Flavonoids as inducers of extracellular proteins and exopolysaccharides of Sinorhizobium fredii

 Some flavonoids present in root exudates are inducers of nod genes in rhizobia-legume symbioses. They also induce changes in the molecular weight, structure, and level of secretion of some extracellular proteins, exopolysaccharides (EPS) and lipopolysaccharides. We showed that incubation of Sinorhizobium fredii USDA 257 with four flavonoids (genistein, naringenin, chrysin, and apigenin) promoted its growth in the late log phase. By contrast, only genistein accelerated the growth of S. fredii TU 6 under the same conditions. When both strains were incubated with naringenin the synthesis of EPS decreased. However, this compound increased the secretion of extracellular proteins in the early log phase. The specific mode of action of naringenin is still not clear. Received: 1 July 1998     

Apigenin inhibits platelet adhesion and thrombus formation and synergizes with aspirin in the suppression of the arachidonic acid pathway

Previous studies using washed platelets demonstrated that certain flavonoids inhibit platelet function through several mechanisms including blockade of TxA(2) receptors (TPs). We aimed to analyze the binding capacity of flavonoids to TPs in platelet rich plasma (PRP), investigated their effect in flowing blood, and evaluated the ability of apigenin to improve the efficacy of aspirin in the inhibition of platelet aggregation. The binding of flavonoids to TPs in PRP was explored using binding assays and the TP antagonist [ (3)H]SQ29548. Effects of flavonoids on platelet adhesion were assessed using arterial subendothelium with annular plate perfusion chambers, and global evaluation of apigenin on high-shear-dependent platelet function was determined by the PFA-100. To evaluate the ability of apigenin to potentiate the effect of aspirin, arachidonic acid-induced platelet aggregation was measured prior to and after consumption of subaggregatory doses of aspirin in the presence or absence of apigenin. Binding assays revealed that apigenin was an efficient competitor of [ (3)H]SQ29548 binding to PRP ( K i = 155.3 +/- 65.4 microM), and perfusion studies showed that apigenin, genistein, and catechin significantly diminished thrombus formation when compared to control (26.2 +/- 3.8, 33.1 +/- 5.2, and 26.2 +/- 5.2 vs 76.6 +/- 2.6%, respectively; p < 0.05). Apigenin, similarly to the TP antagonist SQ29548, significantly prolonged collagen epinephrine-induced PFA-100 closure time in comparison to the control and, when added to platelets that had been exposed in vivo to aspirin, potentiated its inhibitory effect on platelet aggregation. The inhibitory effect of some flavonoids in the presence of plasma, particularly apigenin, might in part rely on TxA(2) receptor antagonism. There is a clear increase in the ex vivo antiplatelet effect of aspirin in the presence of apigenin, which encourages the idea of the combined use of aspirin and certain flavonoids in patients in which aspirin fails to properly suppress the TxA(2) pathway.     

Kinetic study of flavonoid reactions with stable radicals

The antiradical activities of some flavonols (kaempferol, quercetin, robinetin, quercetagetin, and myricetin), flavones (apigenin, baicalein, and luteolin), flavanones (naringenin and dihydroquercetin), and flavanols [(+)-catechin and (-)-epicatechin] were determined by measuring the reaction kinetics with 2,2-diphenyl-1-picrylhydrazyl (DPPH) and alpha,gamma-bisdiphenylene-beta-phenylallyl (BDPA) radicals. The reactions, which follow the mixed second-order rate law, were investigated under pseudo-first-order conditions by use of a large excess of flavonoids, and their stoichiometry was determined by spectrophotometric titration. The results confirm stoichiometric factors of 1, 2, and 3 for flavonoids with one, two, and three hydroxyl groups in the B-ring, respectively, excluding kaempferol, which, despite a single OH group in the B-ring, has a factor of 2, which is explained by the 3-OH group supporting the reaction with free radicals. Structure-activity considerations indicate for the present series of flavonoids the importance of multiple OH substitutions and conjugation. The logarithms of reaction rate constants with the OH, DPPH, and BDPA radicals correlate well with the reduction potential of the flavonoids.     

