Effect of Silymarin on Biliary Bile Salt Secretion in the Rat Fernando A. Crocenzi, Jose ´ M. Pellegrino, Enrique J. Sa ´nchez Pozzi, Aldo D. Mottino, Emilio A. Rodrı ´guez Garay and Marcelo G. Roma* INSTITUTO DE FISIOLOG ´ IA EXPERIMENTAL,FACULTAD DE CIENCIAS BIOQU ´ IMICAS Y FARMAC ´ EUTICAS, CONICET - U.N.R., SUIPACHA 570, 2000 ROSARIO,ARGENTINA ABSTRACT. The effect of the hepatoprotector silymarin on bile secretion, with particular regard to bile salt secretion, was studied in Wistar rats. Silymarin (25, 50, 100, and 150 mg/kg/day, i.p., for 5 days) induced a dose-dependent increase in bile flow and bile salt secretion, the maximal effect being reached at a dose of 100 mg/kg/day (+17 and +49%, for bile flow and bile salt output, respectively; P  0.05). Assessment of bile salt composition in bile revealed that stimulation of the bile salt secretion was accounted for mainly by an increase in the biliary secretion of -muricholate and, to a lesser extent, of -muricholate, chenodeoxycholate, ursodeoxycholate, and deoxycholate. The maximum secretory rate (T m ) of bile salts, as assessed by infusing the non-hepatotoxic bile salt tauroursodeoxycholate i.v. at stepwise-increasing rates, was not influenced by silymarin. The flavonolignan also increased the endogenous bile salt pool size (+53%, P  0.05) and biliary bile acid excretion after bile acid pool depletion (+54%, P  0.05), a measure of de novo bile salt synthesis. These results suggest that silymarin increases the biliary excretion and the endogenous pool of bile salts by stimulating the synthesis, among others, of hepatoprotective bile salts, such as -muricholate and ursodeoxycholate. BIOCHEM PHARMACOL 59;8:1015–1022, 2000. © 2000 Elsevier Science Inc. KEY WORDS. silymarin; hepatoprotection; bile secretion; bile salt output; ursodeoxycholate; muricholate SIL† is a purified extract from the milk thistle Silybum marianum (L.) Gaertn; it is composed of a mixture of three isomeric flavonolignans: silibinin, silidianin, and silicristin, the former being the most active component [1]. 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 (recently reviewed by Flora et al. [1]). In addition, SIL has been shown to protect experimental animals from various hepa- totoxicants, including carbon tetrachloride [2, 3], acet- aminophen [4], phalloidin [5], D-galactosamine [3], and allyl alcohol [3], among others. The mechanisms by which SIL exerts its hepatoprotec- tive action are under intensive investigation, and appear to be multifactorial in origin. SIL stabilizes membranes, mak- ing cells more resistant to osmotic lysis and to the action of detergents [6]. This flavonolignan prevents lipid peroxide formation in liver cells [7], due, at least in part, to its properties as a free radical scavenger [8]. In addition, the active component of SIL, silibinin, has been shown to inhibit the function of Kupffer cells, which are well- recognized sources of fibrogenic mediators [9]. This would help to explain the antifibrotic properties of SIL in biliary cirrhosis secondary to biliary obstruction in rats [10] and, perhaps, the improvement in parenchymal alterations and in portal inflammation observed in patients with chronic hepatitis [11]. Finally, SIL was shown to be a potent protein synthesis inducer via stimulation of RNA synthesis [12]; this could account in large part for its beneficial effect on liver regeneration [13] and its capability to increase activity of the mixed-function oxidation system [14]. Bile salts are chief constituents of bile. They play a crucial role both in bile formation and, once delivered to the intestine, in lipid absorption by the intestinal tract [15]. Hepatic accumulation of bile salts due to secretory alter- ations, either by mechanical or functional causes, is thought to have a crucial role in the initiation and/or perpetuation of liver injury [16]. Indeed, bile salts are amphipathic compounds that, due to their detergent prop- erties, can disrupt membrane structure and alter permeabil- ity to hepatocellular constituents [17]. Furthermore, bile salts are able to uncouple oxidative phosphorylation, thus inducing oxidative stress and other cytotoxic damages [18]. However, it has also become increasingly apparent that, under such conditions, beneficial changes in the pattern of bile salt metabolism may occur either spontaneously, as an adaptive mechanism in liver pathologies, or due to iatro- genic causes, secondary to therapy. * Corresponding author. Tel. (54) 341-430-5799; FAX (54) 341-439- 9473; E-mail: ifise1@citynet.net.ar † Abbreviations: SIL, silymarin; MC, muricholate; CDC, chenodeoxy- cholate; UDC, ursodeoxycholate; TUDC, tauroursodeoxycholate; DC, deoxycholate; HDC, hyodeoxycholate; ASAT, aspartate aminotransfer- ase; ALAT, alanine aminotransferase; and ALP, alkaline phosphatase. Received 27 May 1999; accepted 13 September 1999. Biochemical Pharmacology, Vol. 59, pp. 1015–1022, 2000. ISSN 0006-2952/00/$–see front matter © 2000 Elsevier Science Inc. All rights reserved. PII S0006-2952(99)00407-4