1. Pliny the Elder, Historia Naturalis 77 A.D.

2. Culpeper N. A Physical Directory: or a Translation of the Dispensatory made by the College of Physicians of London. London, England: Peter Cole; 1650.

3. Morazzoni P, Montalbetti A, Malandrino S. Comparative pharmacokinetics of silipide and

silymarin in rats. Eur J Drug Metab Pharmacokinet 1993;18:289-297.

Inverni della Beffa Research and Development Laboratories, Milan, Italy.

The plasma level profile and the biliary excretion of silybin, the main flavanolignan component of silymarin, were evaluated in rats after single equimolar oral doses (200 mg/kg, expressed as silybin equivalents) of the silybin-phosphatidylcholine complex silipide (laboratory code IdB 1016) and of silymarin. Silybin was assayed by using a specific HPLC method which allowed also the determination of other flavanolignans present in the biological fluids after administration of silymarin (i.e. silydianin, silycristin and isosilybin). After oral silipide, silybin reached peak plasma levels within 2 h, with a Cmax of 9.0 +/- 3.0 micrograms/ml for unconjugated drug and 93.4 +/- 16.7 micrograms/ml for total (free + unconjugated drug). Maximum total biliary concentrations of silybin (2989 +/- 568 micrograms/ml) were observed within 2 h and the biliary recovery after 24 h accounted for about 13% of the administered amount. After administration of silymarin, unconjugated and total plasma silybin levels as well as biliary excretion were several-fold lower than those observed after treatment with silipide. Silybin recovered over a 24 h period after silymarin intake accounted for about 2% of the administered dose. Plasma and bile obtained after administration of silymarin contained also silydianin, silycristin and, to a greater extent, isosilybin. The concentrations of the latter compound in plasma and in bile were higher than those of silybin itself. The relative bioavailability of silipide (calculated in the target organ as the ratio between AUCs of cumulative biliary excretion curves) was 10-fold higher than that of silymarin.

4. Schandalik R, Gatti G, Perucca E, et al. Pharmacokinetics of silybin in bile following

administration of silipide and silymarin in cholecystectomy patients. Arzneimittelforschung 1992;42:964-968.

Surgical Department, Regional Hospital, Braunau am Inn, Austria.

The biliary excretion of silybin, the main active component of silymarin, was evaluated by using a specific HPLC method in 9 cholecystectomy patients with T-tube drainage following single oral doses of silipide (CAS 134499-06-2), a lipophilic silybin-phosphatidylcholine complex (IdB 1016), and of silymarin (120 mg, expressed as silybin equivalents). After intake of silipide, the concentration of silybin in bile reached a peak within 4 h and declined thereafter with a mean time of about 10 h. After administration of silymarin, biliary silybin concentrations were several-fold lower than those observed after intake of silipide. The bile collected after silymarin intake also contained considerable amounts of isosilybin (a silybin isomer) and very low levels of silydianin and silycristin. The amount of silybin recovered in bile in free and conjugated form within 48 h accounted for 11% of the dose after silipide and for 3% of the dose after silymarin. Plasma silybin concentrations, determined in 3 subjects, were several-fold lower than those in bile after intake of silipide and mostly undetectable after intake of silymarin. These data indicate that the bioavailability of silybin is much greater after administration of silipide than after administration of silymarin. This results in increased delivery of the compound to the liver, which represents the target organ for pharmacological action.

5. Weyhenmeyer R, Mascher H, Birkmayer J, et al. Study on dose-linearity of the pharmacokinetics of silibinin diastereomers using a new stereospecific assay. Int J Clin Pharmacol Ther Toxicol 1992;30:134-138.

MADAUS AG, Koln, Germany.

Silibinin in single doses of 102, 153, 203 and 254 mg was applied as silymarin in capsules (Legalon 140) to 6 healthy male volunteers. Using a newly developed HPLC method, both diastereomers of silibinin were assayed in plasma as unconjugated compounds as well as total isomers after hydrolysis. In the dose range studied, the areas under the curves correlate linearly with the dose. On average, only 10% of total silibinin in plasma is in the unconjugated form. The ratio of the silibinin isomers is reversed, if unconjugated and total isomers are compared. For unconjugated silibinin, the half-lives are less than one hour, but the terminal half-life has probably not been observed, because already after 4-6 hours the levels fell below the limit of determination of 2.5 ng diastereomer/ml. For total silibinin, an elimination half-life of approximately 6 h is estimated. About 5% of the dose is excreted into urine as total silibinin, corresponding to a renal clearance of approximately 30 ml/min. No adverse events were noted, showing that silymarin even in high doses, up to 5 capsules of Legalon 140, is well tolerated.

6. Vogel G, Tuchweber B, Trost W. Protection by silibinin against Amanita phalloides intoxication in beagles. Toxicol Appl Pharmacol 1984;73:355-362.

A single oral dose of the lyophilized deathcap fungus Amanita phalloides (85 mg/kg body wt) caused gastrointestinal signs of diarrhea, retching, and vomiting in beagles after a latent period of 16 hr. The pathologic lesions; the increases in serum transaminase (GOT, GPT), alkaline phosphatase, and bilirubin, as well as the fall in prothrombin time all indicated that liver damage was maximal at about 48 hr after poisoning. Four of twelve dogs given A. phalloides died with signs of hepatic coma within 35 to 54 hr with the biochemical values in the survivors reverting to normal by the ninth day. Silibinin administration (50 mg/kg) 5 and 24 hr after intoxication suppressed the serum changes and the fall in prothrombin time. The degree of hemorrhagic necrosis in the liver was markedly reduced, and none of the silibinin-treated dogs died.

7. Desplaces A, Choppin J, Vogel G. The effects of silymarin on experimental phalloidine

poisoning. Arzneimittelforschung 1975;25:89-96.

The hepatoprotective action of silymarin, the active principle extracted from the fruit of Silybum marianum (L.) Gaertn., in animals (dogs, rabbits, rats, mice) intoxicated with phalloidine is evident, both after protective and curative treatment. A dose of 15 mg/kg of silymarin protects every animal when given 60 min before the toxin. When injected 10 mim after phalloidine, a dose of 100 mg/kg of silymarin again provides total protection. However, as the time span between administration of the toxic substance and start of treatment increases, so the efficacy of silymarin decreases; after 30 min its curative effect is negligible. The histochemical and histoenzymological studies show that during intoxication of the mice by phalloidine, silymarin inhibits the effect of the toxic substance and regulates the functions of the hepatocyte, when given either 60 min before or 10 min after phalloidine.

