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.     

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.     

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.     

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.     

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.     

Separation and Purification of Arctiin,Arctigenin, Matairesinol,and Lappaol F from Fructus Arctii by High-Speed Counter-Current Chromatography

Arctiin (I), arctigenin (II), matairesinol (III), and lappaol F (IV) were isolated and purified from the traditional Chinese medicine Fructus Arctii by high-speed counter-current chromatography (HSCCC). The crude extracts from Fructus Arctii were treated with D101 macroporous resin first and divided into two parts: fraction 1 and fraction 2. Fraction 1 was separated by ethyl acetate-n-butanol-water (4:0.5:5, v/v/v) and yielded 164 mg of I from 250 mg of fraction 1. Fraction 2 was separated by n-hexane-ethyl acetate-methanol-water (2:3:2:3, v/v/v/v) and yielded 27 mg of II, 5 mg of III, and 3 mg of IV from 150 mg of fraction 2. The purities of the four compounds were 99.64%, 98.48%, 96.16%, and 91.41%, respectively, as determined by HPLC-DAD. The chemical structures of the isolated compounds were identified by MS, UV, ¹H NMR, and ¹³C NMR analysis.     

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).     

Response Surface Optimized Ultrasonic-Assisted Extraction of Quercetin and Isolation of Phenolic Compounds From Hypericum perforatum L. by Column Chromatography

The effects of ultrasonic-assisted extraction factors for the main phenolic compound (quercetin) from Hypericum perforatum L. were optimized using the Box–Behnken design (BBD) combined with response surface methodology. The BBD was employed to evaluate the effects of extraction temperature (30–70°C), extraction time (20–80 min), methanol concentration (20–80%, v/v), and HCl concentration (0.8–2.0 M) on the content of one of the major phenolic compounds of quercetin. The extracts were analyzed by high performance liquid chromatography (HPLC). The major phenolic compounds of H. perforatum were isolated and the antioxidant capacity and total phenol content were determined in crude extract and fractions. The optimum conditions were determined as extraction temperature 67°C, extraction time 67 min, methanol concentration 77% (v/v), and HCl concentration 1.2 M. The predicted content of quercetin was 10.81 mg/g dried plant under the optimal conditions and the subsequent verification experiment with 11.09 mg/g dried plant confirmed the validity of the predicted model. The isolated compounds were identified as quercetin, cyanidin, protocatechuic acid, and kaempferol.     

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.     

Preparative Isolation of Bioenhancer Loganetin from sf Alstonia scholaris by Fast Centrifugal Partition Chromatography

Fast centrifugal partition chromatography (FCPC) was successfully applied in the separation of close R f complex bioactive iridoid, loganetin directly from the ethyl acetate extract of Alstonia scholaris . The experiment was performed with a two-phase solvent system composed of methyl tert- butyl ether (MtBE)/ACN/Water (3:1.5:3 v/v/v) where the lower phase of the biphasic system, the aqueous layer containing 8 mM HCl, was the stationary phase, while the upper organic layer supplemented with 15 mM triethylamine TEA was designated as the mobile phase. From 1.5 g of EtOAc extract, 48 mg of loganetin (1) was obtained in 94.4% purity as determined by HPLC. The isolated compound (1) was characterized on the basis of its ¹ H, ¹³ C–NMR, and ESI-MS spectroscopic data. Although loganetin does not possess antibacterial activity of its own, but in combination, it appreciably reduces the minimum inhibitory concentration (MIC) of nalidixic acid (NA) against nalidixic acid resistant (NAREC) and nalidixic acid sensitive (NASEC) strains of Escherichia coli . Loganetin, a very common, inexpensive, and non-toxic natural product may finds its application in the antibacterial drug development for treating multidrug-resistant Gram negative infections.     

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.     

Activity-Guided Isolation and Purification of Three Flavonoid Glycosides from Neo-Taraxacum siphonanthum by High-Speed Counter-Current Chromatography

DPPH (1,1-diphenyl-2-picryhydrazyl) radical scavenging assay was used to screen different fractions of Neo-Taraxacum siphonanthum ethanol extracts. The potent active fraction was isolated and purified by preparative high-speed counter-current chromatography (HSCCC) with a solvent system composed of n-hexane-n-butanol-water (3:4:7, v/v/v). The flow rate was 1.5 mL/min and resolution speed was 800 rpm. Three flavonoid glycosides with the purity over 99% were obtained and identified as luteolin- 3′-O-β-D-glucopyranoside (I), luteolin-7-O-β-D-glucopyranoside (II), and luteolin-4′-O-β-D-glucopyranoside (III) by ESI-MS, ¹H NMR and ¹³C NMR analysis. Antioxidant activity of three flavonoid glycosides was assessed by DPPH assay, all of which showed potent activity.     

