Toshihiko Shoji

Dietary unripe apple polyphenol inhibits the development of food allergies in murine models

The incidence of type I allergic disorders has been increasing worldwide, particularly, the hypersensitivity to food. We first showed that apple condensed tannin (ACT) intake would inhibit the development of the oral sensitization and that the inhibition could correlate with the rise in the population of TCRγδ‐T cells in the intestinal intraepithelial lymphocytes (IEL) using W/WV mice and B10A mice which were ovalbumin (OVA)‐orally sensitized. Serum OVA‐specific immunoglobulin E and immunoglobulin G1 titers in the OVA‐orally sensitized W/WV and B10A mice ad libitium fed ACT were extremely inhibited compared to those of the control. The ACT intakes of OVA‐sensitized W/WV and B10A mice inhibited the immediate reduction of the body temperature or the rise in serum histamine induced by active systemic anaphylaxis. The proportions of the TCRγδ‐T cells in the IEL of the OVA‐orally sensitized W/WV and B10A mice ad libitium fed ACT were significantly greater than that in the controls. Furthermore, ACT feeding by itself could induce the rise in the percentage of the TCRγδ‐T cells among the IEL of the W/WV and B10A mice. This suggests that the ACT intake may prevent the development of food allergies and this effect could be correlated with the rise in the percentage of TCRγδ‐T cells among the IEL.

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.

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.

Regulation of Vascular Endothelial Function by Procyanidin-Rich Foods and Beverages†

Flavonoid-rich diets are associated with a lower mortality from cardiovascular disease. This has been linked to improvements in endothelial function. However, the specific flavonoids, or biologically active metabolites, conferring these beneficial effects have yet to be fully defined. In this experimental study of the effect of flavonoids on endothelial function cultured endothelial cells have been used as a bioassay with endothelin-1 (ET-1) synthesis being measured an index of the response. Evaluation of the relative effects of extracts of cranberry juice compared to apple, cocoa, red wine, and green tea showed inhibition of ET-1 synthesis was dependent primarily on their oligomeric procyanidin content. Procyanidin-rich extracts of cranberry juice triggered morphological changes in endothelial cells with reorganization of the actin cytoskeleton and increased immunostaining for phosphotyrosine residues. These actions were independent of antioxidant activity. Comparison of the effects of apple procyanidin monomers through heptamer showed a clear structure-activity relationship. Although monomer, dimer, and trimer had little effect on ET-1 synthesis, procyanidin tetramer, pentamer, hexamer, and heptamer produced concentration-dependent decreases with IC(50) values of 5.4, 1.6, 0.9, and 0.7 microM, respectively. Levels of ET-1 mRNA showed a similar pattern of decreases, which were inversely correlated with increased expression of Kruppel-like factor 2 (KLF2), a key endothelial transcription factor with a broad range of antiatherosclerotic actions including suppression of ET-1 synthesis. Future investigations of procyanidin-rich products should assess the role KLF2 induction plays in the beneficial vascular effects of high flavonoid consumption.

Orally administered apple procyanidins protect against experimental inflammatory bowel disease in mice

Apple procyanidins (ACT) is a natural biologically active compound extracted from apple. Our recent studies have shown that ACT ameliorates the symptoms of atopic dermatitis and inhibits food-allergen-induced oral sensitization. The aim of this study was to investigate the potential protective effect and mechanism of action of ACT in a murine model of inflammatory bowel disease. We investigated the preventive effects of ACT in experimental models of colitis induced by dextran sulfate sodium (DSS) or oxazolone. Oral administration of ACT before DSS treatment attenuated the DSS-induced mortality rate and decreased body weight loss. ACT also prevented the body weight loss associated with oxazolone-induced colitis. Next we examined the effect of ACT on intraepithelial lymphocytes (IEL), which is a major T cell population in the intestine. Oral administration of ACT increased the proportions of TCRgammadelta and TCRalphabeta-CD8alphaalpha T cells in IEL and suppressed interferon gamma synthesis in stimulated IEL. In addition, ACT inhibited phorbol 12-myristate 13-acetate-induced secretion of interleukin 8 (IL-8) in intestinal epithelial cells. The combined anti-inflammatory and immunomodulatory effects of ACT on intestinal epithelial cells and IEL suggest that it may be an effective oral preventive agent for inflammatory bowel diseases.