8. Hruby K, Csomos G, Fuhrmann M, Thaler H. Chemotherapy of Amanita phalloides poisoning with intravenous silibinin. Hum Toxicol 1983;2:183-195.

1 A total of 18 cases of Amanita phalloides intoxication was treated by combined chemotherapy during 1980 and 1981. After attempted primary elimination of the toxin all patients received silibinin as basic therapy mainly by infusion and in two instances orally. 2 In order to evaluate the effect of silibinin therapy a retrospective study of the followed-up case records was made. The cases were arbitrarily classified into three groups according to the severity of liver damage (light, medium and severe). 3 There was found a close relationship between the severity of liver injury and the delay between mushroom ingestion and the onset of silibinin therapy. With the exception of one fatality in a particularly high dosage suicidal intoxication, all patients survived. 4 Administration of silibrinin even up to 48 h after mushroom ingestion appears to be an effective measure to prevent severe liver damage in Amanita phalloides poisoning. Contrarily, the onset of general supportive treatment together with penicillin therapy which was throughout several hours before silibinin administration did not correlate with the severity of liver damage.

9. Sabeel AI, Kurkus J, Lindholm T. Intensive hemodialysis and hemoperfusion treatment of Amanita mushroom poisoning. Mycopathologia 1995;131:107-114.

Department of Nephrology, University Hospital, Lund, Sweden.

Over a period of fifteen years, 41 patients including 23 males and 18 females with Amanita mushroom poisoning were treated at the University Hospital of Lund, Sweden. The intensity of poisoning was graded according to serum transaminase elevations and prothrombin time reductions. Severity was mild in 16 patients (Group A), moderate in 14 (Group B) and severe in 11 (Group C). Members of Group C reported shorter latency periods before the onset of symptoms, (10 +/- 1 hours, P < 0.05) and longer delays in treatment, (34 +/- 4 hours), than did the other patients. Intensive treatment was begun before the results of urine amatoxin assay were reported. Treatment consisted of: fluid and electrolyte replacement, oral activated charcoal and lactulose, i.v. penicillin, combined hemodialysis and hemoperfusion in two 8 hour sessions, some received i.v. thioctic acid, other i.v. silibinin, all received a special diet. This combination of treatment modalities was used to accelerate the elimination of amatoxin from the patients' bodies. The longest period of hospitalization, 13 +/- 2 days, was required by the patients of Group C (p < 0.01). All patients improved and were discharged from the hospital asymptomatic. No sequelae were later reported for the majority of those moderately and severely poisoned. We have concluded that intensive combined treatment applied in these cases is effective in relieving patients with both moderate and severe amanitin poisoning.

10. Carducci R, Armellino NIF, Volpe C, et al. Silibinin and acute poisoning with Amanita phalloides . Minerva Anestesiol 1996;62:187-193.

Cattedra di Tossicologia Ospedale A. Cardarelli, Universita degli Studi di Napoli Federico II.

The aim of the present study was to show the therapeutic effect of silibinin dihemisuccinate in a case of intoxication by mushrooms of Amanita gender. We report a clinical case of a 4-person family intoxicated by ingestion of mushrooms Amanita phalloides and admitted to the center for poisoning treatment of the Hospital "A. Cardarelli" in Naples. Although all were treated with standard therapy, there was a worsening of the clinical picture till the third day, when it was decided to add silibinin dihemisuccinate by the intravenous route to the therapy. After the beginning of silibinin administration the patients showed a favourable course with a rapid resolution of the clinical picture, although the prognosis appeared severe on the basis of hematochemical examination results. On day 9 silibinin dihemisuccinate was replaced with silibinin betacyclodextrine per os. All patients were discharged on day 10-13. After two months all hematological parameters are in the normal range also a hepatobiliopancreatic echography does not show any morphological alteration. As in the case of polytherapies and because of the lack of comparative studies, it seems difficult to establish which therapeutic component had the major role in the resolution of the clinical picture. However, on the basis of our experience, and of the literature data, we think that silibinin may play a significant role in protecting hepatic tissue not yet injured. However we believe that other studies are necessary to confirm our hypothesis.

11. Rambousek V, Janda I, Sikut M, et al. Severe Amanita phalloides poisoning in a 7-year-old girl. Cesk Pediatr 1993;48:332-333.

I. detska klinika FN v Motole, Praha.

Authors describe a case of a very severe poisoning, the prothrombin-time was less than 10% of the normal value and the child developed hepatic coma. We suppose, the favourable outcome may have been influenced by the treatment with Silymarin in combination with high doses of G-Penicillin.

Publication Types:

•  Case Reports

12. Salmi HA, Sarna S. Effect of silymarin on chemical, functional, and morphological alterations of the liver; A double blind controlled study. Scand J Gastroenterol 1982;17:517-521.

One hundred and six consecutive patients with liver disease were selected on the basis of elevated serum transaminase levels. The patients were randomly allocated into a group treated with silymarin (treated) and a group receiving placebo (controls). Ninety-seven patients complete the 4-week trial-47 treated and 50 controls. In general, the series represented a relatively slight acute and subacute liver disease, mostly induced by alcohol abuse. There was a statistically highly significantly greater decrease of S-SGPT (S-ALAT) and S-SGOT (S-ASAT) in the treated group than in controls. Serum total and conjugated bilirubin decreased more in the treated than in controls, but the differences were not statistically significant. BSP retention returned to normal significantly more often in the treated group. The mean percentage decrease of BSP was also markedly higher in the treated. Normalization of histological changes occurred significantly more often in the treated than in controls.

Publication Types:

•  Clinical Trial

•  Randomized Controlled Trial

13. Buzzelli G, et al. A pilot study on the liver protective effect of silybin-phosphatidylcholine complex (1dB 1016) in chronic active hepatitis. Int J Clin Pharmacol Ther Toxicol 1993;31:456-460.

Istituto di Clinica Medica II, Universita degli Studi di Firenze, Italy.

