Article Figures & Data
Figures
- Fig. 1.
Typical multiple reaction monitoring chromatograms of luteolin and its metabolites (Lut-7-G, Lut-4′-G, Lut-3′-G, Chr-7-G, Dio-7-G, Chr-4′-G, Dio-3′-G, chrysoeriol, and diosmetin) catalyzed by UGTs and COMTs in plasma (A) and bile samples (B) after oral administration of luteolin (5 mg/kg) to rats. Green peak: the chromatogram of luteolin. Black peak: the chromatogram of luteolin glucuronides (Lut-7-G, Lut-4′-G, and Lut-3′-G). Red peak: the chromatograms of luteolin-methylated glucuronides (Chr-7-G, Dio-7-G, Chr-4′-G, and Dio-3′-G). Blue peak: the chromatograms of luteolin-methylated metabolites (chrysoeriol and diosmetin).
- Fig. 2.
Concentrations of luteolin glucuronides (Lut-7-G, Lut-4′-G, and Lut-3′-G) and luteolin-methylated glucuronides (Chr-7-G, Dio-7-G, Chr-4′-G, and Dio-3′-G) in plasma at 20, 60, and 360 minutes, as well as their cumulative excretion in bile samples within 24 hours after the oral administration of luteolin (5 mg/kg) to rats. (A and B) Plasma concentrations of glucuronides and methylated glucuronides at 20, 60, and 360 minutes after gavage. The symbol “*” shows the significant differences of plasma concentration among the different glucuronides or methylated glucuronides (P < 0.05). (C and D) Cumulative excretion of glucuronides and methylated glucuronides in bile samples within 24 hours. Significant differences of cumulative excretion were found among the different glucuronides (*P < 0.05 versus Lut-3′-G). Only two methylated glucuronides (Chr-7-G and Dio-7-G) were detected in rat bile, and no significant differences were found between Chr-7-G and Dio-7-G (P > 0.05). Each point or column represents the mean of four determinations. The error bar represents the S.D. of the mean (n = 4, mean ± S.D.).
- Fig. 3.
UHPLC chromatograms of luteolin (A), chrysoeriol (B), diosmetin (C), and their respective glucuronides after incubation with UDPGA-supplemented RLS9.
- Fig. 4.
Kinetics of luteolin (A), chrysoeriol (B), and diosmetin (C) glucuronidation by RLS9 at different concentrations (0.3125–80 μM for luteolin and diosmetin; 0.0781–20 μM for chrysoeriol). Each inset shows the Eadie–Hofstee plot. For luteolin glucuronidation, the formations of Lut-7-G (Aa) and Lut-4′-G (Ac) were fitted using the substrate inhibition model; Lut-3′-G (Bb) formation followed the Michaelis–Menten equation. For chrysoeriol glucuronidation, the formations of Chr-7-G (Bd) and Chr-4′-G (Be) were fitted using the Michaelis–Menten equation. For diosmetin glucuronidation, Dio-7-G (Cf) and Dio-3′-G (Cg) formation followed the autoactivation and the substrate inhibition model, respectively. The RLS9 concentrations were 0.005–0.01 mg/mL, and the incubation time was 30 minutes. All incubations were performed in triplicate. Each point represents the mean of three determinations. The error bar represents the S.D. of the mean (n = 3, mean ± S.D.).
- Fig. 5.
UHPLC chromatograms of luteolin (A), Lut-7-G (B), and their respective methylated metabolites after incubation with SAM-supplemented RLS9.
- Fig. 6.
Kinetics of luteolin (A) and Lut-7-G (B) methylation by RLS9 at different concentrations (0.125–80 μM for luteolin; 1.25–60 μM for Lut-7-G). Each inset shows the Eadie–Hofstee plot. For luteolin methylation, the formation of chrysoeriol (Aa) and diosmetin (Ab) was fitted using the biphasic model. For Lut-7-G methylation, the formation of Chr-7-G (Bc) and Dio-7-G (Bd) was fitted using the Michaelis–Menten equation. The RLS9 concentration was 0.25 mg/mL, and the incubation times for luteolin and Lut-7-G were 20 minutes and 30 minutes, respectively. All incubations were performed in triplicate. Each point represents the mean of three determinations. The error bar represents the S.D. of the mean (n = 3, mean ± S.D.).
- Fig. 7.
Kinetics of luteolin glucuronidation and methylation by RLS9 in UGT–COMT coreactions at different concentrations (0.125–600 μM). (A–I) Kinetic curves for the formation of Lut-7-G, Lut-4′-G, Lut-3′-G, Chr-7-G, Dio-7-G, Chr-4′-G, Dio-3′-G, chrysoeriol, and diosmetin in reaction mixture. Each inset shows the Eadie–Hofstee plot. Lut-7-G, Lut-4′-G, and Lut-3′-G formation were fitted using the Michaelis–Menten equation; Chr-7-G and Chr-4′-G formation were fitted using the substrate inhibition model. Other metabolites were fitted using the autoactivation equation. The RLS9 concentration was 0.25 mg/mL, and the incubation time was 30 minutes. All incubations were performed in triplicate. Each point represents the mean of three determinations. The error bar represents the S.D. of the mean (n = 3, mean ± S.D.).
