Inhibition of rat liver UDP-glucuronosyltransferase by silymarin and the metabolite silibinin-glucuronide
Introduction
Silymarin is a purified extract from the milk thistle Silybum marianum (L.) Gaertn, composed mainly of four isomeric flavonolignans: silibinin, isosilibinin, silidianin and silichristin (Kvasnicka et al., 2003). Extracts of milk thistle have been used as medical remedies for almost 2000 years, and continue to be used as therapeutic agents for many types of acute and chronic liver diseases (reviewed by Flora et al., 1998). In addition, silymarin was shown to protect experimental animals from various hepatotoxicants, including carbon tetrachloride, acetaminophen and phalloidin (Schuppan et al., 1999) and from experimental cholestasis (Crocenzi et al., 2001, Crocenzi et al., 2003).
In vitro, silymarin has been reported to inhibit certain hepatic enzymes such as aminopyrine demethylase, benzopyrene hydroxylase, ethoxy coumarin O-deethylase (Lettéron et al., 1990) and glutathione S-transferase (Bartholomaeus et al., 1994). The effect of flavonolignans on UDP-glucuronosyltransferase (UGT) is less known. This enzyme system is a superfamily of enzymes that catalyze the conjugation of glucuronic acid to both endogenous compounds, such as bilirubin and steroid hormones, and exogenous compounds, including food additives, drugs, and environmental pollutants (for review, see Tephly and Burchell, 1990). Based on the nucleotide and amino acid sequences, mammalian UGT isozymes are grouped into two major families termed UGT1A and UGT2B (Burchell et al., 1991, Mackenzie et al., 1997). Silymarin or its main constituent, silibinin, has been reported to decrease the glucuronidation of two family 1A substrates: 4-methylumbelliferone (Venkataramanan et al., 2000) and 3-OH-benzo(a)pyrene (Chrungoo et al., 1997) both in vitro and in intact cells, pointing the experimental evidence to a direct effect of the flavonolignans or their metabolites on the enzyme molecule (Venkataramanan et al., 2000). In this work we further investigate, in vitro, the characteristics of the eventual inhibitory effect of silymarin and silibinin on UDP-glucuronosyltransferase using bilirubin and p-nitrophenol, substrates of the main expressed family 1A isoforms in liver, UGT1A1 and 1A6, respectively (Ritter et al., 1992), and ethinylestradiol, substrate of two family 2B isozymes 2B1 and 2B3 (Mackenzie, 1987, Mackenzie et al., 1997). Since silibinin is metabolized by UGTs (Kren et al., 2000), the effect of silibinin-glucuronide was additionally studied.
Section snippets
Chemicals
Bilirubin, p-nitrophenol, ethinylestradiol, microsomes from male human liver, UDP-glucuronic acid (UDP-GA, ammonium salt), silymarin, silibinin, digitonin, palmitoyl-lisophosphatidylcholine (PLPC) and D-saccharic acid 1,4-lactone were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All the other reagents were of the highest analytical grade. Silymarin composition determined by HPLC analysis (Kvasnicka et al., 2003) was: Silidianin (4.2%), Silichristin (14.7%), Isosilybin (5.3%) and
Effect on p-nitrophenol UGT activity
Silymarin and silibinin-glucuronide significantly inhibited p-nitrophenol glucuronidation (Fig. 1A). Neither silibinin nor the silichristin-silidianin mixture affected p-nitrophenol conjugation indicating that other component of the extract was responsible for the inhibitory effect of silymarin. Incubation with different concentrations of both silymarin and silibinin-glucuronide produced double reciprocal plots that intercepted in the 1/v axis, suggesting competitive inhibition (SM: Vmaxapp:
Discussion
In this study, we described the inhibitory effect of silymarin on glucuronidation that may help to the management of this natural product in the treatment of hepatic diseases. Since silymarin is a complex mixture of compounds containing more than 30 % of unidentified polymeric and oxidation products of flavonolignans and whose activity vs. reproducibility has been questioned (Simanek et al., 2000), the composition of the mixture used in this study was analyzed by HPLC for the sake of
Acknowledgments
Supported by Research Grants from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), and Agencia Nacional de Promoción Científica y Tecnológica. The authors express their gratitude to Dr. Fernando A. Crocenzi for critically reviewed of the manuscript, Dr. José M. Pellegrino and Dr. Marcelo G. Luquita for performing HPLC analyses and Dr. Viviana A. Catania for technical assistance.
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