In order to assess the liver protective activity and the antioxidant properties of a new silybin complex (IdB1016), we carried out a short-term pilot study on 20 patients with chronic active hepatitis (CAH), randomly assigned to 240 mg of silybin b.i.d. (10 patients, 4 m/6 f, mean age: 50 years) or placebo (10 patients, 2 m/8 f, mean age: 55 years). Blood samples were collected before and after 7 days of treatment for liver function tests (LFTs), malonaldehyde (MDA) as an index of lipid peroxidation, and copper (Cu) and zinc (Zn), two trace elements involved in protecting cells against free radical-mediated lipid peroxidation. In the treated group, there was a statistically significant reduction of mean (+/- SEM) serum concentrations of aspartate aminotransferase (AST) from 88.0 (+/- 13.3) to 65.9 (+/- 7.5) u/l, (p < 0.01), of alanine aminotransferase (ALT) from 115.9 (+/- 12.9) to 82.5 (+/- 10.6) u/l (p < 0.01), of gamma-glutamyltranspeptidase (gamma-GT) from 51.4 (+/- 9.3) to 41.3 (+/- 4.2) u/l (p < 0.02) and of total bilirubin (TB) from 0.76 (+/- 0.08) to 0.53 (+/- 0.04) mg/dl (p < 0.05). Alkaline phosphatase (AP) fell slightly from 143.4 (+/- 6.4) to 137.5 (+/- 7.8) u/l. There were no significant changes in MDA, Cu or Zn serum concentrations. These results show that IdB1016 may improve LFTs related to hepatocellular necrosis and/or increases membrane permeability in patients affected by CAH.

Publication Types:

•  Clinical Trial

•  Randomized Controlled Trial

14. Feher I, Deak G, Muzes G. Liver-protective action of silymarin therapy in chronic alcoholic liver diseases. Orv Hetil 1989;130:2723- 2727.

The effects of silymarin (Legalon) therapy on liver function tests, serum procollagen III peptide level and liver histology were studied in 36 patients with chronic alcoholic liver disease in a six month double blind clinical trial. During silymarin treatment serum bilirubin, aspartate aminotransferase and alanin-aminotransferase values have been normalized, while gamma-glutamyl transferase activity and procollagen III peptid level decreased. The changes were significant, and there was a significant difference between post-treatment values of the two groups, as well. In the placebo group only gamma-glutamyl transferase values decreased significantly but to a lesser extent than that in the silymarin group. The histological alterations showed an improvement in the silymarin group, while remained unchanged in the placebo group. These results indicate that silymarin exerts hepatoprotective activity and is able to improve liver functions in alcoholic patients.

Publication Types:

•  Clinical Trial
•  Controlled Clinical Trial
•  Review
•  Review, Tutorial

15. Ferenci P, Dragosics B, Dittrich H, et al. Randomized controlled trial of silymarin treatment in patients with cirrhosis of the liver. J Hepatol 1989;9:105-113.

Silymarin, the active principle of the milk thistle Silybum marianum, protects experimental animals against various hepatotoxic substances. To determine the effect of silymarin on the outcome of patients with cirrhosis, a double blind, prospective, randomized study was performed in 170 patients with cirrhosis. 87 patients (alcoholic 46, non-alcoholic 41; 61 male, 26 female; Child A, 47; B, 37; C, 3; mean age 57) received 140 mg silymarin three times daily. 83 patients (alcoholic 45, non-alcoholic 38; 62 male, 21 female; Child A, 42; B, 32; C, 9: mean age 58) received a placebo. Non-compliant patients and patients who failed to come to a control were considered as 'drop outs' and were withdrawn from the study. All patients received the same treatment until the last patient entered had finished 2-years of treatment. The mean observation period was 41 months. There were 10 drop outs in the placebo group and 14 in the treatment group. In the placebo group, 37 (+2 drop outs) patients had died, and in 31 of these, death was related to liver disease. In the treatment group, 24 (+4 drop outs) had died, and in 18 of these, death was related to liver disease. The 4-year survival rate was 58 +/- 9% (S.E.) in silymarin-treated patients and 39 +/- 9% in the placebo group (P = 0.036). Analysis of subgroups indicated that treatment was effective in patients with alcoholic cirrhosis (P = 0.01) and in patients initially rated 'Child A' (P = 0.03). No side effects of drug treatment were observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Publication Types:

•  Clinical Trial
•  Randomized Controlled Trial

16. Trinchet IC, Coste T, Levy VG. Treatment of alcoholic hepatitis with silymarin. A doubleblind comparative study in 116 patients. Gastroenterol Clin Biol 1989;13:120-124.

Services d'Hepato-Gastroenterologie et d'Anatomopathologie, Hopital Jean Verdier, Bondy.

A randomized double-blind trial of silymarin versus placebo was carried out in 116 patients with histologically proven alcoholic hepatitis, 58 of them with cirrhosis. Patients were not included in case of hepatic encephalopathy, contraindication to percutaneous liver biopsy, hepatocellular carcinoma, evident lack of discipline or refusal to enter the trial. Fifty-seven patients received silymarin orally 420 mg/day and 59 received placebo during 3 months. Biologic parameters were assessed in the serum, and a percutaneous liver biopsy was obtained at the start of the trial and 3 months later. Histologic scores of alcoholic hepatitis and fibrosis were established on each biopsy specimen by two independent pathologists. The 2 groups were comparable at inclusion; 26 p. 100 of patients were lost to follow-up at 3 months, abstinence was obtained in 46 p. 100 of patients at the end of the trial. These percentages were similar in the two groups. Four patients died of hepatic failure during the trial, 3 in the placebo group. Significant improvement in the score of alcoholic hepatitis and serum amino transferase activity, was noted in both groups during the trial, irrespective of treatment with silymarin or placebo. No side-effects were noted. Our results suggest that silymarin 420 mg/d is not clinically relevant in the treatment of moderate alcoholic hepatitis.

Publication Types:

•  Clinical Trial
•  Randomized Controlled Trial

17. Varga M, Buris L, Fodor M. Ethanol elimination in man under influence of hepatoprotective silibinin. Blutalkohol 1991;28:405-408.

Institute of Forensic Medicine, Medical University of Debrecen, Hungary.

The effect of a single dose of hepatoprotective silibinin on blood alcohol elimination was investigated. Neither influence on the blood alcohol curve, nor detectable increase in the beta 60 value was found, although biochemical considerations suggest such an effect. Though silibinin has a protective effect on chronic alcohol liver injury, it does not influence acute alcohol elimination, and is therefore not suitable for use as a "sobering-up" agent.

18. Muriel P, Mourelle M. Prevention by silymarin of membrane alterations in acute CCl4 liver damage. J Appl Toxicol 1990;10:275-279.

Departamento de Farmacologia y Toxicologia, Centro de Investigacion yde Estudios Avanzados del Instituto Politecnico Nacional, Mexico, D.F.

