Akio Yanagida

Identification of Catechin Oligomers from Apple (Malus pumila cv. Fuji) in Matrix‐assisted Laser Desorption/Ionization Time‐of‐flight Mass Spectrometry and Fast‐atom Bombardment Mass Spectrometry

Molecular size information for polymerized catechin larger than the decamer in unripe apple was obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and by fast-atom bombardment mass spectrometry. Matrix-assisted laser desorption/ionization time of flight mass spectrometry provided evidence for the pentadecamer using trans-3-indoleacrylic acid as the matrix in the presence of silver ion. Even in the absence of silver ion, the dodecamer and undecamer were observed in the positive- and negative-ion modes, respectively. Fast-atom bombardment mass spectrometry also afforded evidence for the undecamer in both positive- and negative-ion modes.

Fractionation of apple procyanidins by size-exclusion chromatography

Oligomeric constituents of apple procyanidins were fractionated by size-exclusion chromatography using a TSKgel Toyopearl HW-40F column. The best separation was obtained using a mobile phase of acetone-8 M urea (6:4; adjusted to pH 2) at a flow-rate of 1.0 ml/min. In this chromatographic system, the use of 8 M urea in the mobile phase resulted in a molecular sieve effect without any surface affinity interaction between the gel beads and the procyanidin molecules. Each fraction obtained was examined by reversed-phase high-performance liquid chromatography and time-of-flight mass spectrometry. The order of elution of the procyanidins from the column was coincident with their degree of polymerization.

Fractionation of apple procyanidins according to their degree of polymerization by normal-phase high-performance liquid chromatography

A new method was developed for the fractionation of procyanidin oligomers according to their degree of polymerization. Monomeric flavan-3-ols and low molecular mass procyanidins were selectively extracted from the lyophilized powder of apple condensed tannins (ACTs) by methyl acetate extraction. Sequentially, the separation of each oligomer from dimer to pentamer in this extract was carried out by normal-phase high-performance liquid chromatography using a silica-beads packed column. The best separation was achieved with a mobile phase system containing hexane; (1) hexane-methanol-ethyl acetate, (2) hexane-acetone. These sequential treatments can be easily adapted to large-scale fractionation.

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.

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.

Characterization of Polymerized Polyphenols by Size-exclusion HPLC

Various kinds of high-molecular-mass polyphenols such as condensed tannins, hydrolyzable tannins, and polymerized anthocyanins, were readily characterized by a new size-exclusion HPLC method. This rapid analytical method may also be useful for the profiling of molecular mass distribution of polyphenolic constituents in many kinds of food materials.

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.