Yoichi Shibusawa

Separation of proanthocyanidins by degree of polymerization by means of size-exclusion chromatography and related techniques.

The molecular masses of polyphenols in plants and food vary greatly up to the order of 10 kDa. Polymerized polyphenols are not only natural antioxidants but also strong inhibitors of numerous physiological enzymatic activities. Several useful methods for the determination and separation of these high-molecular-mass polyphenols have recently been developed. In this review, details of the methods and applications of size-exclusion chromatographic separation of polymerized polyphenols, particularly those of proanthocyanidins, are described and compared with other related chromatographic or mass spectrometric analyses.

Separation of apple procyanidins into different degrees of polymerization by high-speed counter-current chromatography.

Apple procyanidins were fractionated by high-speed counter-current chromatography in a one-step operation from apple condensed tannins using a type-J multilayer coil planet centrifuge. The separation of procyanidins was performed with a two-phase solvent system composed of methyl acetate-water (1:1) by eluting the upper phase at a flow-rate of 1.0 ml/min. Each fraction was examined by time-of-flight mass spectrometry. Procyanidins were separated according to their degrees of polymerization.

One-step purification of proteins from chicken egg white using counter-current chromatography

Proteins present in chicken egg white are separated by counter-current chromatography (CCC) in one step using a cross-axis coil planet centrifuge (X-axis CPC). The separation was performed with an aqueous polymer two-phase system composed of 16% (w/w) poly(ethylene glycol) 1000 and 12.5% (w/w) dibasic potassium phosphate by eluting the lower phase at a flow-rate of 1.0 ml/min. From about 20 g of the crude egg white solution, lysozyme, ovalbumin, and ovotransferrin were resolved within 5.5 h. Each component was identified by 12% SDS gel electrophoresis with Coomassie brilliant blue staining.

High-speed counter-current chromatography of apple procyanidins

Apple procyanidins were separated by high-speed counter-current chromatography using a type-J multilayer coil planet centrifuge. Several two-phase solvent systems with a wide range of hydrophobicities from a non-polar hexane system to polar n-butanol systems were evaluated their performance in terms of the partition coefficient and the retention of the phase. The best separation of procyanidins B and C was achieved with a two-phase solvent system composed of n-butanol-methyl tert.-butyl ether-acetonitrile-0.1% trifluoroacetic acid (2:4:3:8) using the lower phase as a mobile at a flow-rate of 1.0 ml/min.

Separation of Apple Catechin Oligomers by CCC

Abstract Catechin oligomers with different degrees of polymerization were separated from apple condensed tannins (ACTs) by countercurrent chromatography using a type‐J multilayer coil planet centrifuge. Partition coefficient values for oligomers up to pentamers of catechin and/or epicatechin were determined on hydrophilic two‐ or three‐phase solvent systems. The best separation was achieved using a three‐phase solvent system composed of hexane/methyl acetate/acetonitrile/water at a volume ratio of 1/1/1/1, which formed three layers. After the elution of monomers, dimers, trimers, and a part of tetramers using the middle phase, methyl acetate was used as a mobile phase to facilitate elution of the oligomers from tetramers to decamers. These oligomers eluted from the column according to their degree of polymerization. The higher polymerized oligomers over 11‐mers were collected from the column with the elution by the lower phase. The masses of the fractionated oligomers were determined by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry analysis using 2′,4′,6′‐trihydroxy acetophenone monohydrate as a matrix.

Countercurrent Chromatography of Proteins with Polyethylene Glycol-Dextran Polymer Phase Systems Using Type-XLLL Cross-Axis Coil Planet Centrifuge

Abstract The performance of a new model of countercurrent chromatograph called the XLLL cross-axis synchronous coil planet centrifuge was evaluated for protein separation. The apparatus produced a satisfactory retention of the stationary phase for the polymer phase systems composed of dextran T500, polyethylene glycol 8000 and potassium phosphate buffers. The capability of the apparatus was demonstrated in separations of histones and serum proteins.

Surface affinity chromatographic separation of blood cells. II. Influence of mobile phase composition on the chromatographic behaviour of human peripheral blood cells on polyethylene glycol-bonded sepharose.

The influence of mobile phase composition on the chromatographic behaviour of human platelets, granulocytes, lymphocytes and erythrocytes has been studied by using a bisoxirane-coupled polyethylene glycol 20M-Sepharose 6B column at pH 7.5. Lowering of the concentration of dextran T40 from 8 to 2% (w/w) produced the highest separation factor between platelets and granulocytes. Addition of 0.5% (w/w) of DEAE-dextran to a mobile phase containing 2% dextran T40 or T500 increased the retention of platelets, and discriminated the cells from erythrocytes. Addition of sodium chloride increased the retention volumes of lymphocytes, granulocytes and platelets. These blood cells were adsorbed to the column in isotonic phosphate-buffered eluent, whereas in imidazole-buffered eluent about 0.15-0.154 M sodium chloride improved their resolution.

Analysis of polyphenols from hop bract region using CCC

Abstract Polyphenols derived from hop (Humulus lupulus L.) bract region (HBP) can be used as food materials, thereby preventing dental caries. Chemical details of the active substances need to be elucidated. The polyphenols from hop bract (HBP) region were purified by countercurrent chromatography (CCC). The fractions were analyzed by high‐performance size‐exclusion chromatography (HPSEC) and reversed phase high‐performance liquid chromatography (RP‐HPLC). From HBP fractions by HPSEC, some low‐molecular‐ weight polyphenols (glycosides of flavonoids, catechins, and proanthocyanidins) were identified by RP‐HPLC. However, a very hydrophilic fraction was found to have the most potent cavity‐preventive activity, but it showed no peak in its RP‐HPLC chromatogram (absence of small polyphenols). HPSEC analysis showed that the major components of this fraction were high‐molecular weight substances, which were supposed to be proanthocyanidins, consisting of approximately 22 catechin units in its structure.

Determination of Log Po/w for Catechins and Their Isomers, Oligomers, and Other Organic Compounds by Stationary Phase Controlled High‐Speed Countercurrent Chromatography

Abstract A new technique, stationary phase volume controlled high‐speed counter‐current chromatography (HSCCC), was used to determine the octanol‐water partition coefficients (P o/w) of catechins and their isomers, oligomers, and other organic compounds. The stationary phase volume in the CCC column was effectively controlled under hydrodynamic equilibrium system. The log P o/w values ranging from −1.35 to +3.60 were measured within 21 min using this new HSCCC technology. The linear relationship (correlation coefficient value, r=0.993) was observed between log P o/w values obtained by the shake‐flask method and those values by the HSCCC method. In this technique, it is possible to inject multiple samples successively into the CCC column at short intervals to measure their retention times without renewing the stationary phase volume.

Purification of lactic acid dehydrogenase from bovine heart crude extract by counter-current chromatography

To test the utility of counter-current chromatography in purifying proteins, lactic acid dehydrogenase (LDH) was extracted from a crude bovine heart filtrate using a cross-axis coil planet centrifuge. The purification was performed with several polymer phase systems composed of 16% (w/w) poly(ethylene glycol) (PEG) 1000-12.5% (w/w) potassium phosphate buffers and 4.4% (w/w) PEG 8000-7.0 (w/w) dextran T500 at pH values ranging from 6.5 to 11.0. The best purification was achieved using PEG 1000-potassium phosphate buffer system at pH 7.3 by eluting the upper phase at 1.0 ml/min. Fractions were analyzed by LDH enzymatic activity and sodium dodecyl sulfate slab gel electrophoresis (SDS-PAGE). The LDH was purified directly from bovine heart crude extract within 3 h.