The effect of silymarin on liver lipid peroxidation and membrane lipid alterations induced by an acute dose of CCl4 was studied. Four groups of animals were treated with CCl4, CCl4 + silymarin, silymarin and its vehicles. CCl4 was given orally (0.4 g 100 g-1 body wt.) and silymarin was administered i.p. All animals were sacrificed 24 h after the treatments. Liver lipid peroxidation was measured and plasma membranes were isolated. Alkaline phosphatase (AP) and gamma-glutamyl transpeptidase (GGTP) were measured in plasma membranes. Membrane lipids were extracted and then analysed by thin-layer chromatography by measuring the phosphorus of the phospholipids in each spot. Liver lipid peroxidation was increased about three times in the group receiving CCl4 only. Silymarin cotreatment prevented this increase. Phosphatidylethanolamine (PEA) decreased, while phosphatidylinositol (PI) increased in the plasma membranes isolated from the CCl4-treated group. Animals that received CCl4 + silymarin showed no decrease in PEA content. A partial prevention of the decrease in phosphatidylinositol content was also observed in plasma membranes of animals treated with silymarin in addition to CCl4. CCl4 decreased gamma-glutamyl transpeptidase (GGTP) and alkaline phosphatase (AP) membrane activities. Silymarin cotreatment prevented the AP (completely) and the GGTP (partially) falls caused by CCl4. Silymarin by itself increased AP membrane activity. A significant relationship between the membrane content of phosphatidylethanolamine (PEA) and the AP activity was observed in plasma membranes of treated animals and in normal liver membranes enriched with PEA. These results indicate that silymarin can protect against the alterations induced by CCl4 on the liver plasma membrane through its antioxidant properties by modifying the plasma membrane phospholipid content.

19. Bosisio E, Benelli C, Pirola O, et al. Effect of the flavanolignans of Silybum marianum L . on lipid peroxidation in rat liver microsomes and freshly isolated hepatocytes. Pharmacol Res 1992;25:147-154.

The effect of several flavanolignans (silicristin, silidianin, silybin and isosilybin) present in silymarin, the extract of Silybum marianum fruits, was tested on lipid peroxidation in rat liver microsomes and freshly isolated hepatocytes. In microsomes lipid peroxidation was generated by ADP/Fe2+ and NADPH. All flavanolignans inhibited peroxidation in a concentration dependent manner. In hepatocytes lipid peroxidation was induced by ADP/Fe3+ complex and cell damage was evaluated as LDH activity released in the medium. The inhibition of the peroxidative process by flavanolignans was also evident in this model, even if with a potency order different from that found in microsomes. In contrast, the effect on LDH release was significant only for silybin and isosilybin, the other compounds being inactive on this parameter.

20. Muriel P, Garciapina T, Perez-Alvarez V, et al. Silymarin protects against paracetamol induced lipid peroxidation and liver damage. J Appl Toxicol 1992;12:439-442.

Departamento de Farmacologia y Toxicologia, Instituto Politecnico Nacional, Mexico D.F.

The effect of silymarin on liver damage induced by acetaminophen (APAP) intoxication was studied. Wistar male rats pretreated (72 h) with 3-methylcholanthrene (3-MC) (20 mg kg-1 body wt. i.p.) were divided into three groups: animals in group 1 were treated with acetaminophen (APAP) (500 mg kg-1 body wt. p.o.), group 2 consisted of animals that received APAP plus silymarin (200 mg kg-1 body wt. p.o.) 24 h before APAP, and rats in group 3 (control) received the equivalent amount of the vehicles. Animals were sacrificed at different times after APAP administration. Reduced glutathione (GSH), lipid peroxidation and glycogen were measured in liver and alkaline phosphatase (AP), gamma-glutamyl transpeptidase (GGTP) and glutamic pyruvic transaminase (GPT) activities were measured in serum. After APAP intoxication, GSH and glycogen decreased very fast (1 h) and remained low for 6 h. Lipid peroxidation increased three times over the control 4 and 6 h after APAP treatment. Enzyme activities increased 18 h after intoxication. In the group receiving APAP plus silymarin, levels of lipid peroxidation and serum enzyme activities remained within the control values at any time studied. The fall in GSH was not prevented by silymarin, but glycogen was restored at 18 h. It was concluded that silymarin can protect against APAP intoxication through its antioxidant properties, possibly acting as a free-radical scavenger.

21. Halim AB, et al. Biochemical effect of antioxidants on lipids and liver function in experimentally-induced liver damage. Ann Clin Biochem 1997;34:656-663.

Tumor Marker Oncology Research Center, Al-Azhar University, Cairo, Egypt.

Recent studies demonstrated the role of antioxidants in preventing organ damage caused by free radicals. The present study was conducted to find out the modulatory effect of some antioxidants on lipid patterns in experimentally-induced liver damage. Rats chronically intoxicated with carbon tetrachloride (CCl4) were used as a model of liver injury terminating with fibrosis or cirrhosis. One hundred and sixty six albino rats were classified into five groups: one served as a control group; the second was subjected to oral administration of CCl4 (200 microL/100 g body weight) twice a week; the other three groups, in addition to CCl4, received oral doses of silymarin (30 mg/kg), vitamin E (200 IU/kg) and vitamin C (50 mg/kg) respectively. At the end of the experiment, the animals were killed, blood was collected and liver was taken for histopathological examination. Liver function tests, disturbed by CCl4 were significantly modulated by antioxidants, and histopathological examination showed that antioxidants ameliorated the necrotic and fibrotic changes caused by CCl4. Treatment with antioxidants was also shown to modulate the toxic effect of CCl4 on the lipid profile and malondialdehyde content. Administration of antioxidants could play an important role in prophylaxis against lipid peroxidation and consequently liver fibrosis caused by free radicals.

22. Chrungoo VJ, et al. Silymarin mediated differential modulation of toxicity induced by carbon tetrachloride, paracetamol and D-galactosamine in freshly isolated rat hepatocytes. Indian J Exp Biol 1997;35:611-617.

Biochemistry Section, Regional Research Laboratory (CSIR), Jammu Tawi, India.

Influence of silymarin on the modulation of hepatotoxicity induced by carbon tetrachloride (CCl4), paracetamol (AAP) and D-galactosamine (GalN) was examined in freshly isolated rat hepatocytes in suspension culture. While the three hepatotoxicants produced differential biochemical response, the flavone was able to restore biochemical alterations only in hepatocytes exposed to CCl4 and AAP induced toxicity. Silymarin at 0.4 mM was able to counteract lipid peroxidation and enzyme leakage induced by 3 mM CCl4 The flavone also offered protection by more than 60% in hepatocytes isolated from PB pre-treated rats where CCl4 at 2 mM produced enhanced toxicity over hepatocytes isolated from untreated control rats. Similarly, the flavone protected AAP-induced GSH depletion by more than 75% in hepatocytes isolated from untreated and 3-methylcholanthrene treated rats. However, instead of protecting GalN-induced depletion of UDP-glucuronic acid in hepatocytes, the flavone itself reduced the nucleotide content very rapidly compared to GalN, the later exerted time dependent effect. Silymarin at 0.4 mM reduced UDPGA by more than 60%. The results suggested that freshly isolated hepatocytes in suspension culture offer a simple and convenient method for evaluation of pharmaceutical agents of antihepatotxic potentials against various hepatotoxicants.