Gas-phase ligation of Fe+ and Cu+ ions with some flavonoids

It was assumed that gas-phase ligation of metal monocations by flavonoids might provide some insight on the intrinsic antioxidant activity of the latter. Thus, the ligation of Fe+ and Cu+ ions by apigenin (1), luteolin (2), kaempferol (3), quercetin (4), myricetin (5), and naringenin (6) was investigated in the gas phase in a Fourier transform mass spectrometer (FTMS). Both of the metal ions, which were produced by laser desorption ionization (LDI), bind consecutively to two neutral flavonoid molecules either with or without the simultaneous loss of some part (H, CO, H2O) of the latter. The flavonoids are present in the instrument at steady concentrations. The formation of flavonoid positive ions by charge exchange is also a common observation but is accompanied, in some cases, by a loss of H, CO, or H2O fragments. The reaction paths and observed fragmentations are presented. The results are supported by DFT B3LYP calculations that indicate a preference for metal ion attack at C-ring and not at the B-ring site considered to be mainly responsible for flavonoid antioxidant activity.     

Differential effects of flavonoids on barrier integrity in human intestinal Caco-2 cells

Flavonoids, present in fruits, vegetables, and teas, provide beneficial effects for our health. We investigated the effect of a number of flavonoids on tight junction (TJ) barrier integrity in human intestinal Caco-2 cells. Transepithelial electrical resistance (TER; a TJ integrity marker) across cell monolayers was measured in cells incubated with flavonoids for 24 h. Chrysin decreased the TER, indicating a decrease in TJ integrity. Daidzein, hesperetin, naringenin, and morin increased the TER, indicating increased TJ integrity. Luteolin and genistein increased or normalized the TER after a transient decrease. Immunoblot analysis revealed that these changes in TER were caused by modification of the cytoskeletal association and expression of TJ proteins, zonula occludens (ZO)-1, ZO-2, occludin, junctional adhesion molecule-1, and/or claudins. Our results suggest that various flavonoids participate in the regulation of intestinal TJ barrier integrity and that this regulation may partially contribute to the flavonoid-mediated biological effects on our health.     

Properties of chalconaringenin and rutin isolated from cherry tomatoes

Fresh cherry tomatoes cv. 'Susanne' contain more of the two flavonoids chalconaringenin (CN) and rutin than lycopene. Therefore some properties including antioxidant behavior of the flavonoids were studied. The two flavonoids were extracted from peel and isolated by use of different chromatographic methods. Molecular absorbtivities were found to be 26907 for CN and 20328 abs M(-1) cm(-1) for rutin. Both compounds exhibited properties as antioxidants through several assays, and rutin was found to be the strongest antioxidant except in one assay. None of the assays revealed pro-oxidative effects. As naringenin rather than CN is frequently reported as a tomato constituent, the stability of CN was investigated in order to detect potential ways of isomerization during sample preparation. CN isomerized slowly both under UVB radiation and in alkaline solutions. Thus, such factors do not explain the occurrence of naringenin in tomato samples. The deficiency in reports on CN may be explained by the similarity in chromatographic behaviors of CN and naringenin, and due to the fact that they have same molecular weights.     

Accumulation of isoflavone genistin in transgenic tomato plants overexpressing a soybean isoflavone synthase gene

Isoflavones are legume-specific flavonoids best known for their potential cancer preventive and phytoestrogenic properties. In this study, we attempted to engineer the isoflavone pathway in the popular fruit crop tomato (Solanum lycopersicum L). Tomato plants were transformed with a soybean (Glycine max L) isoflavone synthase (GmIFS2) cDNA under the control of the cauliflower mosaic virus 35S promoter. LC-MS/MS analysis demonstrated the presence of genistin (genistein 7-O-glucoside) as the major isoflavone metabolite in the transgenic plants. Substantial amounts of genistin (up to 90 nmol/g FW) were found in leaves, while the levels were marginally detectable (less than 0.5 nmol/g FW) in fruit peels. In either case, no drastic variations in endogenous phenolic contents were observed. Fruit peels were found to accumulate high levels of naringenin chalcone, implicating the limitation of naringenin substrates for isoflavone synthesis. Our results suggested that tomato plants could be engineered to produce isoflavones without comprising the levels of endogenous flavonols, which are also health-beneficial, but it may be necessary to enhance the expression levels of chalcone isomerase simultaneously to achieve significant yields in edible tissues such as fruit peels.     

