Consumption of 1-Kestose Upregulates MicroRNA-200 and -192/215 Families in Lamina Propria Leukocytes of the Murine Large Intestine.

By comparing germ-free mice and specific pathogen-free mice, we recently demonstrated that the presence of gut commensals upregulates microRNA-200 family members in lamina propria leukocytes (LPL) of the murine large intestine. The present study tested whether the consumption of 1-kestose (KES), an indigestible oligosaccharide that alters gut microbiota composition, influences the microRNA expression in the LPL. Supplementation of KES (4%) in drinking water for 2 wk increased the levels of miR-182-5p, -205-5p, -290a-5p, miR-200 family members (miR-141-3p, -200a-3p, -200b-3p, -200c-3p, and -429-3p) as well as miR-192/215 family members (miR-192-5p, -194-5p, and -215-5p) as determined by microarray analysis in large intestinal LPL of C57BL/6 mice. Quantitative reverse transcription-PCR further confirmed the increase in miR-192-5p, -194-5p, -200a-3p, -200b-3p, -200c-3p, -205-5p, and 215-5p. KES consumption significantly increased Bifidobacterium pseudolongum in the cecal contents. In a separate experiment, intragastric administration of B. pseudolongum (109 CFU/d) for 7 d increased the levels of miR-182-5p, -194-5p, and -200a-3p and tended to increase the levels of miR-200b-3p, -215-5p, and -429-3p. These results suggest that dietary KES influences miRNA expression in the large intestinal LPL, which may be associated with the increased population of B. pseudolongum.

Comprehensive separation and structural analyses of polyphenols and related compounds from bracts of hops (Humulus lupulus L.).

A novel sequential chromatographic technique was applied to the comprehensive separation of polyphenols and related compounds from a hop bract extract. Over 100 types of constituents were effectively isolated from only 25 g of extract in high yields by high-speed countercurrent chromatography followed by hydrophilic interaction chromatography and reversed-phase high performance liquid chromatography. Among the materials isolated, the structures of 39 compounds were elucidated on the basis of their spectroscopic data including electrospray ionization time-of-flight mass spectrometry and one-dimensional/two-dimensional nuclear magnetic resonance. Three new compounds, 1 known compound identified for the first time in plants, and 20 known compounds that have not been reported in hops, were found. The hop bract extract also contained an abundance of highly oligomeric proanthocyanidins, which consisted of B-type procyanidin structures.

Chemical analysis and acetylcholinesterase inhibitory effect of anthocyanin-rich red leaf tea (cv. Sunrouge).

BACKGROUND: The purpose of this study was to evaluate the effects of leaf order or crop season on anthocyanins and other chemicals in the anthocyanin-rich tea cultivar 'Sunrouge' (Camellia sinensis x C. taliensis) by using high-performance liquid chromatography, and to study the effect of 'Sunrouge' extract on acetylcholinesterase (AChE) activity in human neuroblastoma SK-N-SH cells. RESULTS: The total anthocyanin content was higher in the third (3.09 mg g⁻¹) than in the second (2.24 mg g⁻¹) or first crop season (1.79 mg g⁻¹). The amount of anthocyanins contained in the stem was high (1.61 mg g⁻¹). In the third crop season, the concentrations of delphinidin-3-O-ß-D-(6-(E)-p-coumaroyl)galactopyranoside (DCGa), cyanidin-3-O-ß-D-(6-(E)-p-coumaroyl)galactopyranoside, delphinidin-3-O-ß-D-galactopyranoside, delphinidin-3-O-ß-D-(6-O-(Z)-p-coumaroyl)galactopyranoside, cyanidin-3-O-ß-D-galactoside, and delphinidin-3-O-ß-D-glucoside were 1.57 mg g⁻¹, 0.52 mg g⁻¹, 0.40 mg g⁻¹, 0.22 mg g⁻¹, 0.14 mg g⁻¹, and 0.11 mg g⁻¹, respectively. DCGa accounted for about 50% of the anthocyanins present. The suppressive effect of 'Sunrouge' water extract on AChE activity in human neuroblastoma SK-N-SH cells was the strongest among the three tea cultivars ('Sunrouge', 'Yabukita' and 'Benifuuki'). CONCLUSION: These results suggested that 'Sunrouge' might protect humans from humans from AChE-related diseases by suppressing AChE activity. To obtain sufficient amounts of anthocyanins, catechins and/or caffeine for a functional food material, 'Sunrouge' from the third crop season should be used.