23. Campos R, et al. Silybin dihemisuccinate protects against glutathione depletion and lipid peroxidation induced by acetaminophen on rat liver. Planta Med 1989;55:417-419.

Acetaminophen hepatotoxicity is characterized by glutathione depletion, cellular necrosis, and, in some instances, by the induction of lipid peroxidation. Silybin dihemisuccinate, a soluble form of the flavonoid silymarin, protects rats against liver glutathione depletion and lipid peroxidation induced by acute acetaminophen intoxication. Other biochemical parameters such as serum transaminases did not show the drastic increase observed under acetaminophen intoxication when animals were treated with the flavonoid. Preliminary results suggest that silybin dihemisuccinate may be another antidote against acetaminophen hepatotoxicity.

24. Wu CG, Chamuleau RA, Bosch KS. Protective effect of silymarin on rat liver injury induced by ischemia. Virchows Arch B Cell Pathol Incl Mol Pathol 1993;64:259-263.

Department of Experimental Internal Medicine, Academic Medical Centre, Amsterdam, The Netherlands.

The effect of silymarin on liver cell damage induced by ischemia was studied in rats fasted for 24 h. In the first series of experiments in vitro ischemia was induced by storing tissue blocks in closed vials at 37 degrees C for 15, 30, 45 and 60 min. Cell injury was detected by the cytophotometrical measurement of glycogen phosphorylase activity in unfixed cryostat sections demonstrated by a modified histochemical procedure. In the second series of experiments in vivo ischemia was provoked by clamping the afferent vessels to the median and left lateral lobes of the liver for 60 min, followed by removal of the clamp and reperfusion. The extent of cell damage was determined by measuring the ALAT and ASAT activities in serum at 1, 3, 6 and 24 h after ischemia and by quantifying the extent of necrosis in the liver after 24 h reperfusion by measuring the unstained areas in cryostat sections incubated for lactate dehydrogenase activity. Silymarin (100 mg/kg b.w.) was administered intravenously at 5 min before both the induction of ischemia and the restoration of blood flow (in vivo ischemia) and at 1 h and at 5 min before sacrifice (in vitro ischemia). Controls received an equal amount of saline. The serum amino-transferase activities after 24 h reperfusion were significantly reduced in the silymarin-treated group (n = 10); ALAT 293 +/- 193 U/L, ASAT 343 +/- 229 U/L compared with the control group (n = 7): ALAT 1238 +/- 743 U/L, ASAT 948 +/- 541 U/L (p < 0.03), and the extent of necrosis decreased from 25.6 +/- 16.0% ( n = 7) to 7.8 +/- 8.3% (n = 10) (p < 0.01) after treatment with silymarin.(ABSTRACT TRUNCATED AT 250 WORDS)

25. Kropacova K, Misurova E, Hakova H. Protective and therapeutic effect of silymarin on the development of latent liver damage. Radiats Biol Radioecol 1998;38:411-415.

Department of Cellular and Molecular Biology Faculty of Sciences, University of P. J. Safarik, Kosice, Slovakia.

Radioprotective and therapeutical effect of silymarin (Flavobion) on development and repair of latent injury in rat liver was examined by its application during the continual gamma irradiation (dose rates 0.2 and 0.6 Gy/day) or after acute gamma irradiation (dose 6 Gy). Silymarin influence was evaluated on the basis of mitotic index and chromosomal aberration frequency in the liver regenerating after partial hepatectomy. We have found that silymarin application stimulates the process of liver regeneration in non-irradiated rats as well as in irradiated ones. Positive effect of silymarin (100 mg per kg p.o. ones per day) was manifested at both dose rates of continual irradiation with increase in mitotic activity and mitigation of chromosomal erration frequency in the regenerating liver in comparison with non-protected irradiated animals. Curative effect of silymarin (70 mg/kg p.o., twice per day) was shown especially after 14 days of its postradiation application.

26. Pietrangelo A, Borella F, Casalgrandi G. Antioxidant activity of silybin in vivo during long-term iron overload in rats. Gastroenterology 1995;109:1941-1949.

Dipartimento di Medicina Interna, University of Modena, Italy.

BACKGROUND & AIMS: Hepatic iron toxicity may be mediated by free radical species and lipid peroxidation of biological membranes. The antioxidant property of silybin, a main constituent of natural flavonoids, was investigated in vivo during experimental iron overload. METHODS: Rats were fed a 2.5% carbonyl-iron diet and 100 mg.kg body wt-1.day-1 silybin for 4 months and were assayed for accumulation of hepatic lipid peroxidation by-products by immunocytochemistry, mitochondrial energy-dependent functions, and mitochondrial malondialdehyde content. RESULTS: Iron overload caused a dramatic accumulation of malondialdehyde-protein adducts into iron-filled periportal hepatocytes that was decreased appreciably by silybin treatment. The same beneficial effect of silybin was found on the iron-induced accumulation of malondialdehyde in mitochondria. As to the liver functional efficiency, mitochondrial energy wasting and tissue adenosine triphosphate depletion induced by iron overload were successfully counteracted by silybin. CONCLUSIONS: Oral administration of silybin protects against iron-induced hepatic toxicity in vivo. This effect seems to be caused by the prominent antioxidant activity of this compound.

27. McPartland JM. Viral hepatitis treated with Phyllanthus amarus and milk thistle (Silybum marianum) : A case report. Complementary Medicine International 1996;March:40-42.

28. Basaga H, et al. Free radical scavenging and antioxidative properties of 'silibin' complexes on microsomal lipid peroxidation. Cell Biochem Funct 1997;15:27-33.

Department of Food Engineering, Middle East Technical University, Ankara, Turkey.

