Carbon Monoxide-Releasing Activity of Plant Flavonoids

Abstract

Flavonoids are naturally occurring compounds found in fruits, vegetables, and other plant-based foods, and they are known for their health benefits, such as UV protection, antioxidant, anti-inflammatory, and antiproliferative properties. This study investigates whether flavonoids, such as quercetin and 2,3-dehydrosilybin, can act as photoactivatable carbon monoxide (CO)-releasing molecules under physiological conditions. CO has been recently recognized as an important signaling molecule. Here, we show that upon direct irradiation, CO was released from both flavonoids in PBS with chemical yields of up to 0.23 equiv, which increased to almost unity by sensitized photooxygenation involving singlet oxygen. Photoreleased CO reduced cellular toxicity caused by high flavonol concentrations, partially restored mitochondrial respiration, reduced superoxide production induced by rotenone and high flavonol levels, and influenced the G0/G1 and G2/M phases of the cell cycle, showing antiproliferative effects. The findings highlight the potential of quercetin and 2,3-dehydrosilybin as CO-photoreleasing molecules with chemopreventive and therapeutic implications in human pathology and suggest their possible roles in plant biology.

Keywords: 2,3-dehydrosilybin; carbon monoxide; cell cycle; mitochondrial respiration; oxidative stress; photoCORM; photoinduced release; quercetin.

Scheme 1. CO Photorelease from 3-Hydroxyflavone (a) and Possible Sites of Photooxygenation (b)

Figure 1

Structures of quercetin (QCT) and 2,3-dehydrosilybin (DHS) and their saturated analogs taxifolin and silybin.

Figure 2

Irradiation of quercetin and 2,3-dehydrosilybin. Irradiation of QCT (a) and DHS (b) in PBS solutions (5% DMSO, pH = 7.4, c = 100 mM, I = 100 mM). The spectra were recorded every 5 min, and the spectra before (black line) and after (red line) 90 min of irradiation are highlighted.

Figure 3

CO-releasing ability of quercetin and 2,3-dehydrosilybin. (A) In PBS buffer, QCT or DHS (100 μL of 0.4 mM solution in PBS buffer with 5% DMSO) was irradiated with white light (LED, I = 160 mW/cm²) and the liberation of CO into the headspace was determined over time by gas chromatography. Released CO was expressed as pmol of CO released to the vial headspace. (B) In human HepaRG hepatic cells, cells were incubated with QCT or DHS (50 μmol/L in MEM medium with 5% DMSO) and irradiated with white light for 2 h. For the CO chamber experiment, the cells in the medium were exposed to the atmosphere containing 300 ppm CO for 2 h. CO concentration in the medium was measured by gas chromatography and expressed as pmol of CO per mL of medium. CO, carbon monoxide; DHS, 2,3-dehydrosilybin; QCT, quercetin.

Figure 4

Effect of light irradiation on the viability of human hepatic HepaRG cells exposed to quercetin (A) and 2,3-dehydrosilybin (B). Cells were treated with solutions of QCT or DHS for 24 h in the dark (gray bars) or irradiated for 2 h with white light (LED, I = 160 mW/cm²) and then incubated for 22 h in the dark (colored bars). *p ≤ 0.05. DHS, 2,3-dehydrosilybin; QCT, quercetin.

Figure 5

Effect of light irradiation on respiration of human Jurkat cells exposed to quercetin (A) and 2,3-dehydrosilybin (B). Jurkat cells were treated with quercetin and 2,3-dehydrosilybin (50 μM) and irradiated with white light (120 mW·cm^–2) for 30 min or treated with CO (300 ppm). Basal and maximum respiration (the values expressed as % of the maximum respiration level of untreated controls) were analyzed immediately after incubation. *P-value ≤0.05; n ≥ 4.

Figure 6

Effect of light irradiation on superoxide production by human hepatic HepaRG cells exposed to quercetin and 2,3-dehydrosilybin. HepaRG cells were exposed to quercetin (QCT) (A) or 2,3-dehydrosilybin (DHS) (B) (50 μmol/L) in the dark or irradiated (irrad) with white light (LED, I = 160 mW/cm²) for 30 min. Superoxide production was measured by flow cytometry in live cells using MitoSOX dye. *p ≤ 0.05; n ≥ 6. (C) Fluorescence (left panels) and bright-field (right panels) microscopy images of HepaRG cells. After incubation for 15 min with MitoSOX dye, the cells were visualized using white light (bright field) and the RFP channel (red fluorescence) using JuLI Stage Real-Time Cell Imaging System, NanoEntek, South Korea.

Figure 7

Effect of light irradiation and CO exposure on superoxide production by human Jurkat cells treated with quercetin and 2,3-dehydrosilybin. Jurkat cells were exposed to quercetin (QCT) (A) or 2,3-dehydrosilybin (DHS) (B) (50 μmol/L) and rotenone (ROT, 10 μM) in the dark or irradiated with white light (LED, I = 160 mW/cm²) or treated with CO (300 ppm) for 30 min. Superoxide production was measured by flow cytometry in live cells using MitoSOX dye. *p ≤ 0.05; n ≥ 6.

Figure 8

Effect of light irradiation on the cell cycle of human hepatic HepG2 cells exposed to quercetin and 2,3-dehydrosilybin. HepG2 cells were exposed to quercetin (QCT) or 2,3-dehydrosilybin (DHS) (50 μmol/L) and irradiated with white light (LED, I = 160 mW/cm²) for 2 h. The cell cycle was measured by flow cytometry after another 22 h in the dark. *p ≤ 0.05; n ≥ 6.