Cosupplementation of isoflavones, prenylflavonoids, and lignans alters human exposure to phytoestrogen-derived 17beta-estradiol equivalents.

The microbial metabolism of dietary phytoestrogens varies considerably among individuals and influences the final exposure to bioactive compounds. In view of the increasing number of food supplements combining several classes of phytoestrogens, the microbial potential to activate various proestrogens within an individual was evaluated in 3 randomized dietary crossovers. Treatment allocation was based on participants' eligibility (>45% in vitro bioactivation of >or=2 separate proestrogens by fecal cultures; n = 40/100). After a run-in of >or=4 d, participants were given soy-, hop-, and/or flax-based food supplements dosed either separately (SOY: 2.83 mg daidzein aglycone equivalents/supplement, HOP: 1.20 mg isoxanthohumol (IX)/supplement, or FLAX: 2.08 mg secoisolariciresinol (SECO) aglycone equivalents/supplement; reference intervention) or simultaneously (MIX; test intervention) 3 times/d for 5 d, followed by a wash-out period (>or=7 d) and the second intervention. Before and after each (co)supplementation, spot urine and serum were collected. In total, 22 equol, 19 8-prenylnaringenin (8-PN), and 21 enterolactone (ENL) producers completed the SOY+MIX, HOP+MIX, and FLAX+MIX trials, respectively. The microbial bioactivation of daidzein, IX, and SECO, generally decreased upon coincubation in vitro (equol: 4.4%, P = 0.164; 8-PN: 20.5%, P < 0.001; ENL: 44.3%, P < 0.001) and cosupplementation in vivo (equol: 28.3%, P = 0.009; 8-PN: 35.4%, P = 0.107; ENL: 35.9%, P = 0.003). Although the bioavailabilities of total isoflavones, prenylflavonoids, and lignans were not significantly affected upon coadministration, participants were exposed to lower phytoestrogen-derived 17beta-estradiol equivalents. In conclusion, the bioavailability of phytoestrogens, especially when given in mixtures, is subject to high interindividual variation. These findings support the importance of personalized screening when assessing the efficacy of such products and mixtures.

Hop (Humulus lupulus)-derived bitter acids as multipotent bioactive compounds.

Hop acids, a family of bitter compounds derived from the hop plant (Humulus lupulus), have been reported to exert a wide range of effects, both in vitro and in vivo. They exhibit potential anticancer activity by inhibiting cell proliferation and angiogenesis, by inducing apoptosis, and by increasing the expression of cytochrome P450 detoxification enzymes. Furthermore, hop bitter acids are effective against inflammatory and metabolic disorders, which makes them challenging candidates for the treatment of diabetes mellitus, cardiovascular diseases, and metabolic syndrome. This review summarizes the current knowledge on hop bitter acids, including both phytochemical aspects, as well as the biological and pharmacological properties of these compounds.

Evaluation of a low-cost adsorbent for removal of toxic metal ions from wastewater of an electroplating factory.

In this study, sugar cane residue or bagasse was used for removal of toxic metal ions from wastewater of an electroplating factory located in northeast Brazil. Prior acid treatment increased the adsorption efficacies in batch wise experiments. The microstructure of the material before and after the treatment was investigated by X-ray diffraction, infrared spectroscopy and scanning electron microscopy. Column operations showed that removals of Cu(2+), Ni(2+) and Zn(2+) from wastewater (in the absence of cyanide) were 95.5%, 96.3.0%, and 97.1%, respectively. Regeneration of the adsorbent obtained in acid indicated that the efficiencies decreased only after the fourth cycle of re-use. Acid-treated sugar cane bagasse can be considered a viable alternative to common methods to remove toxic metal ions from aqueous effluents of electroplating industries.

Single Nucleotide Polymorphisms and Biochemical Markers As Complementary Tools To Characterize Hops ( Humulus lupulus L.) in Brewing Practice.