Flavonoid deactivation of excited state flavins: reaction monitoring by mass spectrometry

Flavin mononucleotide (FMN, as a B(2) vitamin model) was shown to induce dimerization of flavonoids (flavanone, apigenin, naringenin, eriodictyol, taxifolin, catechin, kaempferol, luteolin, quercetin, rutin, and seven smaller model phenols studied) as the major photoreaction, when aqueous solutions were exposed to visible light using a new, real-time electrospray ionization mass-spectrometric (ESI-MS) technique supported by LC-MS and MS(2) analysis. Electrophilic intermediates such as transient radical cations, o-quinones, and p-quinone methide were proposed to be involved in the coupling process. The C(3)-OH in flavon-3-ols gave rise to atypical compounds such as a depside or a dioxane-linked dimer. Flavonoid dimers, formed in vegetal extracts added to food during storage in light and for which structures are proprosed based on MS and MS(2), may affect colloidal stability, color, astringency, and antioxidative capacity.     

Soy processing affects metabolism and disposition of dietary isoflavones in ovariectomized BALB/c mice

Soy foods and nutritional supplements are widely consumed for potential health benefits. It was previously shown that isoflavone-supplemented diets, which contained equal genistein equivalents, differentially stimulated mammary tumor growth in athymic mice based on the degree of processing. This paper reports plasma pharmacokinetic analysis and metabolite identification using the parental mouse strain fed the same diets, which contained genistin, mixed isoflavones, Novasoy, soy molasses, or soy flour plus mixed isoflavones. Whereas the degree of soy processing did affect several parameters reflecting isoflavone bioavailability and gut microflora metabolism of daidzein to equol, stimulation of tumor growth correlated significantly with only the plasma concentration of aglycon genistein produced by the diets. This conclusion is consistent with the known estrogen agonist activity of genistein aglycon on mammary tumor growth. Conversely, plasma equol concentration was inversely correlated with the degree of soy processing. Although antagonism of genistein-stimulated tumor growth by equol could explain this result, the very low concentration of aglycon equol in plasma (12-fold lower relative to genistein) is inconsistent with any effect. These findings underscore the importance of food processing, which can remove non-nutritive components from soy, on the pharmacokinetics and pharmacodynamics of isoflavones. Such changes in diet composition affect circulating, and presumably target tissue, concentrations of genistein aglycon, which initiates estrogen receptor-mediated processes required for the stimulation of tumor growth in a mouse model for postmenopausal breast cancer.     

Flavonoid content of U.S. fruits, vegetables, and nuts

Analytical data are reported for 20 flavonoids (as aglycones) determined for more than 60 fresh fruits, vegetables, and nuts collected from four regions across the United States at two times of the year. Sample collection was designed and implemented by the Nutrient Data Laboratory (USDA). Analyses of eight flavan-3-ols (catechin, catechin gallate, epicatechin, epicatechin gallate, epigallocatechin, epigallocatechin gallate, gallocatechin, and gallocatechin gallate), six anthocyanins (cyanidin, delphinidin, malvidin, pelargonidin, peonidin, and petunidin), two flavanones (hesperetin and naringenin), two flavones (apigenin and luteolin), and two flavonols (myricetin and quercetin) were performed by the Food Composition Laboratory (USDA) using a hydrolysis method for the anthocyanidins, flavones, and flavonols and a direct extraction method for the flavan-3-ols and flavanones. Experimental results compare favorably (few statistically significant differences) to literature values in the flavonoid and proanthocyanidin database previously compiled by the Nutrient Data Laboratory. The results of this study showed a seasonal variation only for blueberries. This study also showed that the variation in the flavonoid content of foods, as purchased by the U.S. consumer, is very large. The relative standard deviation, averaged for each flavonoid in each food, was 168%.     

