Pharmacology of Silymarin

F. Fraschini, G. Demartini, D. Esposti,

Disclosures

Clin Drug Invest. 2002;22(1) 

In This Article

1. Pharmacodynamics

1.1 Antioxidant Properties

Flavonoids usually possess good antioxidant activity.

The water-soluble dehydrosuccinate sodium salt of silibinin is a powerful inhibitor of the oxidation of linoleic acid-water emulsion catalysed by Fe2+ salts.[15] It also inhibits in a concentration-dependent way the microsomal peroxidation produced by NADPH-Fe2+ -ADP, a well known experimental system for the formation of hydroxy radicals.[16] In studies performed in rat hepatic microsomes, it has been demonstrated that lipid peroxidation produced by Fe(III)/ascorbate is inhibited by silibinin dihemisuccinate; the inhibition is concentration-dependent.[17,18]

It has been shown that silymarin is as active as quercetin and dihydroquercetin, and more active than quercitrin, in terms of antiperoxidant activity, independent of the experimental model used to produce peroxidation.[19]

It has recently been reported that in rat hepatocytes treated with tert-butyl hydroperoxide (TBH), silymarin reduces the loss of lactate dehydrogenase (LDH), increases oxygen consumption, reduces the formation of lipid peroxides, and increases the synthesis of urea in the perfusion medium. Furthermore, silymarin is able to antagonise the increase in Ca2+ produced by TBH, reducing ion levels down to below 300 nmol/L. The protective effect of silymarin is mediated by the inhibition of lipid peroxidation, and the modulation of hepatocyte Ca2+ content seems to play a crucial role.[20]

1.2 Protective Effects in Models of Oxidative Stress

Oxidative stress is defined as structural and/or functional injury produced in tissues by the uncontrolled formation of pro-oxidant free radicals. Oxidative stress usually develops when the pro-oxidant action of an inducer exceeds the anti-oxidant capacity of the cell defence system, altering its homeostatic capacity. Numerous substances induce oxidative stress, including carbon tetrachloride, TBH, ethanol, paracetamol (acetaminophen) and phenylhydrazine. It has been shown in rats that silibinin protects neonatal hepatocytes from cell damage produced by erythromycin, amitriptyline, nortriptyline and TBH.[21]

Erythrocytes obtained from rats treated with silymarin exhibited high resistance against the haemolysis produced by phenylhydrazine[22,23] and the lysis induced by osmotic shock.[1] This suggests that silymarin may act by increasing the stability of the erythrocyte membrane.

The cytoprotective activity of silymarin has also been shown in hepatocytes of rats subjected to osmotic stress produced by hypotonic saccharose solutions.[24]

The perfused liver is a valid experimental model for the evaluation of the effect of substances that induce oxidative stress and of the protection provided by scavengers. Using this experimental model, it has been shown that phenylhydrazine produces an increase in oxygen consumption in rat liver in vitroand in the release of thiobarbituric acid reactive substances (TBARS) in the perfusate.[25] This stress is associated with a reduction in the amount of reduced glutathione (GSH) in the liver; GSH exerts important protective activity against chemically induced oxidative stress.[26,27] Using liver from rats pretreated in vivo with silibinin 50 mg/kg intravenously, a significant reduction in the oxygen consumption stimulated by phenylhydralazine and in the release of TBARS was observed, without any changes in GSH levels.[22,25]

The antioxidant effect of silibinin was observed in rats with acute intoxication caused by ethanol[1,26] or paracetamol,[28] which are peroxidation inducers that produce marked GSH depletion in the liver. Treatment with silymarin or silibinin was able to protect animals from oxidative stress produced in the liver by ethanol or paracetamol.[2,26,28] Furthermore, it has been reported that treatment with silibinin attenuates the increase in plasma levels of AST, ALT and gammaglutamyl transpeptidase (GGT) observed after intoxication by paracetamol.[1]

The hepatoprotective activity of silibinin has also been studied in rats with liver cirrhosis induced by the long-term administration of carbon tetrachloride. Muriel & Mourelle[29] have shown that silibinin preserves the functional and structural integrity of hepatocyte membranes by preventing alterations of their phospholipid structure produced by carbon tetrachloride and by restoring alkaline phosphatase and GGT activities.