In brewing practice, the use of the appropriate hop variety is essential to produce consistent and high-quality beers. Yet, hop batches of the same variety cultivated in different geographical regions can display significant biochemical differences, resulting in specific taste- and aroma-related characteristics in beer. In this study, we illustrate the complementarity of genetic and biochemical fingerprinting methods to fully characterize hop batches. Using genotyping-by-sequencing (GBS), a set of 1 830 polymorphic single nucleotide polymorphism (SNP) markers generated 48 unique genetic fingerprints for a collection of 56 commercial hop varieties. Three groups of varieties, consisting of somaclonal variants, could not be further differentiated using this set of markers. Biochemical marker information offered added value to characterize hop samples from a given variety grown at different geographical locations. We demonstrate the power of combining genetic and biochemical fingerprints for quality control of hop batches in the brewing industry.

Eubacterium limosum activates isoxanthohumol from hops (Humulus lupulus L.) into the potent phytoestrogen 8-prenylnaringenin in vitro and in rat intestine.

Recently, it was shown that the exposure to the potent hop phytoestrogen 8-prenylnaringenin (8-PN) depends on intestinal bacterial activation of isoxanthohumol (IX), but this occurs in only one-third of tested individuals. As the butyrate-producing Eubacterium limosum can produce 8-PN from IX, a probiotic strategy was applied to investigate whether 8-PN production could be increased in low 8-PN producers, thus balancing phytoestrogen exposure. Using fecal samples from high (Hop +) and low (Hop -) 8-PN-producing individuals, a Hop + and Hop - dynamic intestinal model was developed. In parallel, Hop + and Hop - human microbiota-associated rats were developed, germ-free (GF) rats acting as negative controls. IX and then IX + E. limosum were administered in the intestinal model and to the rats, and changes in 8-PN production and exposure were assessed. After dosing IX, 80% was converted into 8-PN in the Hop + model and highest 8-PN production, plasma concentrations, and urinary and fecal excretion occurred in the Hop + rats. Administration of the bacterium triggered 8-PN production in the GF rats and increased 8-PN production in the Hop - model and Hop - rats. 8-PN excretion was similar in the feces (294.1 +/- 132.2 nmol/d) and urine (8.5 +/- 1.1 nmol/d ) of all rats (n = 18). In addition, butyrate production increased in all rats. In conclusion, intestinal microbiota determined 8-PN production and exposure after IX intake. Moreover, E. limosum administration increased 8-PN production in low producers, resulting in similar 8-PN production in all rats.

Removal of some polycyclic aromatic hydrocarbons from petrochemical wastewater using low-cost adsorbents of natural origin.

Removal of polycyclic aromatic hydrocarbons (PAHs) from petrochemical wastewater was investigated using various low-cost adsorbents of natural origin including sugar cane bagasse, green coconut shells, chitin, and chitosan. Adsorption experiments of mixtures of PAHs (5.0-15.0 mg/L) have been carried out at ambient temperature (28+/-2 degrees C) and pH 7.5. The adsorption isotherms of PAHs were in agreement with a Freundlich model, while the uptake capacity of PAHs followed the order: green coconut shells>sugar cane bagasse>chitin>chitosan. The adsorption properties of green coconut shells were comparable to those of some conventional adsorbents such as Amberlite T. The partition coefficients in acetone:water, the adsorption constants at equilibrium, and the molecular masses of the PAHs could be linearly correlated with octanol-water partition coefficients.

Constitutive expression of a grapevine stilbene synthase gene in transgenic hop (Humulus lupulus L.) yields resveratrol and its derivatives in substantial quantities.

Resveratrol, a well-known phytoalexin and antioxidant, is produced by the action of stilbene synthase (STS) in some plant species. Hop (Humulus lupulus L.) plants of the Tettnang variety were transformed with a gene encoding for STS from grapevine. Under the control of the constitutive 35S cauliflower mosaic virus promoter, expression of the transgene resulted in accumulation of resveratrol and high levels of its glycosylated derivatives in leaves and inflorescences. Piceid, the predominant derivative, reached a concentration of up to 560 microg/g of fresh weight (f.w.) in hop cones, whereas no stilbenes were detected in nontransformed controls (wild-type). In transgenic plants the amounts of alpha- and beta-acids, naringenin chalcone, and prenylated flavonoids did not change significantly when compared with nontransformed plants. Transgenic plants showed normal morphology and flower development as did the nontransformed controls. The results clearly show that in hop constitutive expression of sts interferes neither with plant development nor with the biosynthesis of secondary metabolites relevant for the brewing industry. Since resveratrol is a well-known phytoalexin and antioxidant, sts transgenic hop plants could display enhanced pathogen resistance against microbial pathogens, exhibit new beneficial properties for health, and open new venues for metabolic engineering.