Anthocyanins from new red leaf tea 'Sunrouge'.

New red leaf tea cultivar 'Sunrouge' (Camellia taliensis × Camellia sinensis), for which an application for registration was made in 2009, is an anthocyanin-rich tea. The anthocyanin content of 'Sunrouge' was the highest among 4 tea cultivars, and was 8.4 times higher than that of 'Yabukita'. We purified and isolated 6 anthocyanins from 'Sunrouge' by chromatography, and identified them by LC/MS/MS and NMR analysis. As a result, the four anthocyanins were identified as delphinidin-3-O-ß-D-(6-(E)-p-coumaroyl)galactopyranoside (2), delphinidin-3-O-ß-D-(6-(E)-p-coumaroyl)glucopyranoside (3), cyanidin-3-O-ß-D-(6-(E)-p-coumaroyl)galactopyranoside (4), and cyanidin-3-O-ß-D-(6-(E)-p-coumaroyl)glucopyranoside (5), and the other two were estimated as delphinidin-(Z)-p-coumaroylgalactopyranoside (1), petunidin-(E)-p-coumaroylgalactopyranoside (6). Compound 3 was found in tea for the first time. In general, anthocyanins have various bioactivities, including relieving eyestrain and antioxidative effects, so it is expected that drinking 'Sunrouge' tea brings in similar bioactivities.

Cloning of a novel O-methyltransferase from Camellia sinensis and synthesis of o-methylated EGCG and evaluation of their bioactivity.

The gene of a novel O-methyltransferase was isolated from tea cultivars (Camellia sinensis L.). Using the recombinant enzyme, O-methylated (-)-epigallocatechin-3-O-gallate (EGCG) in all cases were synthesized. EGCG and the synthesized O-methylated EGCGs including (-)-epigallocatechin-3-O-(3-O-methyl)-gallate (EGCG3''Me), (-)-epigallocatechin-3-O- (4-O-methyl)-gallate(EGCG4''Me), (-)-epigallocatechin-3-O-(3,5-O-dimethyl)-gallate (EGCG3'',5''diMe), and (-)-3-O-methyl-epigallocatechin-3-O-(3,5-O-dimethyl)-gallate (EGCG3',3'',5''triMe) were assayed using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay and antibacterial activity. EGCG was the most effective of the O-methylated EGCGs. The antiallergic effects of EGCG and the other O-methylated EGCGs were measured by conducting histamine release assays using bone marrow-derived mouse mast cells, and the order of potency was EGCG3',3'',5''triMe = EGCG3'',5''diMe > EGCG3''Me > EGCG. These results indicated that reducing the number of hydroxyl groups decreases the effectiveness of DPPH radical scavenging and antibacterial activity. In contrast, the inhibition of histamine release was potentiated by an increase in the number of methyl groups in EGCG, especially in the galloyl moiety.

Identifications of inhibitors of IgE production by human lymphocytes isolated from 'Cha Chuukanbohon Nou 6' tea leaves.

BACKGROUND: Tea (Camellia sinensis L.) is consumed all over the world and in especially large quantities in Japan and China, where it has been used not only as a daily beverage but also for medicinal purposes for thousands of years. Tea has been found to exhibit various bioregulatory activities, including antiallergic, anticarcinogenic, antimetastatic, antioxidative, antihypertensive, antihypercholesterolemic, anti-dental caries and antibacterial effects, and to influence intestinal flora. RESULTS: Cha Chuukanbohon Nou 6 is a tea cultivar improved by the National Institute of Vegetable and Tea Science (NIVTS) in Japan. On comparing chemical constituents of 11 varieties of tea leaves by high-performance liquid chromatography, we found two new major compounds in Cha Chuukanbohon Nou 6. Nuclear magnetic resonance spectroscopy revealed these compounds to be theogallin and 1,2-di-O-galloyl-4,6-O-(S)-hexahydroxydiphenoyl-beta-D-glucopyranose. The two were similar in chemical structure to strictinin, an inhibitor of immunoglobulin (Ig) production. Thus their effects on the production of Igs by peripheral blood lymphocytes were tested. Both compounds, like strictinin, inhibited IgE production. CONCLUSION: The results suggest Cha Chuukanbohon Nou 6 to be the basis of an antiallergic beverage.

