90.2%, 98%, 98%, 99.2%, 98.8%, and 98.4%, respectively, were isolated from the water extract of Gentiana macrophylla Pall. The structures were confirmed by UV spectra, MS, as well as NMR measurements.     

Separation and Purification of Three High-Purity Isoflavonoids from Belamcanda chinensis (L.) DC. by Supercritical Fluid Extraction and High-Speed Counter-Current Chromatography

Supercritical fluid extraction (SFE) was used to extract three isoflavonoids including irigenin, irisfloretin and dichtomitin from Belamcanda chinensis (L.) DC. The parameters including pressure, temperature, sample particle size, and flow rate of CO₂ were optimized with an orthogonal test. Under the optimized conditions of 15 MPa, 55°C, a sample particle size of 20–40 mesh and CO₂ flow rate of 40 L h^?1. The process was then scaled up by 10 times using a preparative SFE system. The yield of the crude extract from SFE was 4.1%, which contained irigenin, irisfloretin, and dichtomitin 0.71%, 0.49%, and 0.05%, respectively. To compare the extraction methods, Soxhlet Extraction (SE) was performed. The results indicated that SFE was better than SE. Irigenin, irisfloretin, and dichtomitin in the SFE extract were then separated and purified by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of petroleum ether–ethyl acetate–methanol–water (2:4:3:3, v/v). From 5.0 g of dry crude extract, 27.8 mg irigenin, 16.4 mg irisfloretin, and 2.1 mg dichtomitin were obtained at purities of 97.1%, 96.4%, and 98.0%, respectively, as determined by HPLC-PDA. These results well indicate that SFE and HSCCC are very powerful techniques for the extraction and purification of irigenin, irisfloretin, and dichtomitin from B. chinensis.     

Separation and Purification of Quinolone Alkaloids from the Chinese Herbal Medicine Evodia rutaecarpa (Juss.) Benth by High Performance Counter-Current Chromatography

Preparative separation of quinolone alkaloids in Evodia rutaecarpa (Juss.) Benth was conducted by high performance counter-current chromatography (HPCCC) with a pair of two solvent systems consisting of n-hexane-methanol-water-acetic acid (2:1:1:0.2, v/v) and (5:4:2:0.1, v/v). Consequently, 31.78 mg 1-methyl-2-nonyl-4 (1H)-quinolone (I), 59.25 mg 1-methyl-2-(6-undecenyl)-4 (1H)-quinolone (II), 333.27 mg evocarpine (III), 101.13 mg 1-methyl-2-(6,9-pentadecadienyl)-4(1H)-quinolone (IV), 132.17 mg dihydroevocarpine (V), and 86.99 mg 1-methyl-2-(10-pentadecenyl)-4(1H)-quinolone (VI) were obtained from 1.3 g of the crude extract. The structures of these compounds were identified by mass spectrometer (MS), nuclear magnetic resonance (¹H NMR and ¹³C NMR).     

Separation and Purification of Two Isomeric Saponins from Albiziae Cortex by High-Speed Counter-Current Chromatography and Preparative High-Performance Liquid Chromatography

Following constituents’ enrichment steps with the AB-8 macroporous resin, silica gel, and ODS columns, high-speed counter-current chromatography (HSCCC) and preparative HPLC were successfully used for the isolation and purification of two complex isomeric saponins including a new one from albiziae cortex. The two-phase solvent system used for separation was composed of n -hexane/ n -butanol/water (1:10:5, v/v/v ). A total of 8.2 mg julibroside J _{5 a} and 11.6 mg julibroside J ₅ with purity of higher than 98%, respectively, as determined by HPLC-ELSD were obtained from the constituents enriched fraction (475.4 mg) of albiziae cortex. Their structures were identified by HR-MS, ¹ H NMR ¹³ C NMR, and 2D NMR. This is the first ever report on the separation of complex isomeric saponins from albiziae cortex by HSCCC.