Subtle side-chain modifications of the hop phytoestrogen 8-prenylnaringenin result in distinct agonist/antagonist activity profiles for estrogen receptors alpha and beta.

In search of therapeutic agents for estrogen-related pathologies, phytoestrogens are being extensively explored. In contrast to naringenin, 8-prenylnaringenin is a potent hop-derived estrogenic compound, highlighting the importance of the prenyl group for hormonal activity. We investigated the effects of substituting the prenyl group at C(8) with alkyl chains of varying lengths and branching patterns on estrogen receptor (ER) subtype ERalpha- and ERbeta-binding affinities and transcriptional activities. In addition, features of the ligand-induced receptor conformations were explored using a set of specific ER-binding peptides. The new 8-alkylnaringenins were found to span an activity spectrum ranging from full agonism to partial agonism to antagonism. Most strikingly, 8-(2,2-dimethylpropyl)naringenin exhibited full agonist character on ERalpha, but pronounced antagonist character on ERbeta. Knowledge on how ER-subtype-selective activities can be designed provides valuable information for future drug or tool compound discovery.

Flavin-induced photodecomposition of sulfur-containing amino acids is decisive in the formation of beer lightstruck flavor.

Photooxidation of sulfur-containing amino acids and derivatives readily occurs upon visible-light irradiation in the presence of flavins. The sulfur moiety seems pivotal for interaction, as was determined from kinetic analyses using laser flash photolysis spectroscopy. After photooxidation, the resulting radical intermediates were characterized by addition to a spin trap, followed by electron paramagnetic resonance spectroscopy and evaluation of the coupling constants. The presence of the proposed radical intermediates was strongly supported by the identification of the reaction products using mass spectrometry. Accordingly, feasible degradation pathways for various sulfur-containing amino acids and derivatives were proposed. It was finally proven that flavin-induced photoproduction of sulfhydryl radicals and recombination with a 3-methylbut-2-enyl radical, derived from the photodegradation of hop-derived isohumulones, are decisive in the formation of beer lightstruck flavor.

A first prospective, randomized, double-blind, placebo-controlled study on the use of a standardized hop extract to alleviate menopausal discomforts.

OBJECTIVES: To examine the efficacy of a hop extract enriched in 8-prenylnaringenin (8-PN, the phytoestrogen in hops, Humulus lupulus L.) on relief of menopausal discomforts. METHODS: A prospective, randomized, double-blind, placebo-controlled study over 12 weeks with 67 menopausal women, who were administered a hop extract standardized on 8-PN (100 or 250 microg). The responses were determined by means of a modified Kupperman index (KI) and a patients' questionnaire. RESULTS: All groups, including placebo, showed a significant reduction of the KI both after 6 weeks and after 12 weeks. The hop extract at 100 microg 8-PN was significantly superior to placebo after 6 weeks (P=0.023) but not after 12 weeks (P=0.086). No dose-response relationship could be established, as the higher dose (250 microg) was less active than the lower dose both after 6 weeks and after 12 weeks. Still, a trend for a more rapid decrease of KI was noticed for both active groups as compared to placebo. In particular, the decrease in hot flush score (isolated from the KI) was found significant for both treatment groups after 6 weeks (P<0.01) with respect to placebo. Results of the patients' questionnaire were consistent with those of the KI, with the most pronounced effects being observed for the 100-microg treatment. CONCLUSIONS: Daily intake of a hop extract, standardized on 8-PN as a potent phytoestrogen, exerted favorable effects on vasomotor symptoms and other menopausal discomforts. Hop-derived prenylated flavonoids may provide an attractive addition to the alternative treatments available for relief of hot flushes and other menopausal discomforts.

Sequence analysis of a "true" chalcone synthase (chs_H1) oligofamily from hop (Humulus lupulus L.) and PAP1 activation of chs_H1 in heterologous systems.