The antioxidant properties of silibin complexes, the water-soluble form silibin dihemisuccinate (SDH), and the lipid-soluble form, silibin phosphatidylcholine complex known as IdB 1016, were evaluated by studying their abilities to react with the superoxide radical anion (O2-.), and the hydroxyl radical (OH.). In addition, their effect on pulmonary and hepatic microsomal lipid peroxidation had been investigated. Superoxide radicals were generated by the PMS-NADH system and measured by their ability to reduce NBT. IC50 concentrations for the inhibition of the NBT reduction by SDH and IdB 1016 were found to be 25 microM and 316 microM respectively. Both silibin complexes had an inhibitory effect on xanthine oxidase activity. SDH reacted rapidly with OH radicals at approximately diffusion controlled rate and the rate constant was found to be (K = 8.2 x 10(9) M-1 s-1); it appeared to chelate Fe2+ in solution. In hepatic microsomes, when lipid peroxidation was induced by Fe2+, SDH inhibited by 39.5 per cent and IdB 1016 by 19.5 per cent, whereas when lipid peroxidation was induced by CuOOH, IdB 1016 exerted a better protective effect than SDH (29.4 per cent and 19.4 per cent inhibition, respectively). In both microsomal systems lipid peroxidation proceeded through a thiol depletion mechanism which could be restored in the presence of silibin complexes. Low levels of lipid peroxidation in pulmonary microsomes point out the differences between in-vitro lipid peroxidation occurring in microsomes of different tissues. The results support the free radical scavenger and antioxidative properties of silibin when it is complexed with a suitable molecule to increase its bioavailability.

29. Rui YC. Advances in pharmacological studies of silymarin. Mem Inst Oswaldo Cruz 1991;S2:79-85.

College of Pharmacy, Second Military Medical University, Shanghai, China.

Silymarin is the flavonoids extracted from the seeds of Silybum marianum (L) Gearth as a mixture of three structural isomers: silybin, silydianin and silychristin, the former being the most active component. Silymarin protects liver cell membrane against hepatotoxic agents and improves liver function in experimental animals and humans. It is generally accepted that silymarin exerts a membrane-stabilizing action preventing or inhibiting membrane peroxidation. The experiments with soybean lipoxygenase showed that the three components of silymarin brought about a concentration-dependent non-competitive inhibition of the lipoxygenase. The experiments also showed an analogous interaction with animal lipoxygenase, thus showing that an inhibition of the peroxidation of the fatty acid in vivo was self-evident. Silybin almost completely suppressed the formation of PG at the highest concentration (0.3 mM) and proved to be an inhibitor of PG synthesis in vitro. In our experiments, silybin at lower dose (65 mg/kg) decreased liver lipoperoxide content and microsomal lipoperoxidation to 84.6% and 68.55% of those of the scalded control rats respectively, and prevented the decrease of liver microsomal cytochrome p-450 content and p-nitroanisole-O-demethylase activity 24 h post-scalding. Effects of silymarin on cardiovascular system have been studied in this university since 1980. P. O silymarin 800 mg/kg/d or silybin 600 mg/kg/d reduced plasma total cholesterol, LDL-C and VLDL-C. They however, enhanced HDL-C in hyperlipemic rats. Further studies showed that silymarin enhanced HDL-C but didn't affect HDL-C, a property of this component which is beneficial to treatment of atherosclerosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Publication Types:

•  Review
•  Review, Tutorial

30. Baer-Dubowska W, et al. Inhibition of murine hepatic cytochrome P450 activities by natural and synthetic phenolic compounds. Xenobiotica 1998;28:735-743.

K. Marcinkowski University of Medical Sciences, Department of Pharmaceutical Biochemistry, Poznan, Poland.

1. The effect of the phenolic compounds protocatechuic acid, chlorogenic acid, tannic acid, gallates and silybin on ethoxyresorufin O-dealkylase (CYP1A1), methoxyresorufin O-dealkylase (CYP1A2) and pentoxy-O-dealkylase (CYP2B) was examined in mouse liver microsomes from induced animals. 2. All compounds tested could inhibit cytochrome P450-mediated enzyme activities, but to different extents. Tannic acid was the most potent inhibitor, especially toward EROD activity with an IC50=2.6 microM. Synthetic dodecyl gallate was also relatively selective toward this enzyme activity with an IC50=120 microM. 3. Protocatechuic acid, chlorogenic and silybin were more selective towards PROD and MROD activities. Their relative inhibitory potency for PROD activity was as follows: chlorogenic acid > protocatechuic acid > silybin > dodecyl gallate > propyl gallate. Protocatechuic acid was a more effective inhibitor of MROD activity than chlorogenic acid, and propyl gallate more effective than dodecyl gallate. Thus, no clear structure-activity or selectivity relationship was observed. 4. Analysis of the kinetics of inhibition revealed that the inhibition in most cases was non-competitive in nature.

31. Miguez MP, Anundi I, Sainz-Pardo LA. Hepatoprotective mechanism of silymarin: no evidence for involvement of cytochrome P450 2E1. Chem Biol Interact 1994;91:51-63.

The involvement of the alcohol-inducible cytochrome P450 2E1 in the hepatoprotective mechanism of the plant flavonoid extract silymarin, and its main active component silybin, was investigated in isolated hepatocytes. Allyl alcohol toxicity, associated lipid peroxidation and GSH depletion was efficiently counteracted by silymarin (0.01-0.5 mM), and at higher concentrations by silybin. Cell damage by t-butyl hydroperoxide was also prevented by silymarin and silybin, but less efficiently. However, the covalent binding of the acetaminophen intermediate, formed via P450 2E1, was unaffected by addition of the flavonoids. Silybin did not influence microsomal 2E1-catalyzed demethylation of N-nitrosodimethylamine. Neither did demethylation of N-nitrosodimethylamine or aminopyrine by isolated microsomes affect the in vivo administration of silybin. Addition of silymarin or silybin to primary cultures of isolated hepatocytes did not prevent cell damage induced by exposure to the P450 2E1 substrate CCl4. In contrast, the mere presence of low concentrations (25-50 microM) of these compounds was found to inhibit cell attachment to the matrix and eventually resulted in cell damage. We conclude that contrary to earlier reports we found no evidence for an interaction of silymarin or silybin with cytochrome P450 2E1. This suggests that the antioxidant and free radical scavenging properties may account for most of the therapeutic effect of these compounds. The untoward effect of silymarin on cultured cells may have consequences when considering long-term prescription of this therapeutic agent.

32. Kim DH, Jin YH, Park JB, Kobashi K. Silymarin and its components are inhibitors of beta-glucuronidase. Biol Pharm Bull 1994;17:443-445.

College of Pharmacy, Kyunghee University, Seoul, Korea.

Silymarin, a commercial crude drug used as a hepatoprotective, was found to inhibit 53% of beta-glucuronidase activity at a final concentration of 0.8 mg/ml. Of three compounds A, silybin and C, which were isolated from silymarin, A and silybin potently inhibited the enzyme activity, followed by C. beta-Glucuronidases of intestinal bacteria, HGU-1 and HGU-2, and E. coli HB101 were noncompetitively inhibited by silybin. beta-Glucuronidase of the feces of a healthy human and of a human with colon cancer were also inhibited by silybin, silymarin and saccharic acid 1,4-lactone at 0.03-0.15 mg/ml. Silymarin and silybin protected the increase in enzyme activity in the serum of the rats treated with CCl4.

