Separation and purification of isorhamnetin 3-sulphate from Flaveria bidentis (L.) Kuntze by counter-current chromatography comparing two kinds of solvent systems

The first preparative separation of a flavonoid sulphate isorhamnetin 3-sulphate from Flaveria bidentis (L.) Kuntze by counter-current chromatography (CCC) was presented. Two kinds of solvent systems were used. A conventional organic/aqueous solvent system n-butanol-ethyl acetate-water (4:1:5, v/v) was used, yielding isorhamnetin 3-sulphate 2.0 mg with a purity of 93.4% from 83 mg of pre-enriched crude extract obtained from 553 mg ethanol extract by macroporous resin. A one-component organic/salt-containing system composed of n-butanol-0.25% sodium chloride aqueous solution (1:1, v/v) was also used, and the LC column packed with macroporous resin has been employed for desalination of the target compound purified from CCC. As a result, 2.1 mg of isorhamnetin 3-sulphate with a purity of over 97% has been isolated from 402 mg of crude extract without pre-enrichment. Compared with the conventional organic/aqueous system, the one-component organic/salt-containing aqueous system was more suitable for the separation of isorhamnetin 3-sulphate, and purer target compound was obtained from the crude extract without pre-enrichment using the new solvent system. The chemical structure was confirmed by ESI-MS and (1)H, (13)C NMR. In summary, our results indicated that CCC using one-component organic/salt-containing aqueous solution is very promising and powerful for high-throughput purification of isorhamnetin 3-sulphate from Flaveria bidentis (L.) Kuntze.     

Isolation and purification of chemical constituents from the pericarp of Sophora japonica L. by chromatography on a 12% cross-linked agarose gel

A chromatographic method using 12% cross-linked agarose gel Superose 12 as the separation medium was developed for isolation and purification of the chemical constituents from the pericarp of Sophora japonica L. The mobile phase used for the separation was 2% acetic acid and 7% acetic acid in gradient elution. As a result, eight compounds including four kinds of flavonoids and four kinds of isoflavonoids were obtained in a one-step separation. A straightforward explanation of the separation mechanism of flavonoids and isoflavonoids on Superose 12 is also given. The flavonoids and isoflavonoids are retained on Superose 12 by a combination of hydrogen bonding and hydrophobic interactions between the hydroxyl groups of aglycone and the residues of the cross-linking reagents used in the manufacture of Superose 12.     

Extract with high 1,1-diphenyl-2-picrylhydrazyl (DPPH) inhibitory capability from pericarp and seed of mangosteen (Garcinia mangostana L.) using microwave-assisted extraction (MAE) two-phase solvent technique

The human body needs compounds that are antioxidants to prevent oxidative stress. Some parts of the mangosteen fruit (Garcinia mangostana L.) have been known as sources of bioactive compounds that have antioxidant properties. The pericarp and seeds of mangosteen were extracted using the MAE method to produce the extract with the greatest antioxidant activity. There are two types of solvent mixtures used in the extraction process: single-phase and two-phase solvents. The solvents used were ethanol (EtOH), ethyl acetate (EtOAc), isopropyl alcohol (IPA), and water. First, utilizing dried mangosteen pericarp powder as the raw material, a study was undertaken to determine the ideal operating conditions for the MAE process. A one-factor-at-a-time approach was used to find the best operating conditions. A mixture of solvents with varied ratios (mL/mL), extraction temperature (°C), extraction time (min), and solid to solvent ratio (g/mL) were applied as independent variables. Then, dried mangosteen seed powder extraction was carried out based on the best-operating conditions previously achieved. The DPPH scavenging activity, total phenolic content (TPC) value, and α-mangostin content of the two extracts were compared. It was discovered that the mangosteen pericarp extract showed higher antioxidant activity (IC₅₀ DPPH = 9.40 µg/mL) than the mangosteen seed extract (IC₅₀ DPPH = 37.54 µg/mL), even slightly better than ascorbic acid (IC₅₀ DPPH = 10.47 µg/mL). The best extract was produced from the bottom phase of two-phase solvent system (EtOAc:EtOH:Water 2:1:2), with an MAE temperature of 50 °C, a time of 4 min, and a solid-to-solvent ratio of 1:16. The TPC value of the best extract is 903.54 mgGAE/g extract, with a yield of 16.53 % and an α-mangostin concentration of 0.11 %.     