Screening of a cDNA library of the hop cv. Osvald's 72 and genomic cloning were used to isolate members of an oligofamily of chs_H1 genes that codetermine the biosynthesis of prenylated chalcones known to be valuable medicinal compounds present in hop (Humulus lupulus L.). chs_H1 oligofamily members showed more than 99% and 98% identity on nucleotide and amino acid levels, respectively, and retained all conserved amino acids that form the catalytic center characteristic for "true" chalcone synthases. The chs_H1 promoter exhibited low sequence variability in addition to conservation of all predicted cis-regulatory elements. Possible transactivation of the chs_H1 gene with the transcription factor PAP1 from Arabidopsis thaliana was assayed using Agrobacterium tumefaciens infiltrations of Nicotiana benthamiana and Petunia hybrida plants. Infiltration of N. benthamiana leaves with chs_H1 promoter/GUS chimeras led to a 24.8-fold increase of the GUS activity when coinfiltrated with the pap1 gene. Coinfiltration of the "native" chs_H1 gene with pap1 led to an increased accumulation of chs_H1 mRNA as observed by semiquantitative reverse transcription-polymerase chain reaction. Transgenic lines of P. hybrida expressing the pap1 gene showed unusual patterns of UV-A-inducible pigmentation and anthocyanin accumulation in parenchymatic and medulla cells. Infiltration of transgenic leaves of P. hybrida with chs_H1 and pap1 genes arranged as a tandem led to quick pigmentation within 12 h after UV-A irradiation. It is indicated that the chs_H1 promoter contains functional element(s) mediating an efficient response to PAP1 expression and UV-A irradiation. UV-A also induced chs_H1 mRNA and accumulation of flavonol glycosides in hop leaves. It can be expected that the PAP1 factor could significantly influence the expression of the chs_H1 oligofamily in transgenic hop and modify the hop metabolome.

Metabolism of the food-associated carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine by human intestinal microbiota.

2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine is a putative human carcinogenic heterocyclic aromatic amine formed from meat and fish during cooking. Although the formation of hazardous PhIP metabolites by mammalian enzymes is well-documented, nothing is known about the PhIP transformation potency of human intestinal bacteria. In this study, the in vitro metabolism of PhIP by human fecal samples was investigated. Following anaerobic incubation of PhIP with stools freshly collected from six healthy volunteers, we found that PhIP was extensively transformed by the human intestinal bacteria. HPLC analysis showed that the six human fecal microbiota transformed PhIP with efficiencies from 47 to 95% after 72 h incubation, resulting in one major derivative. ESI-MS/MS, HRMS, 1D (1H, 13C, DEPT) and 2D (gCOSY, gTOCSY, gHMBC, gHSQC) NMR, and IC analysis elucidated the complete chemical identity of the microbial PhIP metabolite as 7-hydroxy-5-methyl-3-phenyl-6,7,8,9-tetrahydropyrido[3',2':4,5]imidazo[1,2-a]pyrimidin-5-ium chloride. At present, no information is available about the biological activity of this newly discovered bacterial PhIP metabolite. Our findings however suggest that bacteria derived from the human intestine play a key role in the activation or detoxification of PhIP, a digestive fate ignored so far in risk assessments. Moreover, the variation in transformation efficiency between the human microbiota indicates interindividual differences in the ability to convert PhIP. This may predict individual susceptibility to carcinogenic risk from this suspected dietary carcinogen.

Regioselective synthesis and estrogenicity of (+/-)-8-alkyl-5,7-dihydroxy-4-(4-hydroxyphenyl)-3,4-dihydrocoumarins.

Nine new (+/-)-8-alkyl-5,7-dihydroxy-4-(4-hydroxyphenyl)-3,4-dihydrocoumarins have been synthesized from 2,4,6-trimethoxybenzaldehyde via a short, efficient, and regioselective pathway, together with the unsubstituted analogue (+/-)-5,7-dihydroxy-4-(4-hydroxyphenyl)-3,4-dihydrocoumarin. The compounds were tested for estrogenic activity using a yeast-based estrogen screen. Weak estrogenicity was determined for seven members of the series.

Activation of proestrogens from hops (Humulus lupulus L.) by intestinal microbiota; conversion of isoxanthohumol into 8-prenylnaringenin.

