Curcumin and other curcuminoids from Curcuma longa are important bioactive compounds exhibiting various pharmacological activities. In addition to the known reductive metabolism of curcuminoids, an alternative biotransformation of curcuminoids by human gut microbiota is reported herein. A curcuminoid mixture, composed of curcumin (1), demethoxycurcumin (2), and bisdemethoxycurcumin (3), was metabolized by the human intestinal bacterium Blautia sp. MRG-PMF1. 1 and 2 were converted to new metabolites by the methyl aryl ether cleavage reaction. Two metabolites, demethylcurcumin (4) and bisdemethylcurcumin (5), were sequentially produced from 1, and demethyldemethoxycurcumin (6) was produced from 2. Until now, sequential reduction of the heptadienone backbone of curcuminoids was the only known metabolism to occur in the human intestine. In this study, a new intestinal metabolism of curcuminoids was discovered. Demethylation of curcuminoids produced three new colonic metabolites that were already known as promising synthetic curcumin analogues. The results could explain the observed beneficial effects of turmeric.

Stereochemical course of isoflavanone dihydrodaidzein (DHD) reduction into the isoflavan (3S)-equol via tetrahydrodaidzein (THD) by the human intestinal anaerobic bacterium

Poylmethoxyflavones (PMFs) are major bioactive flavonoids, which exhibit various biological activities, such as anticancer effects. The biotransformation of PMFs and characterization of a PMF-metabolizing human intestinal bacterium were studied herein for the first time. Hydrolysis of aryl methyl ether functional groups by human fecal samples was observed from the bioconversion of various PMFs. Activity-guided screening for PMF-metabolizing intestinal bacteria under anaerobic conditions resulted in the isolation of a strict anaerobic bacterium, which was identified as Blautia sp. MRG-PMF1. The isolated MRG-PMF1 was able to metabolize various PMFs to the corresponding demethylated flavones. The microbial conversion of bioactive 5,7-dimethoxyflavone (5,7-DMF) and 5,7,4'-trimethoxyflavone (5,7,4'-TMF) was studied in detail. 5,7-DMF and 5,7,4'-TMF were completely metabolized to 5,7-dihydroxyflavone (chrysin) and 5,7,4'-trihydroxyflavone (apigenin), respectively. From a kinetics study, the methoxy group on the flavone C-7 position was found to be preferentially hydrolyzed. 5-Methoxychrysin, the intermediate of 5,7-DMF metabolism by Blautia sp. MRG-PMF1, was isolated and characterized by nuclear magnetic resonance spectroscopy. Apigenin was produced from the sequential demethylation of 5,7,4'-TMF, via 5,4'-dimethoxy-7-hydroxyflavone and 7,4'-dihydroxy-5-methoxyflavone (thevetiaflavone). Not only demethylation activity but also deglycosylation activity was exhibited by Blautia sp. MRG-PMF1, and various flavonoids, including isoflavones, flavones, and flavanones, were found to be metabolized to the corresponding aglycones. The unprecedented PMF demethylation activity of Blautia sp. MRG-PMF1 will expand our understanding of flavonoid metabolism in the human intestine and lead to novel bioactive compounds.

Polymethoxyflavones (PMFs) were biotransformed to various demethylated metabolites in the human intestine by the PMF-metabolizing bacterium, Blautia sp. MRG-PMF1. Because the newly formed metabolites can have different biological activities, the pathways and regioselectivity of PMF bioconversion were investigated. Using an anaerobic in vitro study, 12 PMFs, 5,7-dimethoxyflavone (5,7-DMF), 5-hydroxy-7-methoxyflavone (5-OH-7-MF), 3,5,7-trimethoxyflavone (3,5,7-TMF), 5-hydroxy-3,7-dimethoxyflavone (5-OH-3,7-DMF), 5,7,4'-trimethoxyflavone (5,7,4'-TMF), 5-hydroxy-7,4'-dimethoxyflavone (5-OH-7,4'-DMF), 3,5,7,4'-tetramethoxyflavone (3,5,7,4'-TMF), 5-hydroxy-3,7,4'-trimethoxyflavone (5-OH-3,7,4'-TMF), 5,7,3',4'-tetramethoxyflavone (5,7,3',4'-TMF), 3,5,7,3',4'-pentamethoxyflavone (3,5,7,3',4'-PMF), 5-hydroxy-3,7,3',4'-tetramethoxyflavone (5-OH-3,7,3',4'-TMF), and 5,3'-dihydroxy-3,7,4'-trimethoxyflavone (5,3'-diOH-3,7,4'-TMF), were converted to chrysin, apigenin, galangin, kaempferol, luteolin, and quercetin after complete demethylation. The time-course monitoring of PMF biotransformations elucidated bioconversion pathways, including the identification of metabolic intermediates. As a robust flavonoid demethylase, regioselectivity of PMF demethylation generally followed the order C-7 > C-4' ≈ C-3' > C-5 > C-3. PMF demethylase in the MRG-PMF1 strain was suggested as a Co-corrinoid methyltransferase system, and this was supported by the experiments utilizing other methyl aryl ether substrates and inhibitors.

Two new puerarin-metabolising human intestinal bacteria were isolated and identified, and the deglycosylation activity for various flavonoid glycosides was investigated. The results could facilitate the study of C-glycosidase reaction mechanisms, as well as the pharmacokinetics of bioactive C-glycoside natural products.