Methylation of dietary flavones increases their metabolic stability and chemopreventive effects

Abstract

Dietary flavones have promising chemoprotective properties, in particular with regard to cancer, but problems with low oral bioavailability and sometimes unacceptable toxicity have made their use as protective additives to normal diets questionable. However, methylation of free phenolic hydroxyl groups leads to derivatives not susceptible to glucuronic acid or sulfate conjugation, resulting in increased metabolic stability. Methylation also leads to greatly improved transport through biological membranes, such as in intestinal absorption, and much increased oral bioavailability. Recent studies also indicate that methylation results in derivatives with increasing potency to kill cancer cells. They also show high potency towards inhibition of hormone-regulating enzymes, e.g., aromatase, important in the causation of breast cancer. Methylation of the flavones may also result in derivatives with diminished toxic side-effects and improved aqueous solubility. In conclusion, it appears that methylation of dietary flavones as well as of other food products may produce derivatives with much improved health effects.

Keywords: cancer prevention; flavonoids; methoxyflavones; methylation.

Figure 1.

(a) Basic flavone skeleton; (b) structures of some common dietary and other model flavones.

Figure 2.

Time-dependent metabolic depletion of unmethylated and methylated flavones in pooled human liver S9 fraction (modified from ref. [51]). (a) quercetin; (b) 7-MF (filled symbols) and 7-HF (open symbols); (c) 5,7-DMF (filled symbols) and chrysin (open symbols); (d) 5,7,4’-TMF (filled symbols) and apigenin (open symbols). Human liver S9 fraction was incubated with UDPGA, PAPS and NADPH and 5 μM flavone and analyzed by HPLC. * Significantly higher than the corresponding unmethylated flavone after the same incubation time.

Figure 3.

Caco-2 cell transport of methylated versus unmethylated flavones (modified from ref. [51]). (a) 7-MF (filled squares) and 7-HF (open squares); (b) 5,7-DMF (filled diamonds) and chrysin (open diamonds); (c) 5,7,4’-TMF (filled circles) and apigenin (open circles). A 5 μM concentration of the flavones (10 μM for 5,7-DMF and chrysin) in transport buffer was added to the apical chambers of Transwells. Samples were taken from the basolateral side at 0.5, 1, 3 and 6 hr. * Significantly higher than the corresponding unmethylated flavone after the same incubation time.

Figure 4.

Plasma and tissue levels of 5,7-DMF and chrysin after oral administration of 5 mg/kg in rats. (a) Plasma 5,7-DMF (no chrysin could be detected); (b) tissue 5,7-DMF in liver (○), lung (▪) and kidney (Δ); (mean ± SEM of 5 animals at each time-point) (modified from ref. [54]).

Figure 5.

Effect of methylated flavones (a) 5,7,4’-TMF and (b) 5,7-DMF compared to unmethylated analogs apigenin and chrysin, respectively, on SCC-9 cell proliferation (modified from ref. [54]). Cell proliferation, expressed as percent of control (DMSO-treatment), was measured as BrdU incorporation into cellular DNA after a 24-h exposure of the cells to the flavones. Mean values ± SEM (n = 10). The numbers shown in the figure are the calculated IC₅₀ values. * Significantly lower than control, P < 0.05. # significantly higher than control, P < 0.05.

Figure 6.

Effect of 5,7-DMF (a) compared to chrysin (b) and 5,7,4’-TMF (c) compared to apigenin (d) on SCC-9 cell cycle progression (modified from ref. [54]). Cells were exposed to varying concentrations of flavones for 48 h. The percentage of cells in G1, S and G2/M phase was measured by flow cytometry after propidium iodide staining. Mean values of 3 experiments with duplicate samples are shown. * Significantly different from control, P < 0.05 or better.

Figure 7.

Trout cells after 24-hr exposure to medium, vehicle control, or 25 μM flavones. Dead or dying cells are indicated by arrows (modified from ref. [76]).

Figure 8.

Confluent trout cells treated for 48 hr with various concentrations of chrysin [76].

Figure 9.

Solubility of 5,7-DMF and chrysin in aqueous solution, i.e., deionized water (open symbols) and Hanks’ buffered salt solution (closed symbols). Measurements were made by UV at 265 nm after removal of insoluble material. Each point is the mean of two determinations [81].