Influences of intercropping and nitrogen supply on flavonoid exudation in wheat roots

Abstract

Soil and hydroponic experiments were carried out to examine the influences of intercropping and nitrogen supply on flavonoid exudation in wheat roots. Both experiments comprising three cropping patterns (wheat intercropped with faba bean, monocropped wheat, and monocropped faba bean) and three N supply levels (deficient, adequate, and excessive) with three replicates in a randomized complete block design. Across two experiments, intercropping increased but N fertilization decreased flavonoids of wheat roots frequently. Intercropping variably increased secretion of naringenin from 0.5 to 1.9 folds (P?<?0.5) in wheat roots at all three N levels, but rarely increased secretion of genistein and hesperetin in wheat at the deficient N level. Intercropped wheat secreted more flavonoids than monocropped wheat at its tillering (60^th d) and flowering (95^th d) stages; after the flowering stage, however, the differences between intercropping and monocropping were not significant at any N level. Secretion of flavonoids in wheat roots decreased with increased N supply. Interspecies and N supply altered the contents and proportions of flavonoids in wheat root exudations under wheat and faba bean intercropping. These results indicate facilitative root–root interactions and provide insight into cereal promote nodule of legume in intercropping system.     

Antioxidant and prooxidant actions of prenylated and nonprenylated chalcones and flavanones in vitro

Prenylated flavonoids found in hops and beer, i.e., prenylchalcones and prenylflavanones, were examined for their ability to inhibit in vitro oxidation of human low-density lipoprotein (LDL). The oxidation of LDL was assessed by the formation of conjugated dienes and thiobarbituric acid-reactive substances (TBARS) and the loss of tryptophan fluorescence. At concentrations of 5 and 25 microM, all of the prenylchalcones tested inhibited the oxidation of LDL (50 microg protein/ml) induced by 2 microM copper sulfate. The prenylflavanones showed less antioxidant activity than the prenylchalcones, both at 5 and 25 microM. At 25 microM, the nonprenylated chalcone, chalconaringenin (CN), and the nonprenylated flavanone, naringenin (NG), exerted prooxidant effects on LDL oxidation, based on TBARS formation. Xanthohumol (XN), the major prenylchalcone in hops and beer, showed high antioxidant activity in inhibiting LDL oxidation, higher than alpha-tocopherol and the isoflavone genistein but lower than the flavonol quercetin. When combined, XN and alpha-tocopherol completely inhibited copper-mediated LDL oxidation. These findings suggest that prenylchalcones and prenylflavanones found in hops and beer protect human LDL from oxidation and that prenylation antagonizes the prooxidant effects of the chalcone, CN, and the flavanone, NG.     

Urolithins, ellagic acid-derived metabolites produced by human colonic microflora, exhibit estrogenic and antiestrogenic activities

Urolithins A and B (hydroxy-6H-dibenzo[b,d]pyran-6-one derivatives) are colonic microflora metabolites recently proposed as biomarkers of human exposure to dietary ellagic acid derivatives. Molecular models suggest that urolithins could display estrogenic and/or antiestrogenic activity. To this purpose, both urolithins and other known phytoestrogens (genistein, daidzein, resveratrol, and enterolactone) were assayed to evaluate the capacity to induce cell proliferation on the estrogen-sensitive human breast cancer MCF-7 cells as well as the ability to bind to alpha- and beta-estrogen receptors. Both urolithins A and B showed estrogenic activity in a dose-dependent manner even at high concentrations (40 microM), without antiproliferative or toxic effects, whereas the other phytoestrogens inhibited cell proliferation at high concentrations. Overall, urolithins showed weaker estrogenic activity than the other phytoestrogens. However, both urolithins displayed slightly higher antiestrogenic activity (antagonized the growth promotion effect of 17-beta-estradiol in a dose-dependent manner) than the other phytoestrogens. The IC(50) values for the ERalpha and ERbeta binding assays were 0.4 and 0.75 microM for urolithin A; 20 and 11 microM for urolithin B; 3 and 0.02 for genistein; and 2.3 and 1 for daidzein, respectively; no binding was detected for resveratrol and enterolactone. Urolithins A and B entered into MCF-7 cells and were metabolized to yield mainly urolithin-sulfate derivatives. These results, together with previous studies regarding absorption and metabolism of dietary ellagitannins and ellagic acid in humans, suggest that the gut microflora metabolites urolithins are potential endocrine-disrupting molecules, which could resemble other described "enterophytoestrogens" (microflora-derived metabolites with estrogenic/antiestrogenic activity). Further research is warranted to evaluate the possible role of ellagitannins and ellagic acid as dietary "pro-phytoestrogens".     