Hop, an essential ingredient in most beers, contains a number of prenylflavonoids, among which 8-prenylnaringenin (8-PN) would be the most potent phytoestrogen currently known. Although a number of health effects are attributed to these compounds, only a few reports are available about the bioavailability of prenylflavonoids and the transformation potency of the intestinal microbial community. To test these transformations, four fecal samples were incubated with xanthohumol, isoxanthohumol (IX), and 8-PN. Upon incubation with IX, present in strong ales up to 4 mg/L, 36% was converted into 8-PN in one fecal sample and the estrogenic properties of the sample drastically increased. In an experiment with 12 fecal cultures, this conversion was observed in one-third of the samples, indicating the importance of interindividual variability in the intestinal microbial community. Eubacterium limosum was identified to be capable of this conversion (O-demethylation) of IX into 8-PN, and after strain selection, a conversion efficiency of 90% was achieved. Finally, strain supplementation to a nonconverting fecal sample led to rapid and high 8-PN production at only 1% (v/v) addition. Up to now, the concentration of 8-PN in beer was considered too low to affect human health. However, these results show that the activity of the intestinal microbial community could more than 10-fold increase the exposure concentration. Because prenylflavonoids are present in many beers with IX being the major constituent, the results raise the question whether moderate beer consumption might contribute to increased in vivo levels of 8-PN and even influence human health.

Photooxidative degradation of beer bittering principles: a key step on the route to lightstruck flavor formation in beer.

Isohumulones, dihydroisohumulones, tetrahydroisohumulones, and humulinones, important hop-derived bittering compounds in beer, were shown to give rise to reactive triacylmethyl radicals on interaction with triplet-excited riboflavin after spin trapping by 5,5-dimethyl-1-pyrroline N-oxide or 2-methyl-2-nitrosopropane, followed by electron paramagentic resonance spectroscopy combined with spectral simulation. Electron abstraction from the ionized beta-tricarbonyl chromophore, which is common to all five-membered ring hop derivatives, is the initial event on photoinduced degradation. Radicaloid decomposition of isohumulones leads to precursors for 3-methylbut-2-ene-1-thiol, the lightstruck constituent in beer. Interaction of reduced derivatives of isohumulones with triplet-excited riboflavin furnished radical precursors of volatile aldehydes, which may lead to the development of unpleasant stale or cardboard flavors.

Cloning and molecular analysis of the regulatory factor HlMyb1 in hop (Humulus lupulus L.) and the potential of hop to produce bioactive prenylated flavonoids.

The concentrations of prenylated chalcones and bitter acids were analyzed in Czech hop varieties. The highest levels of (xanthohumol + desmethylxanthohumol) (0.97%, m/m) and of total bitter acids (17.19%, m/m) were observed for cv. Agnus. The concentration ratios of bitter acids to prenylated chalcones varied depending on the genotype, thereby suggesting genetic determination by different set(s) of structural and regulatory genes. Promoter elements of the chs_H1 gene encoding a "true"chalcone synthase, a candidate gene to co-determine the biosynthesis of prenylated chalcones, were analyzed, and several boxes for cis-regulatory elements including Myb transcription factors were discovered. A cDNA library was established from glandular tissue-enriched cones of cv. Osvald's clone 72 and used to screen for Myb regulatory elements. The cDNA of the first Myb regulatory factor from hop, called HlMyb1, was cloned and analyzed. The HlMyb1 open reading frame encodes 272 amino acids (29.8 kDa), and the protein showed highest homology to the light-regulated factor AtMyb68 from Arabidopsis thaliana within the Myb domain, whereas there was no significant homology with known MYB proteins outside this domain. Unlike AtMyb68, which is expressed in mature leaves, HlMyb1 is strongly expressed in hop inflorescences and could participate in the regulation of developmental processes involved in the production of hop cones and bioactive secondary metabolites.

A safety study of oral tangeretin and xanthohumol administration to laboratory mice.