Isolation and purification of flavonoid and isoflavonoid compounds from the pericarp of Sophora japonica L. by adsorption chromatography on 12% cross-linked agarose gel media

A method for isolation and purification of flavonoid and isoflavonoid compounds in extracts of the pericarp of Sophora japonica L. was established by adsorption chromatography on the 12% cross-linked agarose gel Superose 12. The crude extracts were pre-separated to two parts, sample A and sample B, on a D-101 macroporous resin column by elution with 20% ethanol and 40% ethanol, respectively. Samples A and B were then separated by adsorption chromatography on Superose 12 with 40% methanol as the mobile phase. Eight compounds including four kinds of flavonoids and four kinds of isoflavonoids were obtained by the proposed method. The adsorption mechanisms of flavonoids and isoflavonoids on Superose 12 were also discussed.     

A simple and direct isolation of whey components from raw milk by gel filtration chromatography and structural characterization by Fourier transform Raman spectroscopy

A simple and economical method to isolate whey protein from fresh raw milk is developed by serial defatting, casein eliminating, lactose removing, and separating by gel filtration chromatography. Four major whey components, including immunoglobulin (Ig), bovine serum albumin (BSA), β-lactoglobulin (β-Lg) and -lactalbumin (-Lac), and a non-protein of low molecular mass (1.7 kDa) but strong absorbance at 280 nm, are detected simultaneously. The small non-protein molecule is rich in aromatic amino acids and thiol groups as supported by the structural characterization with near infrared Fourier transform Raman spectroscopy (FT-Raman). FT-Raman results show that the secondary structure of Ig is dominated by anti-parallel β-pleated sheet; BSA is mainly in -helix; both β-form and unordered structure are important in β-Lg; while -Lac is mostly in -helix coupling with random coil. Differences in the Raman profile for each whey component reflect their intrinsic compositional differences and distinct spatial arrangement. The S–S linkages diverging around 510–540 cm⁻¹ indicate that the conformation of disulfide bonds in each whey components is different, which may be responsible for their diversified behaviors in solubility, rheological and functional properties.     

Preparative isolation and purification of isobenzofuranone derivatives and saponins from seeds of Nigella glandulifera Freyn by high-speed counter-current chromatography combined with gel filtration

Although the medicinal plant and food Nigella glandulifera Freyn has been researched for decades, isobenzofuranones have never been isolated before. Two isobenzofuranone derivatives and two saponins were successfully separated and purified from seeds of N. glandulifera Freyn by high-speed counter-current chromatography (HSCCC) with the optimized two-phase solvent system, n-hexane-ethyl acetate–methanol–water (7:3:5:5, v/v). Salfredin B₁₁ (22.1 mg, HPLC purity 95.3%), 5, 7-dihydroxy-6-(3-methybut-2-enyl) isobenzofuran-1(3H)-one (18.9 mg, HPLC purity 97.3%) and crude sample 2 (555 mg) were separated from 600 mg of ethyl acetate extract of N. glandulifera Freyn. Following a cleaning-up step by chromatography on Sephadex LH-20, hederagenin (12 mg) and 3-O-[β-d-xylopyranosyl-(1 → 3)-α-l-rhamnopyranosyl-(1 → 2)-α-l-arabinopyranosyl]-hederagenin (45 mg) were separated from sample 2. All of the fractions before peak II were collected and subjected to a Sephadex LH-20 column and eluted by methanol, two of triterpene saponins (12 mg of hederagenin and 45 mg of 3-O-[β-d-xylopyranosyl-(1 → 3)-α-l-rhamnopyranosyl-(1 → 2)-α-l-arabinopyranosyl]-hederagenin) were isolated. The structures of peak fractions were identified by IR, electron ionization MS, ¹H NMR and ¹³C NMR. 5, 7-Dihydroxy-6-(3-methybut-2-enyl) isobenzofuran-1(3H)-one was isolated for the first time from higher plant and salfredin B11 was isolated for the first time in this plant.     

