Isolated Silymarin Flavonoids Increase Systemic and Hepatic Bilirubin Concentrations and Lower Lipoperoxidation in Mice

Abstract

Bilirubin is considered to be one of the most potent endogenous antioxidants in humans. Its serum concentrations are predominantly affected by the activity of hepatic bilirubin UDP-glucuronosyl transferase (UGT1A1). Our objective was to analyze the potential bilirubin-modulating effects of natural polyphenols from milk thistle (Silybum marianum), a hepatoprotective herb. Human hepatoblastoma HepG2 cells were exposed to major polyphenolic compounds isolated from milk thistle. Based on in vitro studies, 2,3-dehydrosilybins A and B were selected as the most efficient compounds and applied either intraperitoneally or orally for seven days to C57BL/6 mice. After, UGT1A1 mRNA expression, serum, intrahepatic bilirubin concentrations, and lipoperoxidation in the liver tissue were analyzed. All natural polyphenols used increased intracellular concentration of bilirubin in HepG2 cells to a similar extent as atazanavir, a known bilirubinemia-enhancing agent. Intraperitoneal application of 2,3-dehydrosilybins A and B (the most efficient flavonoids from in vitro studies) to mice (50 mg/kg) led to a significant downregulation of UGT1A1 mRNA expression (46 ± 3% of controls, p < 0.005) in the liver and also to a significant increase of the intracellular bilirubin concentration (0.98 ± 0.03vs.1.21 ± 0.02 nmol/mg, p < 0.05). Simultaneously, a significant decrease of lipoperoxidation (61 ± 2% of controls, p < 0.005) was detected in the liver tissue of treated animals, and similar results were also observed after oral treatment. Importantly, both application routes also led to a significant elevation of serum bilirubin concentrations (125 ± 3% and 160 ± 22% of the controls after intraperitoneal and oral administration, respectively, p < 0.005 in both cases). In conclusion, polyphenolic compounds contained in silymarin, in particular 2,3-dehydrosilybins A and B, affect hepatic and serum bilirubin concentrations, as well as lipoperoxidation in the liver. This phenomenon might contribute to the hepatoprotective effects of silymarin.

Figure 1

Structures of flavonolignans of the silymarin complex and related flavonols.

Figure 2

The effect of silymarin flavonoids and related compounds on HMOX1 and UGT1A1 expressions in HepG2 cells. mRNA expressions were analyzed after 4 h exposure of HepG2 cells to individual flavonolignans (in corresponding nontoxic concentrations). Data are expressed as percentage of control values. ^∗ P < 0.05.

Figure 3

The effect of silymarin flavonoids and related compounds on (a) HMOX and (b) UGT1A1 activities in HepG2 cells. HMOX and UGT1A1 activities were analyzed after 24 h exposure of HepG2 cells to individual flavonolignans. HMOX activity was calculated as pmol CO/h/mg fresh weight and expressed as a percentage of the control values. UGT1A1 enzyme activity was based on relative light unit values and expressed as a percentage of the remaining activity. ^∗ P < 0.05.

Figure 4

The effect of silymarin flavonoids and related compounds on intracellular concentrations of bilirubin in HepG2 cells. Control values are expressed as 100% and correspond to 2 pmol of bilirubin per mg of protein. ^∗ P < 0.05; ^∗∗ P < 0.005.

Figure 5

Serum concentrations of 2,3-dehydrosilybins after intraperitoneal and oral administration. 2,3-Dehydrosilybins (DH) were administered at a dose of 50 mg/kg b.wt. i.p.: intraperitoneal; p.o.: oral; b.wt.: body weight.

Figure 6

The effect of 2,3-dehydrosilybins on hepatic HMOX1 and UGT1A1 mRNA expressions and intrahepatic and systemic bilirubin concentrations. (a) HMOX1 and UGT1A1 mRNA expressions in mouse livers after intraperitoneal administration of a mixture of 2,3-dehydrosilybins A and B (50 mg/kg b.wt. for 7 days). (b) HMOX1 and UGT1A1 mRNA expressions in mouse livers after oral administration of either 2,3-dehydrosilybin A or B (50 mg/kg b.wt. for 7 days). (c) Intrahepatic bilirubin concentrations after intraperitoneal administration of a mixture of 2,3-dehydrosilybins A and B (50 mg/kg b.wt. for 7 days). (d) Intrahepatic bilirubin concentrations after oral administration of either 2,3-dehydrosilybin A or B (50 mg/kg b.wt. for 7 days). (e) Systemic bilirubin concentrations after intraperitoneal administration of a mixture of 2,3-dehydrosilybins A and B (50 mg/kg b.wt. for 7 days). (f) Systemic bilirubin concentrations after oral administration of either 2,3-dehydrosilybin A or B (50 mg/kg b.wt. for 7 days). ^∗ P < 0.05. b.wt.: body weight.

Figure 7

The effect of intraperitoneal and oral administration of 2,3-dehydrosilybins on lipoperoxidation in the liver tissue. (a) MDA concentrations in the mouse liver tissue after intraperitoneal administration of a mixture of 2,3-dehydrosilybins A and B (50 mg/kg b.wt. for 7 days). (b) MDA concentrations in the mouse liver tissue after oral administration of either 2,3-dehydrosilybin A or B (50 mg/kg b.wt. for 7 days). Data are expressed as a percentage of the control values. ^∗ P < 0.05. b.wt.: body weight.