Search

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.

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.

Two new triterpenes named 7-oxodihydrokarounitriol (1) and 7,11-dioxodihydrokarounidiol (2), and one known triterpene, 7-oxodihydrokarounidiol (3), were isolated from the unsaponifiable matter of the seeds of Trichosanthes cucumeroides. The structures of 1 and 2 were elucidated as (3alpha, 11beta, 13alpha, 14beta, 20alpha)-3,11,29-trihydroxy-13-methyl-26-norolean-8-ene-7-one, and (3alpha,13alpha,14beta,20alpha)-3,29-dihydroxy-13-methyl-26-norolean-8-ene-7,11-dione on the basis of extensive NMR (1H, 13C, 1H-1H COSY, DEPT, HMQC, HMBC and NOESY) and MS studies.

Glucosyltransferase (GTF), a cariogenic factor of Streptococcus sobrinus (SS), was extracted from a large quantity of cell culture medium by batch extraction with aqueous polymer two‐phase systems (APTPs). The small quantity of the extract was further purified by countercurrent chromatography (CCC) using polyethylene glycol (PEG) 8000‐dextran T500 system at pH 9.2, by eluting the impurities with the upper mobile phase at 1.0 mL/min. After the elution, the GTF still remaining in the dextran‐rich lower stationary phase was collected. The GTF fractions were subjected to hydroxyapatite chromatography to eliminate polymers, such as PEG 8000 and dextran T500. Purified fractions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and GTF enzyme activity, which rose over 270 fold from that in the original culture medium.

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 la...

Polysaccharides were purified from a high molecular weight complex of Achyranthes bidentata Bl. (Fam. Amaranthaceae) root by high speed countercurrent chromatography (HSCCC) using a cross‐axis coil planet centrifuge (X‐axis CPC). The purification was performed with an aqueous polymer two‐phase system composed of 12.5% (w/w) polyethylene glycol (PEG) 1000, 8% (w/w) potassium dihydrogen phosphate, and 8% (w/w) dipotassium hydrogen phosphate buffer at pH 6.8. The preparative separation of the crude sample (2 g) was successfully performed, yielding 1.1 g of polysaccharides at high purity.

Chicken egg white proteins were fractionated by high-speed counter-current chromatography (HSCCC) using a cross-axis coil planet centrifuge (X-axis CPC). The separation was performed with an aqueous polymer two-phase system composed of 16% polyethylene glycol (PEG) 1000 and 12.5% potassium phosphate buffer at different pH of 6.8, 8.0, and 9.2. From 20 g of the crude egg white solution, the best separation was achieved with a polymer phase system at pH 8.0 by eluting the lower phase at 1.0 mL/min. After the sequential elution of ovotransferrin, ovalbumin, and lysozyme from the column, ovomucin still retained in the column was eluted in the reverse direction with upper PEG-rich phase. All four proteins in the CCC fractions were identified by SDS gel electrophoresis with Coomassie brilliant blue staining.

The type XL cross-axis coil planet centrifuge (CPC) was successfully utilized for purification of cholinesterase (ChE) from human serum by countercurrent chromatography (CCC). Aqueous-aqueous polymer phase systems composed of polyethylene glycol-potassium phosphate buffers were tested at various pHs, ranging from 6.8 to 9.2 for the distribution ratios (D) of ChE, human serum albumin, α- and γ-globulins. The CCC separation was performed with optimized polymer phase systems in two different coiled columns. The best purification was achieved with 16.0% PEG 600–12.5% potassium phosphate at pH 9.2, by eluting the lower phase at 0.5 mL/min through the small capacity column in which ChE was purified directly from human serum within 2h. The ChE fractions confirmed by the enzymatic activity were free of serum proteins by SDS PAGE analysis.

Antigen primed macrophage and thymic RNAs hybridize in an antigen restricted fashion. Thymic RNA complementary to the antigen-stimulated macrophage RNA can be obtained from mice inoculated with antigen three days earlier. The ratio of thymic and macrophage RNAs for the optimal hybridization was 50 : 1. The optimal incubation time for hybridization was 32 hours at 60 degrees C. Hybrid RNAs were insensitive to various kinds of RNase. The 31p Fourier transform nuclear magnetic resonance spectra identified hybrid RNA molecules form single stranded RNS's.