Another interesting property of silibinin and silymarin is their role as regulators of the content of GSH in various organs. In rats treated with silibinin intravenously or silymarin intraperitoneally, a significant increase in the amount of the GSH contained in the liver, intestine and stomach was found, whereas there were no changes in the lungs, spleen and kidneys ( Table 1 ).[30]

1.3 Activity against Lipid Peroxidation

Lipid peroxidation is the result of an interaction between free radicals of diverse origin and unsaturated fatty acids in lipids. Lipid peroxidation involves a broad spectrum of alterations, and the consequent degeneration of cell membranes may contribute towards the development of other disorders of lipoprotein metabolism, both in the liver and in peripheral tissues.

Silymarin appears to act as an antioxidant not only because it acts as a scavenger of the free radicals that induce lipid peroxidation,[17,31] but also because it influences enzyme systems associated with glutathione and superoxide dismutase.[30]

It has been shown that all the components of silymarin inhibit linoleic acid peroxidation catalysed by lipoxygenase[37] and that silymarin protects rat liver mitochondria and microsomes in vitro against the formation of lipid peroxides induced by various agents.[38]

1.4 Effects on Liver Lipids

The influence of silymarin on cellular permeability is closely associated with qualitative and quantitative alterations of membrane lipids (both cholesterol and phospholipids).[29,39,40] This suggests that silymarin may also act on other lipid compartments in the liver; this may influence lipoprotein secretion and uptake. It has been shown that silymarin and silibinin reduce the synthesis and turnover of phospholipids in the liver of rats. Furthermore, silibinin is able to neutralise two effects of ethanol in rats: the inhibition of phospholipid synthesis and the reduction in labelled glycerol incorporation into lipids of isolated hepatocytes.[14,27,32] In addition, silibinin stimulates phosphatidylcholine synthesis and increases the activity of cholinephosphate cytidyltransferase in rat liver both in normal conditions and after intoxication by galactosamine.[41]

Data on the influence of silymarin on triglyceride metabolism in the liver are scanty. It is known that in rats silibinin is able to partly antagonise the increase in total lipids and triglycerides produced in the liver by carbon tetrachloride[12] and, probably, to activate fatty acid ß-oxidation.[1] It has also been suggested that silymarin may diminish triglyceride synthesis in the liver.[14]

Letteron et al.[31] studied the mechanisms of action of silymarin that provide protection against lipid peroxidation and the hepatotoxicity of carbon tetrachloride in mice, and came to the conclusion that silymarin works by reducing metabolic activation by carbon tetrachloride and by acting as an antioxidant that prevents chain rupture.

Other authors have shown that silymarin affords hepatoprotection against specific injury induced by microcystin (a hepatotoxin), paracetamol, halothane and alloxan in several experimental models ( Table 1 ).[11,35,36,42]

1.5 Effects on Plasma Lipids and Lipoproteins

The administration of silymarin reduces plasma levels of cholesterol and low-density lipoprotein (LDL) cholesterol in hyperlipidaemic rats, whereas silibinin does not reduce plasma levels of cholesterol in normal rats; however, it does reduce phospholipid levels, especially those transported in LDL.[14]

Data obtained in experimental models of hepatic injury have shown that silymarin is able to normalise the increase in plasma lipids observed after administration of carbon tetrachloride and to antagonise the reduction in serum free fatty acids induced by thioacetamide. In the experimental model of hepatic injury produced by thioacetamide, silymarin did not appear to be able to normalise the reduction in triglycerides in serum. In the experimental model of hepatic injury produced by paracetamol in rats, it was evident that silymarin improves LDL binding to hepatocytes, an important factor for the reduction of LDL in plasma.[14]

1.6 Stimulation of Liver Regeneration

One of the mechanisms that can explain the capacity of silymarin to stimulate liver tissue regeneration is the increase in protein synthesis in the injured liver. In in vivo and in vitro experiments performed in the liver of rats from which part of the organ had been removed, silibinin produced a significant increase in the formation of ribosomes and in DNA synthesis, as well as an increase in protein synthesis.[43] Interestingly, the increase in protein synthesis was induced by silibinin only in injured livers, not in healthy controls.[44] The mechanism whereby silibinin stimulates protein synthesis in the liver has not been defined; it may be the physiological regulation of RNA polymerase I at specific binding sites, which thus stimulates the formation of ribosomes.[13] In rats with experimental hepatitis caused by galactosamine, treatment with intraperitoneal silymarin 140 mg/kg for 4 days completely abolished the inhibitory effect of galactosamine on the biosynthesis of liver proteins and glycoproteins.[34]

