Biomedical subjects

Annett Braune

Publications and source records attributed to Annett Braune.

10 recordsLinked to original sources

PubMed2006

Mutagenicity of arbutin in mammalian cells after activation by human intestinal bacteria.

Arbutin (hydroquinone-beta-D-glucopyranoside) is present in various food plants. Its aglycone, hydroquinone, is mutagenic and carcinogenic. We investigated whether hydroquinone may be released under conditions encountered in the human gastrointestinal tract. Arbutin was stable in artificial gastric juice. Fecal slurries from nine human subjects completely converted arbutin (2 mM) into hydroquinone. Four of nine representative human intestinal species investigated, namely Eubacterium ramulus, Enterococcus casseliflavus, Bacteroides distasonis, and Bifidobacterium adolescentis, deglycosylated arbutin at rates of 21.08, 16.62, 8.43 and 3.59 nmol x min(-1) x (mg protein)(-1), respectively. In contrast, homogenates from small intestinal mucosa and cytosolic fractions from colon mucosa deglycosylated arbutin at substantially lower rates: 0.50 and 0.09 nmol x min(-1) x (mg protein)(-1), respectively. Arbutin, unlike hydroquinone, did not induce gene mutations in Chinese hamster V79 cells in the absence of an activating system. However, in the presence of cytosolic fractions from E. ramulus or B. distasonis, arbutin was strongly mutagenic. Cytosolic fraction from Escherichia coli, showing no arbutin glycosidase activity, was not able to activate arbutin in this model system. The release of the proximate mutagen hydroquinone from arbutin by intestinal bacteria in the immediate vicinity of the colon mucosa may pose a potential risk.

Adult↗

PubMed2005

Occurrence and activity of human intestinal bacteria involved in the conversion of dietary lignans.

The human intestinal microbiota is necessary for the production of enterolignans from the dietary lignan secoisolariciresinol diglucoside (SDG). However, little is known about the bacteria that contribute to SDG conversion. Therefore, we aimed at describing the occurrence and activity of SDG metabolising bacteria. The data showed differences in conversion efficiency between SDG deglycosylating species, but SDG was completely deglycosylated within 20 h by five of six strains. The strain Clostridium sp. SDG-Mt85-3Db showed the highest initial rate of SDG deglycosylation. Furthermore, we found that Bacteroides distasonis and B. fragilis made up 0.5% and 3.3% of total faecal bacteria, respectively. However, Clostridium sp. SDG-Mt85-3Db was detected within the dominant microbiota of only two out of 20 faecal samples. Bacteria involved in the demethylation step of SDG conversion also demethylated a variety of compounds other than SDG. In particular, Peptostreptococcus productus demethylated the lignans pinoresinol, lariciresinol and matairesinol. Finally, Eggerthella lenta catalysed the reduction of pinoresinol and lariciresinol to secoisolariciresinol.

Bacteroides↗

PubMed2005

Degradation of neohesperidin dihydrochalcone by human intestinal bacteria.

The degradation of neohesperidin dihydrochalcone by human intestinal microbiota was studied in vitro. Human fecal slurries converted neohesperidin dihydrochalcone anoxically to 3-(3-hydroxy-4-methoxyphenyl)propionic acid or 3-(3,4-dihydroxyphenyl)propionic acid. Two transient intermediates were identified as hesperetin dihydrochalcone 4'-beta-d-glucoside and hesperetin dihydrochalcone. These metabolites suggest that neohesperidin dihydrochalcone is first deglycosylated to hesperetin dihydrochalcone 4'-beta-d-glucoside and subsequently to the aglycon hesperetin dihydrochalcone. The latter is hydrolyzed to the corresponding 3-(3-hydroxy-4-methoxyphenyl)propionic acid and probably phloroglucinol. Eubacterium ramulus and Clostridium orbiscindens were not capable of converting neohesperidin dihydrochalcone. However, hesperetin dihydrochalcone 4'-beta-d-glucoside was converted by E. ramulus to hesperetin dihydrochalcone and further to 3-(3-hydroxy-4-methoxyphenyl)propionic acid, but not by C. orbiscindens. In contrast, hesperetin dihydrochalcone was cleaved to 3-(3-hydroxy-4-methoxyphenyl)propionic acid by both species. The latter reaction was shown to be catalyzed by the phloretin hydrolase from E. ramulus.

Bacteria↗

PubMed2005

Xanthohumol does not affect the composition of rat intestinal microbiota.