QUANTIFICATION OF GALLIC ACID AND ELLAGIC ACID FROM THE SEED OF CORNUS OFFICINALIS BY UHPLC METHOD AND THEIR ANTIOXIDANT ACTIVITY

Gallic acid and ellagic acid have been identified in the seed of Cornus officinalis by the use of an ultrahigh performance liquid chromatography (UHPLC) method coupled with a diode array detector (DAD). The water extract of C. officinalis seed contained the highest gallic acid content (3.03 ± 0.10 mg/g seed), which was followed by aqueous methanol extract (2.43 ± 0.10 mg/g seed) and aqueous ethanol extract (1.53 ± 0.25 mg/g seed). But a higher concentration (12.32 ± 0.45 mg/g seed) of ellagic acid was obtained from extraction with aqueous methanol than with aqueous ethanol (11.03 ± 0.42 mg/g seed) and water (7.28 ± 0.16 mg/g seed). After heat treatment and acid hydrolysis, C. officinalis seed had higher concentrations of gallic acid and ellagic acid, contributing to more potent antioxidant activity. The results demonstrated a rich source of gallic acid and ellagic acid in C. officinalis seed, which might provide a novel source of these natural antioxidants.     

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).     

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.     

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.     

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.     

Rapid Detection of Antioxidant Flavonoids in Azalea (Rhododendron mucronulatum) Flowers using On-line HPLC-ABTS+ System and Preparative Isolation of Three Flavonoids by Centrifugal Partition Chromatography

High-performance liquid chromatography coupled to an on-line ABTS⁺-based assay (on-line HPLC-ABTS⁺) system was used to determine the principle antioxidants in azalea flowers. Three flavonoids, myricetin, quercetin, and kaempferol, recovered in ethyl acetate extracts of azalea flowers were determined to have antioxidant activities. These three flavonoids were isolated and purified by successive centrifugal partition chromatography (CPC) using two different biphasic solvent systems, consisting of n-hexane-ethyl acetate-methanol-water (3:5:3:5 or 4:5:4:5, v/v). A total of 46.2 mg of myricetin, 28.9 mg of quercetin, and 10.6 mg of kaempferol with purities of 97.0%, 95.4%, and 93.9%, respectively, were purified from 500 mg of ethyl acetate soluble material from azalea flowers. The structures were identified by their retention time, UV spectra, and ESI-MS in the positive ion mode and were confirmed by NMR experiments.     

Extraction and HPLC Characterization of Chlorogenic Acid from Tobacco Residuals

Abstract

Chlorogenic acid is a highly valuable natural polyphenol compound used in medicine and industries. Its current commercial sources are from plant extracts of Lonicera japonica Thunb and Eucommia ulmoides Oliver. These sources are limited and expensive. On the other hand, tobacco residuals contain chlorogenic acid and other natural polyphenol compounds. Large quantities of tobacco residuals are produced each year as waste materials from tobacco manufacturing, potentially providing an alternative commercial source of chlorogenic acid and other valuable compounds. In this paper, microwave and ultrasound extractions of chlorogenic acid with mixed solvent were studied. Total polyphenol concentrations in extract solutions obtained with different extraction methods were analyzed with the method of ferrous tartrate and UV‐Vis spectrophotometry and compared. The extraction solutions were also characterized for polyphenol compositions with the method of HPLC. Experimental results indicated that high extract concentrations of chlorogenic acid were obtained with a mixed solvent of acetone and water (1:2 v/v). A total polyphenol concentration of up to 4.87 mg/ml and a chlorogenic acid concentration of up to 2.12 mg/ml were achieved. The application of microwave and ultrasound significantly increased the extract concentrations. The extraction time needed was also much reduced. HPLC analysis indicated that acetone water mixed solvent extraction achieved much higher relative concentrations of chlorogenic acid to other compounds in the extract solutions. These results indicted that fast and effective extraction of chlorogenic acid from tobacco residuals were achieved.     

Combination of supercritical fluid extraction with high-speed countercurrent chromatography for extraction and isolation of ethyl p-methoxycinnamate and ethyl cinnamate from Kaempferia galanga L.

Supercritical fluid extraction (SFE) with high-speed countercurrent chromatography (HSCCC) was successfully used for the extraction and isolation of ethyl p-methoxycinnamate (EPMC) and ethyl cinnamate (EC) from Kaempferia galanga L. The SFE parameters including extraction temperature, extraction pressure and entrainer volume were optimized by central composite design (CCD). Then the crude extract was separated by HSCCC with a two-phase solvent system composed of n-hexane:ethyl acetate:methanol:water (7:3:8:2, v/v/v/v) in one-step within 60 min. As a result, 13 mg of EPMC and 2 mg of EC were isolated from 100 mg of crude extract with purities of 98.4% and 98.1%, as determined by HPLC. The structural identification was carried out by UV, MS and NMR spectra.