These data support the results of previous experiments in a similar model of acute hepatitis in the rat, in which silymarin protected hepatic structures, liver glucose stores and enzyme activity in vivo from injury produced by galactosamine.[9]

The capacity of silymarin to stimulate protein synthesis has also been studied in neoplastic cell lines, in which no increase in protein synthesis, ribosome formation or DNA synthesis has been found after treatment with silymarin.[44]

1.7 Effects during Experimental Intoxication with

Amanita phalloides

The therapeutic activity of silymarin against mushroom poisoning is worthy of particular attention. The hepatoprotective properties of silymarin have been tested in dogs, rabbits, rats and mice. A dose of 15 mg/kg of silymarin was administered intravenously 60 minutes before intraperitoneal administration of a lethal dose of phalloidin, and was able to protect all animal species tested (100% survival) from the action of the toxin.

When it is injected 10 minutes after phalloidin, silymarin affords similar protection only at doses of 100 mg/kg. The longer the time that has elapsed after administration of the toxin, the less effective the drug becomes, and after 30 minutes it is no longer effective even at high doses. Histochemical and histoenzymological studies have shown that silymarin, administered 60 minutes before or no longer than 10 minutes after induction of acute intoxication with phalloidin, is able to neutralise the effects of the toxin and to modulate hepatocyte function.[6,7]

Similar results were obtained in dogs treated with sublethal oral doses of A phalloides, in which hepatic injury was monitored by measuring enzymes and coagulation factors. Amongst the numerous substances tested (prednisolone, cytochrome c, benzylpenicillin, silymarin), only benzyl-penicillin (1000 mg/kg intravenous infusion after 5 hours) and silymarin (50 mg/kg intravenous infusion after 5 hours and 30 mg/kg after 24 hours) were able to prevent the increase in hepatic enzymes and the fall in coagulation factors induced by experimental intoxication ( Table 2 ).[45]

The cyclopeptides of fungi of the genus Amanita, including amatoxins and fallotoxins, are captured by hepatocytes through the sinusoidal system, which is also involved in the mediation of liver uptake of biliary salts. It has been demonstrated that silibinin is able to inhibit uptake of amanitin in isolated preparations of hepatocyte membranes, and the same effect has been shown for taurocholate, antamanide, prednisolone and phalloidin. The effect of silibinin appears to be competitive.[2]

Recently, the role of tumour necrosis factor- (TNF ) in hepatic injury produced by -amanitin has been investigated in primary cultures of rat hepatocytes. At a concentration of 0.1 µmol/L, the toxin inhibits RNA and protein synthesis within 12 hours, but cytotoxicity appears only much later (36 hours). TNF is not indispensable for the development of cytotoxicity, but exacerbates it and markedly increases lipid peroxidation. The addition of silibinin at a concentration of 25µmol/L to the culture medium prevented the effects of TNF (50µg/L)

1.8 Anti-Inflammatory and Anticarcinogenic Properties

A significant anti-inflammatory effect of silymarin has been described in liver tissue. Studies have shown that silymarin exerts a number of effects, including inhibition of neutrophil migration, inhibition of Kupffer cells, marked inhibition of leukotriene synthesis and formation of prostaglandins.[13,47,48,49]

The protection afforded by silymarin against carcinogenic agents has been studied in various experimental animal models. A series of experiments have been performed in nude mice with nonmelanoma skin cancer produced by UVB radiation, studying its initiation, promotion and complete carcinogenesis. In all the stages studied, silymarin applied onto the skin at different doses appeared to reduce significantly the incidence, multiplicity and volume of tumours per animal. Furthermore, in a short-term experiment (using the same experimental model), the application of silymarin significantly reduced apoptosis, skin oedema, depletion of catalase activity and induction of cyclo-oxygenase and ornithine decarboxylase activity. This effect provides protection against photocarcinogenesis.[50] Similar results were also obtained in the model of skin carcinogenesis produced by chemical carcinogenic agents in carcinogenesis-sensitive (SENCAR) mice.[51,52]

The molecular bases of the anti-inflammatory and anticarcinogenic effects of silymarin are not yet known; they might be related to the inhibition of the transcription factor NF- B, which regulates the expression of various genes involved in the inflammatory process, in cytoprotection and carcinogenesis.[53,54,55] It has also been hypothesised that silymarin may act by modulating the activation of regulating substances of the cellular cycle and of mitogen-activated protein kinase.[56]

