Research Articles
Hepatic Metabolism and Biliary Excretion of Valerenic Acid in Isolated Perfused Rat Livers: Role of Mrp2 (Abcc2)

https://doi.org/10.1002/jps.21671Get rights and content

Abstract

The study was designed to investigate the hepatic metabolism and transport system of valerenic acid, a main active constituent of valerian, in isolated perfused livers from Wistar and Mrp2-deficient TR rats. After administration of 20 µM valerenic acid, the formation of seven valerenic acid glucuronides (M1–M7), namely two glucuronides of valerenic acid (M6, M7), four glucuronides of hydroxylated valerenic acid (M1, M3, M4, M5), and one glucuronide of hydroxylated dehydro-valerenic acid (M2) in bile and perfusate was quantified by HPLC. The hepatic extraction ratio and clearance of valerenic acid were very high in Wistar and TR rats (E: 0.983 ± 0.006 vs. 0.981 ± 0.004; Cl: 35.4 ± 0.21 mL/min vs. 35.3 ± 0.14 mL/min). However, biliary excretion and efflux of conjugates differed greatly in TR rats. While cumulative biliary excretion of unconjugated valerenic acid and the glucuronides M1–M7 dropped dramatically to 1–9%, their efflux into perfusate increased 1.5- to 10-fold. This indicates that valerenic acid and its glucuronides are eliminated into bile by Mrp2. In summary, valerenic acid was metabolized to several conjugates, whereby the canalicular transporter Mrp2 mediated biliary excretion of the parent drug and its glucuronides. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3839–3849, 2009

Introduction

Extracts from the roots of valerian (Valeriana officinalis L., Valerianaceae) have long been used in phytotherapy for the treatment of insomnia. These extracts are widely accepted as herbal sedatives throughout the world. Several clinical trials have confirmed that treatment with valerian extracts improves the latency and quality of sleep in healthy individuals as well as in patients suffering from sleep disorders, without producing the well known side effects of sedatives.1, 2, 3, 4 One major active constituent of valerian is valerenic acid, whose central nervous depressant activity has been shown in mice and rats by involvement in the GABA-ergic system.5,6 Recently valerenic acid was also identified as a potent and subunit-specific allosteric modulator of GABAA receptors7,8 and a partial agonist of the 5-HT5a receptor that plays a major role in the serotonergic regulation of circadian time-keeping.9

Although valerian is one of the most widely used herbal agents throughout the world (one of the top ten best-selling herbals in the United States10), the pharmacokinetics and metabolism of valerian constituents have been scarcely investigated.11 However, some authors have demonstrated in vitro inhibition of cytochrome P450 and glucuronosyl transferases by valerian extracts and valerenic acids, indicating affinity to drug-metabolizing enzymes.12, 13, 14 Using the in vitro model of the isolated perfused rat liver, we investigated the hepatic metabolism of valerenic acid, which is one of the major active compounds of valerian. The European Pharmacopoeia refers to valerenic acid as a marker for qualitative and quantitative analysis of Valeriana officinalis L.15 The majority of commercially available products are standardized to its content. Carboxylic acids such as diflunisal and naproxen were clearly shown to be eliminated as glucuronides in the liver.16, 17, 18 We hypothesized that valerenic acid is also likely to be glucuronized in the liver.

Based on recently published data from our laboratory, which showed that hepatobiliary transport of glucuronides is mediated almost exclusively by the multidrug resistance-associated protein 2 (MRP2, ABCC2),19,20 we further hypothesized that MRP2 might also have a major impact on biliary excretion of potential metabolites of valerenic acid. As the substrate specificity of MRP2 has been described in detail with the use of the mutant rat strain TR, which lacks a functional Mrp2 at the canalicular membrane,21,22 we also investigated the metabolism and distribution of valerenic acid in perfused isolated livers of Mrp2-deficient TR rats. Contrary to in vivo studies, the isolated perfused rat liver is a better model to study the metabolism and biliary elimination of drugs because pharmacokinetic variables such as renal clearance and drug distribution are eliminated in this setting.

Section snippets

Materials

Valerenic acid was purchased from Phytolab GmbH & Co KG (Vestenbergsgreuth, Germany). Sulfatase type V from limpets (Patella vulgata), and β-glucuronidase type B-3 from bovine liver were obtained from Sigma (Munich, Germany). Methanol and water were of HPLC grade (Merck, Darmstadt, Germany). All other chemicals and solvents were of analytical grade and used without further purification.

Liver Perfusion

The study was approved by the Animal Experimentation Ethics Committee of the Austrian Ministry for Education,

Metabolism and Disposition of Valerenic Acid in Wistar Rats

Application of valerenic acid (20 µM) to the liver of Wistar rats stimulated bile flow by 61.5 ± 2.8%. Choleresis reached its peak value of 0.84 ± 0.03 µg/g liver min at 10–15 min after addition of valerenic acid to the perfusion medium, indicating moderate solvent drag by the concentrated excretion of valerenic acid and its metabolites into bile (mean concentrations of valerenic acid and M1–M7 in bile and perfusate: 276 ± 83 µmol/L and 39 ± 12 µmol/L). In TR rats, valerenic acid did not significantly

Discussion

In the present study, hepatic biotransformation and hepatobiliary transport of valerenic acid and its metabolites were investigated for the first time by perfusing isolated rat livers of male Wistar rats with 20 µM of valerenic acid, which was chosen based on daily intakes of 500–1000 mg valerian (5–10 mg valerenic acid/day). During 90 min the perfused liver extracted more than 98% of applied valerenic acid; steady-state concentrations were achieved within 10 min after commencement of infusion.

Acknowledgements

This study was supported by a grant from the Jubiläumsfonds der Österreichischen Nationalbank 12600 (W.J.).

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