Fig 1.
Analysis of diosmetin, chrysoeriol, and their metabolites by UHPLC—MS/MS.
Fig 1a and 1b show the chromatogram of the incubation samples of diosmetin and chrysoeriol by HLMs, respectively.
Fig 2.
Kinetics of diosmetin and chrysoeriol glucuronidation by HLMs.
The curves were estimated on the basis of fitted parameters generated using the Michaelis-Menten kinetics (B, C) or substrate inhibition (A, D) in HLMs. The Eadie—Hofstee plots are shown in right panal (a-d). Each data point corresponds to the average of three determinations with error bars representing S.D.
Table 1.
Kinetic parameters of diosmetin and chrysoeriol glucuronidation by HLMs.
Fig 3.
Glucuronidation of diosmetin and chrysoeriol by human expressed UGT isoforms.
Experiments were conducted at concentrations 1.25, 2.5, and 10 μM. The concentration of each human expressed UGT isoform was 0.0053 mg/mL. The experiments were conducted at 37°C for 30 min, and the amount of glucuronides formed were measured by UHPLC—MS/MS. The rates of glucuronidation were calculated as nmol/mg/min. Each column corresponds to the average of three determinations with error bars representing the S.D.
Fig 4.
Effects of chemical inhibitors on diosmetin and metabolism and UGT1A9- and UGT1A1-mediated chrysoeriol metabolism in HLMs.
Fig 4 A shows the inhibitory effects of phenylbutazone on the 7-O-glucuronide of diosmetin in HLMs and UGT1A6. Fig 4 B displays the inhibitory effects of carvacrol on the 3′-O-glucuronide of diosmetin in HLMs and UGT1A9. Fig 4 C presents the inhibitory effects of carvacrol on the 7-O-glucuronide of chrysoeriol in HLMs and UGT1A9. Fig 4 D shows the inhibitory effects of bilirubin on the 4′-O-glucuronides of chrysoeriol in HLMs and UGT1A1. Each column corresponds to the average of three determinations with error bars representing the S.D. The “*” symbol means a statistically significant difference compared with control at p < 0.05; “**” means p < 0.01; “***” means p < 0.001.
Fig 5.
Kinetics of diosmetin and chrysoeriol glucuronidation by human expressed UGT enzymes.
The curves are estimated on the basis of fitted parameters generated using the substrate inhibition (A, D) or Michaelis-Menten kinetics (B, C) in UGT1A6, UGT1A1, UGT1A9, and UGT1A9, respectively. The Eadie—Hofstee plots are shown in the right panal (a-d). Each data point corresponds to the average of three determinations with error bars representing the S.D.
Table 2.
Kinetic parameters of diosmetin and chrysoeriol glucuronidation by human expressed UGT enzymes.
Fig 6.
Effect of substrate concentrations on excretion rates (a), intracellular amounts (b), CL (c), and fmet (d) of Dio-7-G, Dio-3′-G, Chr-7-G, and Chr-4′-G.
Three samples (500 μL) were obtained at 15, 30, and 60 min and replaced with fresh loading solution (500 μL) that contains diosmetin or chrysoeriol. The excretion rates of glucuronides were calculated from the slope of the amount-versus-time curves. The intracellular amounts of the glucuronides were determined at the end of the excretion experiments after the cells were washed twice with ice-cold HBSS. Each column corresponds to the average of three determinations with error bars representing the S.D. The “*” (for Dio-7-G or Chr-7-G) or “#” (for Dio-3′-G or Chr-4′-G) symbol means a statistically significant difference between-group, at p < 0.05; “**” or “##” means p < 0.01; “***” or “###” means p < 0.001.
Fig 7.
Effect of BCRP-specific inhibitor Ko143 on the excretion rate of glucuronides (Dio-7-G, Dio-3′-G, Chr-7-G, and Chr-4′-G) (a), total intracellular glucuronide amounts (Dio-7-G, Dio-3′-G, Chr-7-G, and Chr-4′-G) (b), CL (c), and fmet (d).
Engineered HeLa cells stably overexpressing UGT1A9 grown on six-well plates (1 × 105 cells/well) were treated with 2.5 μM of diosmetin or chrysoeriol in the absence or presence of Ko143 at 5 or 10 μM. Three samples (500 μL) were obtained at 20, 40, and 60 min and replaced with fresh loading solution (500 μL) containing 2.5 μM diosmetin or chrysoeriol. Each data point (panel a) and each column (panel b-d) corresponds to the average of three determinations with error bars representing the S.D. The “*” (for Dio-7-G or Chr-7-G) or “#” (for Dio-3′-G or Chr-4′-G) symbol means a statistically significant difference between-group at p < 0.05; “**” or “##” means p < 0.01; “***” or “###” means p < 0.001.
Fig 8.
The effects of diosmetin and chrysoeriol on cell viability in A549 and HepG2 cells with untreated and treated Ko143.
5 μM Ko143, 10 μM Ko143, 10 μM diosmetin, 10 μM diosmetin with 5 μM Ko143 or 10 μM Ko143, 10μM chrysoeriol, 10μM chrysoeriol with 5 μM Ko143 or 10 μM Ko143 were used as substrates to determine the effects of diosmetin and chrysoeriol on cell viability in A549 and HepG2 cells with untreated and treated Ko143. Each column corresponds to the average of three determinations with error bars representing the S.D. The “*” symbol means a statistically significant difference compared with control at p < 0.05; “**” means p < 0.01; “***” means p < 0.001).