BACKGROUND: The detection of molecular targets for flavonoids in cell signalling has opened new perspectives for their application in medicine. Both tangeretin, a citrus methoxyflavone, and xanthohumol, the main prenylated chalcone present in hops (Humulus lupulus L.), act on the mitogen-activated protein kinase pathway and await further investigation for administration in vivo. MATERIALS AND METHODS: A safety study was designed in laboratory mice orally administered concentrates of purified tangeretin (1 x 10(-4) M) or xanthohumol (5 x 10(-4) M) at libitum for 4 weeks. Blood samples were collected for the analysis of a variety of haematological and biochemical parameters. RESULTS: A reduction of the circulating lymphocyte number was noticed for tangeretin, while all other parameters were unaffected by treatment with either tangeretin or xanthohumol. The parameters encompassed an integrity check of the following tissues and organs: bone marrow, liver, exocrine pancreas, kidneys, muscles, thyroid, ovaries and surrenal cortex. Furthermore, no differences were noted in the metabolism of proteins, lipids, carbohydrates and uric acid, as well as in ion concentrations. CONCLUSION: All data indicate that oral administration of tangeretin or xanthohumol to laboratory mice does not affect major organ functions and opens the gate for further safety studies in humans.

Estrogenic activity of a polyphenolic extract of the leaves of Epimedium brevicornum.

The estrogenic activity of a polyphenolic extract of the leaves of Epimedium brevicornum and five fractions obtained by solid-phase extraction were investigated using estrogen-responsive bioassays, a yeast cell assay and the Ishikawa Var-I assay. The extract was found to exhibit significant estrogenic activity in both assays. Furthermore, bioassay-guided fractionation led to localisation of the estrogenicity in the relatively non-polar fractions of the polyphenolic extract.

3'-C-branched-chain-substituted nucleosides and nucleotides as potent inhibitors of Mycobacterium tuberculosis thymidine monophosphate kinase.

Thymidine monophosphate kinase (TMPK) of Mycobacterium tuberculosis (TMPKmt) represents an attractive target for blocking the bacterial DNA synthesis. In an attempt to find high-affinity inhibitors of TMPKmt, a cavity in the enzyme at the 3'-position was explored via the introduction of various substituents at the 3'-position of the thymidine monophosphate (dTMP) scaffold. Various 3'-C-branched chain substituted nucleotides in the 2'-deoxyribo (3-6) and ribo series (7, 8) were synthesized from one key intermediate (23). 2'-Deoxy analogues proved to be potent inhibitors of TMPKmt: 3'-CH(2)NH(2) (4), 3'-CH(2)N(3) (3), and 3'-CH(2)F (5) nucleotides exhibit the highest affinities within this series, with K(i) values of 10.5, 12, and 15 microM, respectively. These results show that TMPKmt tolerates the introduction of sterically demanding substituents at the 3'-position. Ribo analogues experience a significant affinity decrease, which is probably due to steric hindrance of Tyr103 in close vicinity of the 2'-position. Although the 5'-O-phosphorylated compounds have somewhat higher affinities for the enzyme, the parent nucleosides generally exhibit affinities for TMPKmt in the same order of magnitude and display a superior selectivity profile versus human TMPK. This series of inhibitors holds promise for the development of a new class of antituberculosis agents.

Distribution of soy-derived phytoestrogens in human breast tissue and biological fluids.

OBJECTIVE: Soy-derived phytoestrogens may exert several health-beneficial effects. Although plasma and urine levels of these compounds after ingestion have been thoroughly investigated, little is known about their tissue distribution, which is particularly important for tissues with high endogenous estrogen and estrogen receptor concentrations. We aimed to investigate the concentrations of genistein, daidzein, and equol in human breast tissue homogenate and to compare these with the corresponding values in serum and urine. METHODS: A randomized, double-blind, placebo-controlled study was undertaken to evaluate the concentrations of soy-derived phytoestrogens achieved in breast tissue homogenate, serum, and urine after ingestion of either a soy-based food supplement (n = 9) or a placebo tablet (n = 19) for 5 consecutive evenings before aesthetic breast surgery. To account for the heterogeneity of the breast tissue samples, markers for cellularity, epithelial content, blood vessel content, and total fat were determined. RESULTS: Urine concentrations of genistein, daidzein, and equol were significantly higher in the soy-supplemented subjects than in the subjects ingesting the placebo (P <.05). Only genistein was found to be significantly higher in serum of the soy group than in the placebo group, and no significant differences were found in breast tissue homogenate concentrations of all analytes between the 2 groups. CONCLUSION: Intake of soy-based food supplements for 5 consecutive days did not result in significantly higher genistein, daidzein, and equol concentrations in breast tissue homogenate when compared with the placebo group. The concentrations were in the low nanomolar range, whereas in the corresponding serum samples, concentrations were a hundred-fold higher.