33. Cabrera C. "Milk Thistle: A clinician's report." Medical Herbalism 1996;6:1-5.

34. Fiebrich F, Koch H. Silymarin, an inhibitor of lipoxygenase. Experientia 1979;35:1548-1560.

35. Sonnenbichler J, et al. Stimulatory effect of silibinin on the DNA synthesis in partially hepatectomized rat livers: non-response in hepatoma and other malignant cell lines. Biochem Pharmacol 1986;35:538-541.

36. Schopen RD, Lange OK, Panne C. Searching for a new therapeutic principle. Experience with hepatic therapeutic agent legalon. Medical Welt 1969;20:888-893.

37. Zi X, et al. Novel cancer chemopreventive effects of a flavonoid antioxidant silymarin: inhibition of mRNA expression of an endogenous tumor promoter TNF-alpha. Biochem Biophys Res Comun 1997;239:334-339.

In this study we describe exceptionally high protective effects of silymarin, a flavonoid antioxidant isolated from milk thistle, against 12-O-tetradecanoylphorbol 13-acetate (TPA)- and okadaic acid (OA)-caused tumor promotion in SENCAR mouse skin. Pre-application of silymarin to that of TPA in 7, 12-dimethylbenz(a)anthracene (DMBA)-initiated mouse skin resulted in almost complete protection in terms of tumor incidence (85%) as well as multiplicity (94%). In OA-caused tumor promotion studies, application of silymarin prior to that of OA in DMBA-initiated mouse skin resulted in a complete protection against tumorigenicity. We next assessed the effect of silymarin on TPA- and OA-caused induction of mRNA expression of tumor necrosis factor alpha (TNF alpha) which is an endogenous tumor promoter and a central mediator of tumor promotion in vivo in the case of both TPA and OA tumor promotion. Topical application of silymarin on mouse skin prior to that of TPA or OA resulted in a highly significant to complete inhibition in a dose-dependent manner against both TPA- and OA-caused induction of TNF alpha mRNA expression in mouse epidermis. These results indicate that silymarin exerts novel chemopreventive effects against tumorigenicity by inhibiting endogenous tumor promoter TNF alpha. Additional studies are warranted in other tumor models to further evaluate the cancer chemopreventive effect of silymarin and to define the involvement of TNF alpha as a molecular target for such an effect.

38. Fantozzi R, et al. FMLP-activated neutrophils evoke histamine release from mast cells. Agents Actions 1986;18:155-158.

Human neutrophils, having been activated by the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (FMLP), evoke histamine release from rat serosal mast cells. The release is dependent on FMLP concentration and it can be inhibited by disodium cromoglycate and by a flavonoid, silymarin, which displays free radical scavenging properties. Silymarin inhibition of neutrophil-mediated histamine release is dose-dependent. These results further stress the concept of a neutrophil-mast cell interaction, which may be involved in inflammatory processes.

39. Deak G, Muzes G, Lang I. Immunomodulator effect of silymarin therapy in chronic alcoholic liver diseases. Orv Hetil 1990;131:1291-1292, 1295-1296.

Semmelweis Orvostudomanyi Egyetem, II. sz. Belklinika, Budapest.

The effects of the hepatoprotective, antioxidant drug silymarin (Legalon) on some cellular immune parameters of patients with histologically proven chronic alcoholic liver disease were studied in a six month double blind study. The lectin induced proliferative activity of the lymphocytes got enhanced, the originally low T cell percentage and the originally high CD8+ cell percentage have been normalized, the antibody-dependent and natural cytotoxicity of the lymphocytes decreased during silymarin therapy. All these changes were significant, while in the placebo group no significant changes occurred, except for a moderate elevation of the T cell percentage. Thus, the immunomodulatory activity of silymarin might be involved in the hepatoprotective action of the drug and improves the depressed immunoreactivity of the patients.

Publication Types:

•  Clinical Trial
•  Controlled Clinical Trial
•  Review
•  Review Literature

40. Lang I, Nekam K, Gonzalez-Cabello R. Hepatoprotective and immunological effects of antioxidant drugs. Tokai J Exp Clin Med 1990;15:123-127.

Second Department of Medicine, Semmelweis University Medical School, Budapest, Hungary.

The hepatoprotective and immunomodulatory effects of silymarin and amino-imidazole-carboxamide-phosphate were studied in 40 patients with alcoholic cirrhosis of the liver in a one-month double-blind clinical trial. Treatment with either of the drugs normalized the elevated levels of aspartate aminotransferase, alanine aminotransferase and serum bilirubin, markedly reduced the high level of gamma-glutamyl transferase, increased lectin-induced lymphoblast transformation, decreased the percentage of OKT8+ cells and suppressed lymphocytotoxicity. None of these changes occurred in the placebo-treated group. Thus, the hepatoprotective effects of silymarin and amino-imidazole-carboxamide-phosphate in alcoholic cirrhosis can partly be attributed to the immunomodulatory activity of the drugs.

Publication Types:

•  Clinical Trial
•  Controlled Clinical Trial

42. Dehmlow C, Murawski N, de Groot H, et al. Scavenging of reactive oxygen species and inhibition of arachidonic acid metabolism by silibinin in human cells. Life Sci 1996;58:1591-1600.

The effects of the flavonoid silibinin, which is used for the treatment of liver diseases, on the formation of reactive oxygen species and eicosanoids by human platelets, white blood and endothelial cells were studied. Silibinin proved to be a strong scavenger of HOCI (IC50 7 microM), but not of O2- (IC50 > 200 microM) produced by human granulocytes. The formation of leukotrienes via the 5-lipoxygenase pathway was strongly inhibited. In human granulocytes IC50-values of 15 microM and 14.5 microM silibinin were detected for LTB4 and LTC4/D4/E4/F4 formation, respectively. In contrast to this, three- to fourfold silibinin concentrations were necessary to half maximally inhibit the cyclooxygenase pathway. For PGE2 formation by human monocytes an IC50-value of 45 microM silibinin was found. IC50-values of 69 microM and 52 microM silibinin were determined for the inhibition of TXB2 formation by human thrombocytes and of 6-K-PGF1 alpha formation by human omentum endothelial cells, respectively. Thus, the deleterious effects of HOCI that can lead to cell death, and those of leukotrienes that are especially important in inflammatory reactions, can be inhibited by silibinin in concentrations that are reached in vivo after the usual clinical dose. Silibinin is thought not only to display hepatoprotective properties but might also be cytoprotective in other organs and tissues.

