Annett Braune

Anaerobic Degradation of Flavonoids by Clostridium orbiscindens

ABSTRACT 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 × 108 to 2.50 × 109 cells g of fecal dry mass−1, corresponding to a mean count of 4.40 × 108 cells g of dry feces−1.

Degradation of Quercetin and Luteolin by Eubacterium ramulus

ABSTRACT The degradation of the flavonol quercetin and the flavone luteolin by Eubacterium ramulus, a strict anaerobe of the human intestinal tract, was studied. Resting cells converted these flavonoids to 3,4-dihydroxyphenylacetic acid and 3-(3,4-dihydroxyphenyl)propionic acid, respectively. The conversion of quercetin was accompanied by the transient formation of two intermediates, one of which was identified as taxifolin based on its specific retention time and UV and mass spectra. The structure of the second intermediate, alphitonin, was additionally elucidated by1H and 13C nuclear magnetic resonance analysis. In resting-cell experiments, taxifolin in turn was converted via alphitonin to 3,4-dihydroxyphenylacetic acid. Alphitonin, which was prepared by enzymatic conversion of taxifolin and subsequent purification, was also transformed to 3,4-dihydroxyphenylacetic acid. The coenzyme-independent isomerization of taxifolin to alphitonin was catalyzed by cell extract or a partially purified enzyme preparation ofE. ramulus. The degradation of luteolin by resting cells of E. ramulus resulted in the formation of the intermediate eriodictyol, which was identified by high-performance liquid chromatography and mass spectrometry analysis. The observed intermediates of quercetin and luteolin conversion suggest that the degradation pathways in E. ramulus start with an analogous reduction step followed by different enzymatic reactions depending on the additional 3-hydroxyl group present in the flavonol structure.

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.

Isolation of a Human Intestinal Bacterium Capable of Daidzein and Genistein Conversion

ABSTRACT A rod-shaped gram-positive anaerobic bacterium, strain HE8, was isolated from human feces. The isolate was able to convert the isoflavones daidzein and genistein to equol and 5-hydroxy-equol, respectively. Based on phenotypic and phylogenetic analyses, strain HE8 is described as a new species, Slackia isoflavoniconvertens.

Isolation of catechin‐converting human intestinal bacteria

Aims:  To isolate and characterize bacteria from the human intestine that are involved in the conversion of catechins, a class of bioactive polyphenols abundant in the human diet.

Daidzein and Genistein Are Converted to Equol and 5-Hydroxy-Equol by Human Intestinal Slackia isoflavoniconvertens in Gnotobiotic Rats

Intestinal conversion of the isoflavone daidzein to the bioactive equol is exclusively catalyzed by gut bacteria, but a direct role in equol formation under in vivo conditions has not yet been demonstrated. Slackia isoflavoniconvertens is one of the few equol-forming gut bacteria isolated from humans and, moreover, it also converts genistein to 5-hydroxy-equol. To demonstrate the isoflavone-converting ability of S. isoflavoniconvertens in vivo, the metabolization of dietary daidzein and genistein was investigated in male and female rats harboring a simplified human microbiota without (control) or with S. isoflavoniconvertens (SIA). Feces, urine, intestinal contents, and plasma of the rats were analyzed for daidzein, genistein, and their metabolites. Equol and 5-hydroxy-equol were found in intestinal contents, feces, and urine of SIA rats but not in the corresponding samples of the control rats. 5-Hydroxy-equol was present at much lower concentrations than equol and the main metabolite produced from genistein was the intermediate dihydrogenistein. The plasma of SIA rats contained equol but no 5-hydroxy-equol. Equol formation had no effect on plasma concentrations of the insulin-like growth factor I. The concentrations of daidzein and genistein were considerably lower in all samples of the SIA rats than in those of the control rats. Male SIA rats had higher intestinal and fecal concentrations of the isoflavones and their metabolites than female SIA rats. The observed activity in the rat model indicates that S. isoflavoniconvertens is capable of contributing in vivo to the bioactivation of daidzein and genistein by formation of equol and 5-hydroxy-equol.

