Metabolism of Kaempferia parviflora Polymethoxyflavones by Human Intestinal Bacterium Bautia sp. MRG-PMF1

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

Demethylation of Polymethoxyflavones by Human Gut Bacterium, <i>Blautia</i> sp. MRG-PMF1

Polymethoxyflavones (PMFs) were biotransformed to various demethylated metabolites in the human intestine by the PMF-metabolizing bacterium, <i>Blautia</i> 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

Induction of colon carcinoma cell apoptosis by HMF.

<p>(A) After 24 h of incubation, HCT-116 cells were stained with Hoechst 33342, after which cell nuclei were observed under a microscope for detection of apoptosis. The number of apoptotic cells (strong blue staining) significantly increased compared to the control group in a dose-dependent manner. (B) Apoptotic index of HMF-treated cells was significantly higher than the control group. Apoptotic index was calculated as the percentage of apoptotic nuclei compared to the total number of cells and is presented as the mean ± SD (n = 10). Data represent the mean ± SD (n = 3) from three independent experiments.*p<0.05; **p<0.01. Scale bars 0.1 mm.</p

List of Antibodies.

<p>List of Antibodies.</p

Sequences of RT-PCR oligonucleotide primers specific for human <i>bax</i>, <i>bcl-2</i>, <i>β-actin</i> and <i>b2m</i>.

<p>Sequences of RT-PCR oligonucleotide primers specific for human <i>bax</i>, <i>bcl-2</i>, <i>β-actin</i> and <i>b2m</i>.</p

HMF significantly induces ROS generation in HCT-116 cells.

<p>(A) Cytosolic and mitochondrial ROS level was measured using the H₂DCFDA and MitoSOX red fluorescence probe method respectively as described in materials and methods. Mean fluorescent intensity of ROS was analyzed by ImageJ software and presented as the mean ± SD (n = 10). Fluorescence intensity significantly increased in HMF-treated groups. (B) MDA levels were examined by the TBA method. MDA levels were significantly elevated in HMF-treated groups. (C) Cytosolic and mitochondrial ROS generation in time-dependent manner was measured using the H₂DCFDA and MitoSOX red fluorescence probe methods, respectively. Fluorescence intensity significantly increased in time course manner by HMF treatment. (D) Antioxidant marker enzymes including Prx, Trx, glutathione reductase and SOD-2 was analyzed by western blotting. β-actin was utilized as a loading control. Data represent the mean ± SD (n = 3) from three independent experiments.*p<0.05; **p<0.01. Scale bars 0.1 mm.</p

Schematic representation of plausible detailed molecular mechanism of HMF-induced cell death by ROS-mediated intrinsic apoptosis pathway.

<p>HMF treatment leads to ROS generation and Ca²⁺ release, resulting in ER stress induction. Simultaneously, HMF causes alteration of mitochondrial membrane potential (MMP) and reduction of the Bcl-2/Bax ratio, leading to activation of caspase-3 and apoptosis progression. In contrast, ROS inhibition by NAC attenuates HMF-induced mitochondrial apoptosis in HCT-116 cells.</p

Concentration-dependent effect of HMF on colorectal cancer cell viability.

<p>Cells were incubated with various concentrations of HMF for 24 h, after which cell viability was measured using (A) MTT and (B) LDH assays. MTT assay demonstrated fewer viable HCT-116 cells than normal cells at all concentrations. Data represent the mean ± SD (n = 3) from three independent experiments. Asterisk indicates *p<0.05; **p<0.01.</p

Effect of HMF on disruption of mitochondrial membrane potential and Cyt c release in HCT-116 cells.

<p>(A) Cells were treated with the indicated concentrations of HMF for 24 h and stained with Rhodamine-123. Mean fluorescence intensity was quantified using ImageJ software and presented as the mean ± SD (n = 10). (B) Expression profile of <i>bax</i> and <i>bcl-2</i> gene was assayed using real-time quantitative RT-PCR. Data represent fold changes versus control cells. Data were normalized to housekeeping genes, <i>β-actin</i> and <i>b2m</i>. (C) Markers of apoptosis, including BID, Bax, Bcl-2, and Caspase-3, were detected by Western blotting. (D) Alteration of Bcl-2 and Bax in HCT-116 cells after 25, 50, and 100 μM HMF treatment for 24 h. (E) Results of Western blotting were analyzed by ImageJ software. (F) and (G) Cyt c release from mitochondria into cytosol in HCT-116 cells after 25, 50, and 100 μM HMF treatment for 24 h was detected by Western blotting. (H) Release of Cyt c from mitochondria in HCT-116 cells was analyzed by measuring the absorbance at 550 nm. Data represent the mean ± SD (n = 3) from three independent experiments.*p<0.05; **p<0.01. Scale bars 0.1 mm.</p