1.9 Antifibrotic Effects

Stellate hepatocytes have a crucial role in liver fibrogenesis. In response to fibrogenic influences (for example protracted exposure to ethanol or carbon tetrachloride), they proliferate and transform into myofibroblasts responsible for the deposition of collagen fibres in the liver. Recently, the effects of silibinin on the transformation of stellate cells into myofibroblasts have been investigated. The results have shown that silibinin, at a concentration of 100µmol/L reduces the proliferation of stellate cells isolated from fresh liver of rats by about 75%, reduces the conversion of such cells into myofibroblasts, and downregulates gene expression of extracellular matrix components indispensable for fibrosis.[57]

Furthermore, it has been demonstrated that silymarin improves hepatic fibrosis in vivo in rats subjected to complete occlusion of the biliary duct, a manoeuvre that causes progressive hepatic fibrosis without inflammation. Silymarin, administered at a dosage of 50 mg/kg/day for 6 weeks, is able to reduce fibrosis by 30 to 35% as compared with controls. A dose of 25 mg/kg/day is not effective.[58]

Colchicine and silymarin, administered at a dose of 50 mg/kg orally for 55 days, were able to prevent completely all the alterations induced by carbon tetrachloride in rats (peroxidation of lipids, Na+ ,K+ -and Ca2+ -ATPase), except for the hepatic content of collagen, which was reduced only by 55% as compared with controls; moreover, alkaline phosphatase and ALT were unchanged as compared with controls. In the group of rats treated with silymarin, the loss of glycogen was inhibited completely.[59]

1.10 Inhibition of Cytochrome P450

Silymarin can inhibit the hepatic cytochrome P450 (CYP) detoxification system (phase I metabolism). It has been shown recently in mice that silibinin is able to inhibit numerous hepatic CYP enzyme activities,[60] whereas other researchers have not detected any effect of silymarin on the CYP system.[61,62,63]

This effect could explain some of the hepatoprotective properties of silymarin, especially against the intoxication due to A phalloides. The Amanita toxin becomes lethal for hepatocytes only after having been activated by the CYP system. Inhibition of toxin bioactivation may contribute to the limitation of its toxic effects. Additionally, silymarin, together with other antioxidant substances, could contribute towards protection against free radicals generated by enzymes of the CYP system.

1.11 Overview of Mechanisms of Action

The hepatoprotection provided by silymarin appears to rest on four properties:

  • activity against lipid peroxidation as a result of free radical scavenging and the ability to increase the cellular content of GSH;

  • ability to regulate membrane permeability and to increase membrane stability in the presence of xenobiotic damage;

  • capacity to regulate nuclear expression by means of a steroid-like effect; and

  • inhibition of the transformation of stellate hepatocytes into myofibroblasts, which are responsible for the deposition of collagen fibres leading to cirrhosis.

Silymarin and silibinin inhibit the absorption of toxins, such as phalloidin or -amanitin, preventing them from binding to the cell surface and inhibiting membrane transport systems. Furthermore, silymarin and silibinin, by interacting with the lipid component of cell membranes, can influence their chemical and physical properties. Studies in erythrocytes, mast cells, leucocytes, macrophages and hepatocytes have shown that silymarin renders cell membranes more resistant to lesions (figure 2).[1,2,13]

Furthermore, the well documented scavenging activity of silymarin and silibinin can explain the protection afforded by these substances against hepatotoxic agents. Silymarin and silibinin may exert their action by acting as free radical scavengers and interrupting the lipid peroxidation processes involved in the hepatic injury produced by toxic agents. Silymarin and silibinin are probably able to antagonise the depletion of the two main detoxifying mechanisms, GSH and superoxide dismutase (SOD), by reducing the free radical load, increasing GSH levels and stimulating SOD activity.

Furthermore, silibinin probably acts not only on the cell membrane, but also on the nucleus, where it appeared to increase ribosomal protein synthesis by stimulating RNA polymerase I and the transcription of rRNA.[13,34,44] The stimulation of protein synthesis is an important step in the repair of hepatic injury and is essential for restoring structural proteins and enzymes damaged by hepatotoxins.[1,2]

Figure 2.

Mechanism of action of silymarin as proposed by Valenzuela and Garrido.[1]

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