Xanthohumol (XN), a prenylated chalcone, has been proposed to have beneficial effects on human health, including antimicrobial activity. To clarify whether the exposure to XN has an impact on the composition of the intestinal microbiota, 100 mg XN/kg body weight was given daily to rats for 4 wk. Diversity of the fecal microbial community was analyzed using PCR-DGGE. Although intact XN was detected in the feces of the rats at a concentration of up to 2.3 mg/g fecal dry weight, major shifts in the PCR-DGGE patterns in response to this flavonoid were not observed. The similarity index decreased slightly from 70 to 62% for the XN-treated rats and from 71 to 63% for the untreated animals. Thus, changes in the rat fecal microbiota observed in the course of the XN application are most likely due to intraindividual variability. However, the water content of the feces increased significantly during the XN treatment period.

Animals↗

PubMed2004

First bacterial chalcone isomerase isolated from Eubacterium ramulus.

The human fecal anaerobe Eubacterium ramulus is capable of degrading various flavonoids, including the flavone naringenin. The first step in the proposed degradation pathway is the isomerization of naringenin to the corresponding chalcone. Cell-free extracts of E. ramulus displayed chalcone isomerase activity. The enzyme from E. ramulus was purified to homogeneity. Its apparent molecular mass was estimated to be 136 and 129 kDa according to gel filtration and native polyacrylamide gel electrophoresis, respectively. Chalcone isomerase is composed of one type of subunit of 30 kDa. The purified enzyme catalyzed the isomerization of naringenin chalcone, isoliquiritigenin, and butein, three chalcones that differ in their hydroxylation pattern. N-bromosuccinimide, but also naringenin and phloretin, inhibited the purified enzyme considerably. This is the first report on a bacterial chalcone isomerase. The physiological function of the purified enzyme is unclear, but an involvement in the conversion of the flavanone naringenin to the chalcone is proposed.

Bromosuccinimide↗

PubMed2004

Cloning and expression of a phloretin hydrolase gene from Eubacterium ramulus and characterization of the recombinant enzyme.

Phloretin hydrolase catalyzes the hydrolytic C-C cleavage of phloretin to phloroglucinol and 3-(4-hydroxyphenyl)propionic acid during flavonoid degradation in Eubacterium ramulus. The gene encoding the enzyme was cloned by screening a gene library for hydrolase activity. The insert of a clone conferring phloretin hydrolase activity was sequenced. Sequence analysis revealed an open reading frame of 822 bp (phy), a putative promoter region, and a terminating stem-loop structure. The deduced amino acid sequence of phy showed similarities to a putative protein of the 2,4-diacetylphloroglucinol biosynthetic operon from Pseudomonas fluorescens. The phloretin hydrolase was heterologously expressed in Escherichia coli and purified. The molecular mass of the native enzyme was approximately 55 kDa as determined by gel filtration. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of phy indicated molecular masses of 30 and 30.8 kDa, respectively, suggesting that the enzyme is a homodimer. The recombinant phloretin hydrolase catalyzed the hydrolysis of phloretin to equimolar amounts of phloroglucinol and 3-(4-hydroxyphenyl)propionic acid. The optimal temperature and pH of the catalyzed reaction mixture were 37 degrees C and 7.0, respectively. The K(m) for phloretin was 13 +/- 3 microM and the k(cat) was 10 +/- 2 s(-1). The enzyme did not transform phloretin-2'-glucoside (phloridzin), neohesperidin dihydrochalcone, 1,3-diphenyl-1,3-propandione, or trans-1,3-diphenyl-2,3-epoxy-propan-1-one. The catalytic activity of the phloretin hydrolase was reduced by N-bromosuccinimide, o-phenanthroline, N-ethylmaleimide, and CuCl(2) to 3, 20, 35, and 85%, respectively. Phloroglucinol and 3-(4-hydroxyphenyl)propionic acid reduced the activity to 54 and 70%, respectively.

Amino Acid Sequence↗

PubMed2003

Transformation of flavonoids by intestinal microorganisms.