A NMR-based, non-targeted multistep metabolic profiling revealed L-rhamnitol as a metabolite that characterised apples from different geographic origins.

This study utilises (1)H NMR-based metabolic profiling to characterise apples of five cultivars grown either in Japan (Fuji, Orin, and Jonagold) or New Zealand (Fuji, Jazz, and Envy). Principal component analysis (PCA) showed a clear separation between the Fuji-Orin-Jonagold class and the Jazz-Envy class, primarily corresponding to the differences in sugar signals, such as sucrose, glucose, and fructose. Multistep PCA removed the influence of dominant sugars and highlighted minor metabolites such as aspartic acid, 2-methylmalate, and an unidentified compound. These minor metabolites separated the apples into two classes according to different geographical areas. Subsequent partial least squares discriminant analysis (PLS-DA) indicated the importance of the unidentified metabolite. This metabolite was isolated using charcoal chromatography, and was identified as L-rhamnitol by 2D NMR and LC/MS analyses. The remarkable contribution of L-rhamnitol to geographic discrimination suggests that apples may be characterised according to various factors, including storage duration, cultivation method, and climate.

Non-absorbable apple procyanidins prevent obesity associated with gut microbial and metabolomic changes

Several studies have suggested that flavan-3-ols/procyanidins are associated with a reduced risk of developing obesity and metabolic syndrome. However, the role of highly polymeric procyanidins (PP), which are major non-absorbable flavonoids, in the biological effects, is not completely understood. Here, we show that 0.5% PP administration for 20 weeks alleviated obesity and regulate expression of genes related to lipid metabolism in C57BL/6J mice fed a high-fat/high-sucrose diet. PP-treatment attenuated weight gain and inflammatory effects including lipopolysaccharide and gut permeability. Additionally, metabolic urine profiling using high-performance liquid chromatography–quadrupole time-of-flight/mass spectrometry demonstrated that PP-treatment decreased the levels of endogenous metabolites associated with insulin resistance. Furthermore, microbial 16S rRNA gene sequencing of the cecum demonstrated that PP administration markedly decreased the Firmicutes/Bacteroidetes ratio and increased eight times the proportion of Akkermansia. These data suggest that PPs influence the gut microbiota and the intestinal metabolome to produce beneficial effects on metabolic homeostasis.

Characterization and activity of anthocyanins in Zijuan tea (Camellia sinensis var. kitamura).

Zijuan tea is a new cultivar produced in Yunnan province of China. Unlike most tea cultivars, Zijuan tea is anthocyanin-rich. The composition and antioxidant activities of anthocyanins of Zijuan tea were studied for the first time in this paper. Anthocyanins were extracted with acidified methanol and quantified as 707 ± 28 μg/g of dry weight (cyanidin-3-O-β-D-glucoside equivalent) by high-performance liquid chromatography (HPLC) analysis. Four anthocyanins were successfully identified after Amberlite XAD-7HP adsorption column chromatography and octadecyl silane (ODS) flash chromatography. Among the four, delphinidin-3-O-β-D-galactoside (1) and cyanidin-3-O-β-D-galactoside (2) were confirmed by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) and HPLC. Delphinidin-3-O-β-D-(6-(E)-p-coumaroyl) galactopyranoside (3) and cyanidin-3-O-β-D-(6-(E)-p-coumaroyl) galactopyranoside (4) were characterized by the high-resolution time-of-flight-mass spectrometry (HRTOF-MS) and nuclear magnetic resonance (NMR) spectrometry. The antioxidant activities of compounds 3 and 4, which composed approximately 75% of the total anthocyanins content in HPLC analysis, were evaluated by DPPH and FRAP assays. Results showed that both had higher antioxidant activities than commercial antioxidants butylated hydroxytoluene (BHT) used as one of controls in these assays.

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.