Nonlinear calibrations on the assay of dilitiazem and two of its metabolites from plasma samples by means of liquid chromatography and ESI/MS(2) detection: application to a bioequivalence study

The assay of diltiazem (DLTZ) and its active metabolites desacetyldiltiazem (DAcD) and desmethyldiltiazem (DMeD) in plasma samples was achieved by means of an HPLC/(ESI)MS(2) method. The diastereoisomer of diltiazem, namely {(2R,3S)-5-[2-(dimethylamino)ethyl]-2-(4-methoxyphenyl)-4-oxo-2,3,4,5-tetrahydro-1,5-benzothiazepin-3-yl acetate} was used as internal standard (IS). Sample preparation was based on protein precipitation by means of organic solvent addition (acetonitrile). The isocratic elution based on a reversed-phase mechanism allows good separation of the analytes within 15 min. Atmospheric pressure electrospray ionization was used. All analytes were monitored in the MS(2)-MRM mode. A fragmentation schema is proposed for the target compounds. As the method was designed for bioequivalence purposes, a full validation procedure was considered. On validation, nonlinear calibrations were observed. Consequently, concentration intervals requiring nonlinear calibrations are discussed. Low limits of quantification in the 0.6-1 ng/mL concentration range were obtained. The analytical method was successfully applied to a single dose (120 mg), open-label, randomized, two-period, two-sequence, crossover bioequivalence study of two commercially available solid oral dosage pharmaceutical formulations (tablets).     

Separation of triterpenoid saponins from the root of Bupleurum falcatum by counter current chromatography: The relationship between the partition coefficients and solvent system composition

A separation method using counter current chromatography coupled with an evaporative light‐scattering detection system was developed to purify five triterpenoid saponins from the roots of Bupleurum falcatum. The methanol extract was loaded onto a Diaion® HP20 column and fractionated by a methanol and water gradient elution. The saikosaponin‐enriched fraction was obtained by elution with 100% methanol. The two‐phase solvent systems used for separation were composed of chloroform/methanol/isopropanol/water at a volume ratio of 60:60:1:60 and 6:6:1:6. The relationship between the isopropanol ratio of each phase and the partition coefficients of the target compounds was investigated by calculating partition coefficient by high‐performance liquid chromatography and measuring the accurate composition of each phase by ¹H NMR spectroscopy. Each fraction obtained was collected and dried, which yielded the following five saikosaponins from 700 mg of injected sample: saikosaponin B₁ (8.7 mg), saikosaponin A (86 mg), saikosaponin B₃ (17 mg), saikosaponin B₂ (41 mg), and saikosaponin C (33 mg). Saikosaponin A showed the most potent cytotoxicity against human cancer cells (gastric cancer, AGS cells; breast cancer, MCF‐7 cells; and hepatoma, HepG2 cells) after 24 h. The IC₅₀ values for the above three cell types were 34.6, 33.3, and 23.4 μmol/L, respectively.     

Separation and purification of two taxanes and one xylosyl‐containing taxane from Taxus wallichiana Zucc.: A comparison between high‐speed countercurrent chromatography and reversed‐phase flash chromatography

10‐Deacetylbaccatin III, an important semisynthetic precursor of paclitaxel and docetaxel, can be extracted from Taxus wallichiana Zucc. A process for the isolation and purification of 10‐deacetylbaccatin III ( 1 ), baccatin III ( 2 ), and 7β‐xylosyl‐10‐deacetyltaxol ( 3 ) from the leaves and branches of Taxus wallichiana Zucc. via macroporous resin column chromatography combined with high‐speed countercurrent chromatography or reversed‐phase flash chromatography was developed in this study. After fractionation by macroporous resin column chromatography, 80% methanol fraction was selected based on high‐performance liquid chromatography and liquid chromatography with mass spectrometry qualitative analysis. A solvent system composed of n‐hexane, ethyl acetate, methanol, and water (1.6:2.5:1.6:2.5, v/v/v/v) was used for the high‐speed countercurrent chromatography separation at a flow rate of 2.5 mL/min. The reversed‐phase flash chromatography separation was performed using methanol/water as the mobile phase at a flow rate of 3 mL/min. The high‐speed countercurrent chromatography separation produced compounds 1 (10.2 mg, 94.4%), 2 (2.1 mg, 98.0%), and 3 (4.6 mg, 98.8%) from 100 mg of sample within 110 min, while the reversed‐phase flash chromatography separation purified compounds 1 (9.8 mg, 95.6%) and 3 (4.9 mg, 97.9%) from 100 mg of sample within 120 min.