Fruit, vegetables and cereals contain a wealth of secondary plan metabolites which have been implicated in the promotion of health. To understand the mechanism of their action it is necessary to gain more information on their fate in the body following ingestion. A certain proportion of ingested secondary plant constituents may escape absorption in the small intestine and therefore undergo transformation by intestinal microorganisms or enterohepatic circulation. To study the transformation of secondary plant metabolites by bacteria, Eubacterium ramulus was isolated from human feces and incubated with selected flavonoids. E. ramulus is a strictly anaerobic bacterium which was found to be present in the gastrointestinal tract of most individuals investigated. E. ramulus cleaves the ring system of several flavonols and flavones giving rise to the corresponding hydroxyphenylacetic and hydroxyphenylpropionic acids, respectively, as well as acetate and butyrate. Degradation pathways were proposed based on the intermediates detected by high performance liquid chromatography (HPLC) and HPLC coupled with mass spectrometry (LC-MS) and the detection of enzymes that catalyze reactions such as taxifolin isomerization, phloretin hydrolysis and phloroglucinol reduction. The dearomatizing phloroglucinol reductase, presumably part of all flavonoid degradation pathways, was purified and characterized. The gene encoding phloretin hydrolase was cloned from a E. ramulus gene library taking advantage of a newly developed fluorescence test for activity screening. Moreover, a new intermediate was discovered and identified by MS and 1H and 13C NMR analysis as alphitonin. To investigate the degradational potential of E. ramulus under in vivo conditions, germfree rats were associated with E. ramulus. Following the intragastric application of quercetin-3-glucoside, urine and feces of gnotobiotic rats were analyzed for degradational products originating from quercetin-3-glucoside. In feces of rats monoassociated with E. ramulus, 3,4-dihydroxyphenylacetic acid was found, indicating that this organism is able to cleave quercetin under in vivo conditions. To investigate in which way the dietary flavonoid content affects the cell counts of E. ramulus in the human intestinal tract, twelve human subjects consumed a flavonoid-free diet for one week and at one point during this period a large dose of flavonoids. Fecal samples from both phases of the study were analyzed by in-situ hybridization for total bacterial counts and counts of E. ramulus. Total cell counts and the cell counts of E. ramulus decreased significantly during the flavonoid-free period, while there was an increase in the E. ramulus counts of up to 10-fold during the flavonoid-rich period indicating that dietary secondary plant metabolites may have an influence on the intestinal microflora. E. ramulus is also capable of converting the isoflavonoids genistein and daidzein to the products 2-(4-hydroxyphenyl)-propionic acid and O-desmethylangolensin, respectively.

Animals↗

PubMed2003

Anaerobic degradation of flavonoids by Clostridium orbiscindens.

An anaerobic, quercetin-degrading bacterium was isolated from human feces and identified as Clostridium orbiscindens by comparative 16S rRNA gene sequence analysis. The organism was tested for its ability to transform several flavonoids. The isolated C. orbiscindens strain converted quercetin and taxifolin to 3,4-dihydroxyphenylacetic acid; luteolin and eriodictyol to 3-(3,4-dihydroxyphenyl)propionic acid; and apigenin, naringenin, and phloretin to 3-(4-hydroxyphenyl)propionic acid, respectively. Genistein and daidzein were not utilized. The glycosidic bonds of luteolin-3-glucoside, luteolin-5-glucoside, naringenin-7-neohesperidoside (naringin), quercetin-3-glucoside, quercetin-3-rutinoside (rutin), and phloretin-2'-glucoside were not cleaved. Based on the intermediates and products detected, pathways for the degradation of the flavonol quercetin and the flavones apigenin and luteolin are proposed. To investigate the numerical importance of C. orbiscindens in the human intestinal tract, a species-specific oligonucleotide probe was designed and tested for its specificity. Application of the probe to fecal samples from 10 human subjects proved the presence of C. orbiscindens in 8 out of the 10 samples tested. The numbers ranged from 1.87 x 10(8) to 2.50 x 10(9) cells g of fecal dry mass(-1), corresponding to a mean count of 4.40 x 10(8) cells g of dry feces(-1).

Adult↗

PubMed2002

Purification and characterization of an NADH oxidase from Eubacterium ramulus.

An NADH oxidase from the strictly anaerobic Eubacterium ramuluswas purified to homogeneity. The enzyme is composed of two types of subunits with molecular masses of 40 and 30 kDa. The molecular mass of the native enzyme is 450 kDa according to gel filtration and PAGE analysis. Six to eight mol of FAD were found per mol of native enzyme. The NADH-specific enzyme was inhibited by N-bromosuccinimide and sulfhydryl reagents such as N-ethylmaleimide, CuCl(2) or ZnCl(2). The physiological function of the purified enzyme is unclear, but the demonstration of NADH-dependent O(2)-consumption suggests that it plays a role in the scavenging of oxygen.

Amino Acids↗

PubMed2002

Anaerobic C-ring cleavage of genistein and daidzein by Eubacterium ramulus.

Eubacterium ramulus, a flavonoid-degrading anaerobic bacterium from the human gastrointestinal tract, was tested for its ability to transform the isoflavonoids genistein-7-O-glucoside (genistin), genistein and daidzein. Genistein was completely degraded by E. ramulus via 6'-hydroxy-O-desmethylangolensin to 2-(4-hydroxyphenyl)-propionic acid. Dihydrogenistein was neither observed as an intermediate in this transformation nor converted itself by growing cells or cell-free extracts of E. ramulus. Genistein-7-O-glucoside was partially transformed by way of genistein to the product 2-(4-hydroxyphenyl)-propionic acid. Daidzein was in part degraded to O-desmethylangolensin, the corresponding metabolite to 6'-hydroxy-O-desmethylangolensin. The hydroxyl group in position 6' of O-desmethylangolensin is crucial for further degradation.

Biotransformation↗