Conversion of Daidzein and Genistein by an Anaerobic Bacterium Newly Isolated from the Mouse Intestine

ABSTRACT The metabolism of isoflavones by gut bacteria plays a key role in the availability and bioactivation of these compounds in the intestine. Daidzein and genistein are the most common dietary soy isoflavones. While daidzein conversion yielding equol has been known for some time, the corresponding formation of 5-hydroxy-equol from genistein has not been reported previously. We isolated a strictly anaerobic bacterium (Mt1B8) from the mouse intestine which converted daidzein via dihydrodaidzein to equol as well as genistein via dihydrogenistein to 5-hydroxy-equol. Strain Mt1B8 was a gram-positive, rod-shaped bacterium identified as a member of the Coriobacteriaceae. Strain Mt1B8 also transformed dihydrodaidzein and dihydrogenistein to equol and 5-hydroxy-equol, respectively. The conversion of daidzein, genistein, dihydrodaidzein, and dihydrogenistein in the stationary growth phase depended on preincubation with the corresponding isoflavonoid, indicating enzyme induction. Moreover, dihydrogenistein was transformed even more rapidly in the stationary phase when strain Mt1B8 was grown on either genistein or daidzein. Growing the cells on daidzein also enabled conversion of genistein. This suggests that the same enzymes are involved in the conversion of the two isoflavones.

The Bioavailability of Apigenin-7-Glucoside Is Influenced by Human Intestinal Microbiota in Rats

We investigated the impact of human intestinal microbiota on bioavailability of the flavone apigenin-7-glucoside (A7G) by comparing germ-free and human microbiota-associated (HMA) rats. First, the ability of the human intestinal microbiota to convert A7G was proven in vitro by incubating A7G with fecal suspensions. Apigenin, naringenin, and 3-(4-hydroxyphenyl)propionic acid were formed as main metabolites. After application of A7G to germ-free rats, apigenin, luteolin, and their conjugates were detected in urine and feces. In HMA rats, naringenin, eriodictyol, phloretin, 3-(3,4-dihydroxyphenyl)propionic acid, 3-(4-hydroxyphenyl)propionic acid, 3-(3-hydroxyphenyl)propionic acid, and 4-hydroxycinnamic acid in their free and conjugated forms were additionally formed. In whole-blood samples from germ-free and HMA rats, only apigenin conjugates and phloretin, respectively, were detected. The total excretion of A7G and its metabolites within 48 h was similarly low in both germ-free and HMA rats, with 11 and 13% of the A7G dose, respectively. In germ-free rats, A7G metabolites dominated by apigenin and its conjugates were mainly excreted with feces. In contrast, the compounds in HMA rats were predominantly recovered from urine, 3-(4-hydroxyphenyl)propionic acid being the main metabolite. The ability of selected gut bacteria and the host intestinal mucosa to deglycosylate A7G was tested using cell extracts. Apigenin was formed by cytosolic extracts of Eubacterium ramulus and Bacteroides distasonis and by the microsomal fraction of the small intestinal mucosa of rats. Overall, human intestinal microbiota largely contributed to A7G metabolism, indicating its influence on the bioactivity of flavones.

Bacterial species involved in the conversion of dietary flavonoids in the human gut.

ABSTRACT The gut microbiota plays a crucial role in the conversion of dietary flavonoids and thereby affects their health-promoting effects in the human host. The identification of the bacteria involved in intestinal flavonoid conversion has gained increasing interest. This review summarizes available information on the so far identified human intestinal flavonoid-converting bacterial species and strains as well as their enzymes catalyzing the underlying reactions. The majority of described species involved in flavonoid transformation are capable of carrying out the O-deglycosylation of flavonoids. Other bacteria cleave the less common flavonoid-C-glucosides and/or further degrade the aglycones of flavonols, flavanonols, flavones, flavanones, dihydrochalcones, isoflavones and monomeric flavan-3-ols. To increase the currently limited knowledge in this field, identification of flavonoid-converting bacteria should be continued using culture-dependent screening or isolation procedures and molecular approaches based on sequence information of the involved enzymes.

Conversion of Dehydrodiferulic Acids by Human Intestinal Microbiota

Plant cell wall associated dehydrodiferulic acids (DFA) are abundant components of cereal insoluble dietary fibers ingested by humans. The ability of human intestinal microbiota to convert DFA was studied in vitro by incubating 8-O-4- and 5-5-coupled DFA with fecal suspensions. 8-O-4-DFA was completely degraded by the intestinal microbiota of the majority of donors, yielding homovanillic acid, 3-(3,4-dihydroxyphenyl)propionic acid, and 3,4-dihydroxyphenylacetic acid as the main metabolites. The transient formation of ferulic acid and presumably 3-(3-hydroxy-4-methoxyphenyl)pyruvic acid suggests an initial cleavage of the ether bond. In contrast to 8-O-4-DFA, the 5-5-coupled DFA was not cleaved into monomers by any of the fecal suspensions. Only the side chains were hydrogenated and the methoxy groups were demethylated. The cleavage of DFA by human intestinal microbiota, which depended on their coupling type, may affect both the bioavailability of DFA and the degradability of DFA-coupled fiber in the gut.