Absorption and excretion of luteolin and apigenin in rats after oral administration of Chrysanthemum morifolium extract

Chrysanthemum morifolium extract (CME) has the protective effect on cardiovascular diseases. Luteolin and apigenin are two major bioactive components in vivo when CME is orally administrated to experimental animal. The present paper shows the study of the absorption and excretion of luteolin and apigenin in rats after a single oral dose of CME (200 mg/kg). The levels of luteolin and apigenin in plasma, urine, feces, and bile were measured by HPLC after deconjugation with hydrochloric acid or beta-glucuronidase/sulfatase. The results showed that the plasma concentrations of luteolin and apigenin reached the highest peak level at 1.1 and 3.9 h after dosing, respectively. The area under the concentration-time curves (AUC) for luteolin and apigenin were 23.03 and 237.6 microg h mL-1, respectively. The total recovery of the dose was 37.9% (6.6% in urine; 31.3% in feces) for luteolin and 45.2% (16.6% in urine; 28.6% in feces) for apigenin. The cumulative luteolin and apigenin excreted in the bile was 2.05% and 6.34% of the dose, respectively. All of the results suggest apigenin may be absorbed more efficiently than luteolin in CME in rats, and both luteolin and apigenin have a slow elimination phase, with a quick absorption, so a possible accumulation of the two flavonoids in the body can be hypothesized.     

Deconjugation and degradation of flavonol glycosides by pig cecal microbiota characterized by Fluorescence in situ hybridization (FISH)

As the bioavailability of flavonoids is influenced by intestinal metabolism, we have investigated the microbial deconjugation and degradation of several flavonols and flavonol glycosides using the pig cecum in vitro model system developed in our group. For this model system the microbiota was directly isolated from the cecal lumen of freshly slaughtered pigs. The characterization of the cecal microbiota by fluorescence in situ hybridization (FISH) with 16S rRNA-based oligonucleotide probes confirmed the suitability of the model system for studying intestinal metabolism by the human microbiota. We have investigated the microbial degradation of quercetin-3-O-beta-d-rutinoside 1, quercetin-3-O-beta-d-glucopyranoside 2, quercetin-4'-O-beta-d-glucopyranoside 3, quercetin-3-O-beta-d-galactopyranoside 4, quercetin-3- O-beta-d-rhamnopyranoside 5, quercetin-3- O-[alpha-l-dirhamnopyranosyl-(1-->2)-(1-->6)-beta-d-glucopyranoside 6, kaempferol-3-O-[alpha-l-dirhamnopyranosyl-(1-->2)-(1-->6)-beta-d-glucopyranoside 7, apigenin 8, apigenin-8- C-glucoside (vitexin) 9, and feruloyl-O-beta-d-glucopyranoside 10 (100 microM), representing flavonoids with different aglycones, sugar moieties, and types of glycosidic bonds. The degradation rate was monitored using HPLC-DAD. The flavonol O-glycosides under study were almost completely metabolized by the intestinal microbiota within 20 min and 4 h depending on the sugar moiety and the type of glycosidic bond. The degradation rates of the quercetin monoglycosides showed a clear dependency on the hydroxyl pattern of the sugar moiety. The degradation of 2 with all hydroxyl groups of the glucose in the equatorial position was the fastest. The intestinal metabolism of di- and trisaccharides was much slower compared to the monoglycosides. The structure of the aglycone has not much influence on the intestinal metabolism; however, the type of glycosidic bond ( C- or O-glycoside) has substantial influence on the degradation rate. The liberated aglycones were completely metabolized within 8 h. Phenolic compounds such as 3,4-dihydroxyphenylacetic acid 12, 4-hydroxyphenylacetic acid 13, and phloroglucinol 18 were detected by GC-MS as main degradation products.     