Identification of hop polyphenolic components which inhibit prostaglandin E2 production by gingival epithelial cells stimulated with periodontal pathogen.

Chronic marginal periodontitis is a destructive inflammatory disease caused by an imbalance between bacterial virulence and host defense ability, resulting in eventual tooth exfoliation. Porphyromonas gingivalis, a major periodontal pathogen, triggers a series of cellular inflammatory responses including the production of prostaglandin E2 (PGE2), which causes periodontal destruction; thus, anti-inflammatory reagents are considered beneficial for periodontal therapy. In the present study, we examined whether hop- and apple-derived polyphenols (HBP and ACT, respectively) inhibit PGE2 production by human gingival epithelial (HGE) cells stimulated with P. gingivalis components. HGE cells were stimulated with P. gingivalis membrane vesicles, and the effects of HBP, ACT and epigallocatechin gallate (EGCg) on PGE2 production by HGE cells were evaluated using an enzyme-linked immunosorbent assay. HBP and EGCg significantly inhibited PGE2 production, whereas ACT did not. By further fractionation steps of HBP to identify the effective components, 3 components of HBP, 2-[(2-methylpropanoyl)-phloroglucinol]1-O-beta-D-glucopyranoside (MPPG), quercetin 3-O-beta-D-glucopyranoside (isoquercitrin), and kaempferol 3-O-beta-glucopyranoside (astragalin), were found to be elements which significantly inhibited cellular PGE2 production. These results suggest that HBP is a potent inhibitor of cellular PGE2 production induced by P. gingivalis, and HBP may be useful for the prevention and attenuation of periodontitis.

Inflammatory responses of gingival epithelial cells stimulated with Porphyromonas gingivalis vesicles are inhibited by hop-associated polyphenols.

BACKGROUND: Periodontitis is induced by an imbalance between bacterial virulence and host defense ability. Porphyromonas gingivalis, a predominant periodontal pathogen, triggers a series of host inflammatory responses that aggravate the destruction of periodontium. Thus, anti-inflammatory reagents are considered desirable for effective periodontal therapy. In the present study, we examined the inhibitory effects of hop bract polyphenol (HBP) on cellular inflammatory responses induced by P. gingivalis membrane vesicles. METHODS: Immortalized human gingival epithelial cells were stimulated with P. gingivalis membrane vesicles, and the effects of HBP on mRNA expression of cyclooxygenase (COX)-2, interleukin (IL)-6 and -8, and matrix metalloproteinase (MMP)-1 and -3 were examined using real-time reverse transcription-polymerase chain reaction. RESULTS: HBP inhibited the mRNA expression of COX-2, IL-6 and -8, and MMP-1 and -3 in a dose-dependent manner, whereas epigallocatechin gallate (a control polyphenol) inhibited COX-2 mRNA expression only. Following further fractionation of HBP to identify the effective components, 2-[(2-methylpropanoyl)-phloroglucinol]1-O-beta-D-glucopyranoside (MPPG) was identified as a significant anti-inflammatory element that completely inhibited the inflammatory mRNA induction. Kaempferol 3-O-beta-glucopyranoside (astragalin) also was found to have anti-inflammatory effects. CONCLUSIONS: HBP is suggested to be a potent inhibitor of cellular inflammatory responses induced by P. gingivalis vesicles. Further, MPPG and astragalin, identified here as effective components of HBP, also may be useful for the prevention and/or attenuation of periodontitis.

Safety evaluation of polyphenols extracted from hop bracts.

Hop bract polyphenols contain polyphenols as promising functional ingredients. To assess the safety of topical hop bract polyphenols, Hopsphenon, we examined acute, 14-day, 28-day and 90-day toxicity tests in rats, and mutagenicity tests using Ames test and micronucleus test in mice. The acute, 14-day, 28-day and 90-day toxicity tests revealed that Hopsphenon produced no symptoms of significant injury. The lethal dose of hop bract polyphenols is greater than 2000 mg/kg. The Ames test in the absence of S9 mix for TA98 and in the presence of S9 mix for TA1537 revealed that Hopsphenon had slight mutagenicity at a high dose of 5000 microg/plate; however, in the micronucleus test, Hopsphenon was negative. These tests demonstrated that hop bract polyphenols are safe and do not cause any detrimental effects in vivo under the conditions investigated in this study.