Tomomasa Kanda

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.

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.

Dose-dependent hypocholesterolemic actions of dietary apple polyphenol in rats fed cholesterol.

The dose-dependent hypocholesterolemic and antiatherogenic effects of dietary apple polyphenol (AP) from unripe apple, which contains approximately 85% catechin oligomers (procyanidins), were examined in male Sprague-Dawley rats (4 wk of age) given a purified diet containing 0.5% cholesterol. Dietary AP at 0.5 and 1.0% levels significantly decreased the liver cholesterol level compared with that in the control (AP-free dietfed) group. Dietary AP also significantly lowered the serum cholesterol level compared with that in the control group. However, the HDL cholesterol level was significantly higher in the 1.0% AP fed group than in the control group. Accordingly, the ratio of HDL-cholesterol/total cholesterol was significantly higher in the 0.5% AP-fed group and 1.0% AP-fed group than in the control group. Moreover, the atherogenic indices in the 0.5 and 1.0% AP-fed groups were significantly lower than those in the control group. The activity of hepatic cholesterol 7α-hydroxylase tended to be increased by dietary AP in a dose-dependent manner. In accord with this observation, dietary AP increased the excretion of acidic steroids in feces. Dietary AP also significantly promoted the fecal excretion of neutral steroids in a dose-dependent manner. These observations suggest that dietary AP at 0.5 or 1.0% level exerts hypocholesterolemic and antiatherogenic effects through the promotion of cholesterol catabolism and inhibition of intestinal absorption of cholesterol.

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.

Procyanidins from Apples (Malus pumila Mill.) Extend the Lifespan of Caenorhabditis elegans

Apple polyphenols (AP) mainly consist of procyanidins (PC), which are composed of (-)-epicatechins and (+)-catechins. In order to investigate the antiageing effects of PC, we measured the lifespan of CAENORHABDITIS ELEGANS worms treated with PC. Treatment with 65 µg/mL PC extended the mean lifespan of wild-type N2 and FEM-1 worms by 12.1 % and 8.4 %, respectively, i.e., to a similar extent as resveratrol. In addition, treatment with 100 µg/mL AP also significantly prolonged the mean lifespan of the same worms by 12.0 % and 5.3 %, respectively, i.e., to a similar extent as PC. In contrast, treatment with (-)-epicatechin did not extend the lifespan of the worms. PC did not modify the growth, food intake, or fecundity of C. elegans. Treatment with PC did not extend the lifespan of MEV-1 worms, which show excessive oxidative stress, indicating that PC had no antioxidant ability in the MEV-1 mutant. Moreover, treatment with PC had no effect on the longevity of SIR-2.1 worms, which lack the activity of SIR-2, a member of the sirtuin family of NAD (+)-dependent protein deacetylases. These results indicated that PC has SIR-2.1-dependent antiageing effects on C. elegans.

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.

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.