- Fig. 8.
The influence of absence of SAM or UDPGA on the glucuronidation and methylation of luteolin by RLS9 in UGT–COMT coreactions. Three different concentrations (1, 5, and 10 μM) of luteolin were used in the experiment. (A1–A3) 1, 5, and 10 μM luteolin was incubated in the absence and presence of SAM, respectively; (B1–B3) 1, 5, and 10 μM luteolin was incubated in the absence and presence of UDPGA, respectively. The RLS9 concentration was 0.25 mg/mL, and the incubation time was 30 minutes. All incubations were performed in triplicate. Each column represents the mean of three determinations. The error bar represents the S.D. of the mean (n = 3, mean ± S.D.). Significant differences (P < 0.05, marked by *) compared with the corresponding control group were described.
- Fig. 9.
Two proposed disposition pathways of luteolin mediated by the interplay of UGTs and COMTs in rats. Glu, glucuronic acid. Red and green lines represent the methyl and glucuronic acid substitutions, respectively.
Tables
- TABLE 1
Apparent kinetic parameters of luteolin, chrysoeriol, and diosmetin glucuronidation obtained from RLS9
Calculations were based on curve fitting using the Michaelis–Menten equation, substrate inhibition, or autoactivation model, as described in Materials and Methods. Data are expressed as the mean ± S.D., n = 3.
Metabolites Km (μmol/L) Vmax (nmol/mg/min) CLint (mL/min/mg) Ki (μmol/L) Luteolin Lut-7-Ga 5.5 ± 1.1 5.3 ± 0.5 1.0 ± 0.3 226 ± 100 Lut-4′-Ga 2.2 ± 0.5 0.9 ± 0.1 0.4 ± 0.1 221 ± 98 Lut-3′-Gb 8.6 ± 1.2 6.0 ± 0.3 0.7 ± 0.1 Chrysoeriol Chr-7-Gb 3.5 ± 0.3 3.9 ± 0.1 1.1 ± 0.1 Chr-4′-Gb 0.3 ± 0.1 2.3 ± 0.1 8.2 ± 2.5 Diosmetin Dio-7-Gc 6.2 ± 1.6 3.8 ± 0.5 0.6 ± 0.1 Dio-3′-Ga 2.5 ± 0.4 6.1 ± 0.4 2.5 ± 0.2 425 ± 246 - TABLE 2
Apparent kinetic parameters of luteolin and Lut-7-G methylation obtained from RLS9
Calculations were based on curve fitting using the Michaelis–Menten equation or biphasic model, as described in Materials and Methods. Data are expressed as the mean ± S.D., n = 3.
Metabolites Km (μmol/L) Vmax (nmol/mg/min) CLint (mL/min/mg) Luteolin Chrysoeriola 33.8 ± 4.1 1.06 ± 0.05 0.03 ± 0.01 Diosmetina 17.9 ± 1.7 0.92 ± 0.14 0.05 ± 0.01 Lut-7-G Chr-7-Gb 8.9 ± 0.6 0.38 ± 0.01 0.04 ± 0.01 Dio-7-Gb 15.1 ± 1.1 0.67 ± 0.02 0.04 ± 0.01 - TABLE 3
Apparent kinetic parameters of luteolin glucuronidation and methylation obtained from RLS9 in UGT–COMT coreactions
Calculations were based on curve fitting using the Michaelis–Menten equation, substrate inhibition, or autoactivation model, as described in Materials and Methods. Data are expressed as the mean ± S.D., n = 3.
Metabolites Km (μmol/L) Vmax (nmol/mg/min) CLint (mL/min/mg) Ki (μmol/L) Lut-7-Ga 9.9 ± 1.6 2.58 ± 0.11 0.26 ± 0.03 Lut-4′-Ga 2.9 ± 0.3 0.69 ± 0.01 0.24 ± 0.02 Lut-3′-Ga 11.5 ± 0.6 3.08 ± 0.05 0.27 ± 0.02 Chr-7-Gb 7.3 ± 1.8 0.17 ± 0.02 0.02 ± 0.01 59 ± 17 Dio-7-Gc 9.4 ± 6.0 0.90 ± 0.40 0.11 ± 0.03 Chr-4′-Gb 12.4 ± 4.5 0.24 ± 0.06 0.02 ± 0.01 18 ± 7 Dio-3′-Gc 44.4 ± 6.8 0.24 ± 0.02 0.01 ± 0.01 Chrysoeriolc 1.3 ± 0.9 1.04 ± 0.18 1.16 ± 0.86 Diosmetinc 6.6 ± 0.4 2.59 ± 0.15 0.39 ± 0.01
In this issue
Interplay of UGTs and COMTs in Disposition of Luteolin