Antioxidant effectiveness of phenolic apple juice extracts and their gut fermentation products in the human colon carcinoma cell line caco-2

Apples represent a major dietary source of antioxidative polyphenols. Their metabolic conversion by the gut microflora might generate products that protect the intestine against oxidative damage. We studied the antioxidant effectiveness of supernatants of fermented apple juice extracts (F-AEs, 6 and 24 h fermentation) and of selected phenolic degradation products, identified by HPLC-DAD-ESI-MS. Cell free antioxidant capacity of unfermented apple juice extracts (AEs) was decreased after fermentation by 30-50%. In the human colon carcinoma cell line Caco-2, F-AEs (containing <0.5% of original AE-phenolics) decreased the reactive oxygen species (ROS) level more efficiently than the F-blank (fermented without AE) but were less effective than the respective AEs. Similarly, antioxidant effectiveness of individual degradation products was lower compared to respective AE constituents. Glutathione level was slightly increased and oxidative DNA damage slightly decreased by fermented AE03, rich in quercetin glycosides. In conclusion, F-AEs/degradation products exhibit antioxidant activity in colon cells but to a lesser extent than the respective unfermented AEs/constituents.     

Metabolism of biochanin A and formononetin by human liver microsomes in vitro

Biochanin A and formononetin are abundant in legumes. These proestrogenic isoflavones can be converted by 4'-O-demethylation to the more potent phytoestrogens genistein and daidzein. Incubation of biochanin A or formononetin with human liver microsomes resulted in 4'-O-demethylation and the production of additional metabolites. Three new hydroxylated formononetin derivatives, 6,7-dihydroxy-4'-methoxyisoflavone, 7,8-dihydroxy-4'-methoxyisoflavone, and 7,3'-dihydroxy-4'-methoxyisoflavone, were isolated and characterized. We surveyed the O-demethylase competence of cytochrome P450 isoforms found in human liver. Human cytochrome P450 isoforms 1A2, 2E1, 2C9*1, 2C19, and 2D6*1 catalyzed biochanin A consumption and genistein production. Human cytochrome P450 isoforms 1A2, 2C9*1, 2A6, 2D6*1, and 2C19 catalyzed formononetin consumption and daidzein production. These isoforms also generated other hydroxylated metabolites. Although O-demethylation of isoflavones has been attributed to metabolism by gut microflora, our study demonstrates that human hepatic microsomal enzymes can perform the same transformation and may play a key role in the conversion of 4'-O-methylated isoflavones to more potent phytoestrogens.     

Stability of ferric complexes with 3-hydroxyflavone (flavonol), 5,7-dihydroxyflavone (chrysin), and 3',4'-dihydroxyflavone

The acid dissociation and ferric stability constants for complexation by the flavonoids 3-hydroxyflavone (flavonol), 5,7-dihydroxyflavone (chrysin), and 3',4'-dihydroxyflavone in 50:50 (v/v) ethanol/water are determined by pH potentiometric and spectrophotometric titrations and the linear least-squares curve-fitting program Hyperquad. Over the entire range of pH and reagent concentrations spanning the titration experiments, the stoichiometry for iron-flavonoid complex formation was 1:1 for all three flavonoids examined. The three flavonoids were chosen for their hydroxy substitution pattern, with each possessing one of the three most commonly suggested sites for metal binding by the flavonoids. On the basis of the calculated stability constants, the intraflavonoid-binding site competition is illustrated as a function of pH via speciation curves. The curves indicate that the binding site comprised of the 3',4'-hydroxy substitutions, the catecholic site, is most influential for ferric complexation at the physiological pH of 7.4. The possibility for antioxidant activity by flavonoid chelation of ferric iron in the presence of other competitive physiological complexing agents is demonstrated through additional speciation calculations.     

Dietary apigenin suppresses IgE and inflammatory cytokines production in C57BL/6N mice

Flavonoids ubiquitously exist in plants, vegetables, fruits, and teas. We evaluated the effect of dietary apigenin, one of the well-known flavonoids, on the immune system in C57BL/6N mice. Mice were fed experimental diets containing apigenin for 2 weeks. After the experimental period, there was no significant difference in body and organ weights between the control and the apigenin group. The total immunoglobulin (Ig) E levels in mice fed apigenin were significantly suppressed, whereas levels of IgG, IgM, and IgA were not affected. We also examined the effect of the apigenin diet on cytokine expression in mice sera using a cytokine array. The production of regulated upon activation normal T cell expressed and secreted (RANTES) and soluble tumor necrosis factor receptor I (sTNFRI) in mice sera was down-regulated by the apigenin diet. These results suggest that a diet containing apigenin can reduce serum IgE and inflammatory cytokines such as RANTES and sTNFRI in mice.