43. Dehmlow C, Erhard J, de Groot H. Inhibition of Kupffer cell functions as an explanation for the hepatoprotective properties of silibinin. Hepatology 1996;23:749-754.

The flavonoid silibinin, the main compound extracted from the milk thistle Silybum marianum, displays hepatoprotective properties in acute and chronic liver injury. To further elucidate the mechanisms by which it acts, we studied the effects of silibinin on different functions of isolated rat Kupffer cells, namely the formation of superoxide anion radical (02-), nitric oxide (NO), tumor necrosis factor alpha (TNF-alpha), prostaglandin E(2) (PGE(2)), and leukotriene B(4) (LTB(4)). Production of 02- and NO were inhibited in a dose-dependent manner, with an 50 percent inhibitory concentration (IC(50)) value around 80 micro mol/L. No effect on TNF-alpha formation was detected. Opposite effects were found on the cyclooxygenase and 5-lipoxygenase pathway of arachidonic acid metabolism. Whereas no influence on PGE(2) formation was observed with silibinin concentrations up to 100 micro mol/L, a strong inhibitory effect on LTB(4) formation became evident. The IC(50)-value for inhibiting the formation of this eicosanoid was determined to be 15 micro mol/L silibinin. The strong inhibition of LTB(4), formation by silibinin was confirmed in experiments with phagocytic cells isolated from human liver. Hence, while rather high concentrations of silibinin are necessary to diminish free radical formation by activated Kupffer cells, significant inhibition of the 5-lipoxygenase pathway already occurs at silibinin concentrations which are achieved in vivo. Selective inhibition of leukotriene formation by Kupffer cells can at least partly account for the hepatoprotective properties of silibinin.

44. Fuchs EC, et al. Effects of silibinin and of a synthetic analogue on isolated rat hepatic stellate cells and myofibroblasts. Arzneimittelforschung 1997;47:1383-1387.

Department of Clinical Chemistry, Philipps-Universitat, Koln, Germany.

Hepatic stellate cells and the derived myofibroblasts play a central pathogenic role in liver fibrogenesis. In order to identify the still unknown hepatoprotective properties of the flavonoid silibinin and the related pyridylchromone NH40 x HCl (2-(3-pyridyl)-4-H-1-benzopyran-4-one hydrochloride), their effects on isolated rat hepatic stellate cells and derived myofibroblasts were determined. Concentrations of 10(-4) mol/l silibinin reduced the proliferation of freshly isolated rat hepatic stellate cells by about 75%, but had no detectable effect on their viability, morphology and their cytoskeletal architecture. It reduced the transformation towards myofibroblasts and down-regulated the gene expression of extracellular matrix components and the profibrogenic transforming growth beta. Whereas silibinin concentrations higher than 10(-4) mol/l were toxic, lower concentrations had no effects on the proliferation and transformation behavior. Although 10(-4) mol/l NH40 x HCl reduced the proliferation rate by about 50%, this substance had no significant effect on the transformation process. The results indicate that one important aspect of the potential antifibrotic properties of silibinin might be the inhibition of hepatic stellate cell proliferation and transformation.

45. Boigk G, Stroedter L, Herbst H. Silymarin retards collagen accumulation in early and advanced biliary fibrosis secondary to complete bile duct obliteration in rats. Hepatology 1997;26:643-649.

Silymarin (SIL), a standardized plant extract containing about 60% polyphenole silibinin, is used as a hepatoprotective agent. Its antifibrotic potential in chronic liver diseases has not been explored. Therefore, we applied SIL to adult Wistar rats that were subjected to complete bile duct occlusion (BDO) by injection of sodium amidotrizoate (Ethibloc). This treatment induces progressive portal fibrosis without significant inflammation. Rats with sham-operation that received SIL at 50 mg/kg/d (n = 10) and rats with BDO alone (n = 20) served as controls, whereas groups of 20 animals were fed SIL at a dose of 25 and 50 mg/kg/d during weeks 1 through 6 or doses of 50 mg/kg/d during weeks 4 through 6 of BDO. Animals were sacrificed after 6 weeks for determination of blood chemistries, total and relative liver collagen (as hydroxyproline [HYP]), and the serum aminoterminal propeptide of procollagen type III (PIIINP). BDO in untreated rats caused an almost ninefold increase in total liver collagen (16.1 +/- 3.1 vs. 1.8 +/- 0.4 mg HYP, P < .001). SIL at 50 mg/kg/d reduced total HYP by 30% to 35%, either when given from week 1 through 6 or from week 4 through 6 after BDO (10.6 +/- 2.7 and 10.2 +/- 3.9 mg HYP, both P < .01 vs. BDO alone), whereas 25 mg/kg/d were ineffective. Because SIL at 50 mg/kg/d also reduced the collagen content per gram of liver tissue, it acted as a true antifibrotic agent. The single value of PIIINP at killing paralleled the antifibrotic activity of SIL with 11.6 +/- 3.8 and 9.9 +/- 3.7 vs. 15.3 +/- 5.2 microg/L in both high-dose groups (P < .05 and P < .01, respectively, vs. rats with BDO alone). Except for a decreased alkaline phosphatase and a lower histological fibrosis score in the groups that received SIL, clinical-chemical parameters were not different among all groups with BDO. We therefore conclude that 1) BDO with Ethibloc is a suitable model to test for pure antifibrotic drugs because it induces progressive rat secondary biliary fibrosis without major inflammation; 2) oral SIL can ameliorate hepatic collagen accumulation even in advanced (biliary) fibrosis; and 3) PIIINP appears to be a suitable serum marker to monitor the inhibition of hepatic fibrogenesis in this model of biliary fibrosis.

46. Favari L, Perez-Alvarez V. Comparative effects of colchicine and silymarin on CCl4-chronic liver damage in rats. Arch Med Res 1997;28:11-17.

The comparative effects of colchicine (10 micrograms day-1, p.o.) and silymarin (50 mg kg-1, p.o.) each given for 5 days a week on the chronic carbon tetrachloride (CCl4) liver damage were studied. Treatment with CCl4 resulted in a marked reduction of Na+, K+, and Ca2(+)-ATPases in plasma liver membranes as compared to vehicles or either silymarin or colchicine alone. Collagen content in livers of animals treated with CCl4 was increased about four-fold as compared to controls and histological examination of liver samples showed that collagen increase distorted the normal liver architecture. Colchicine or silymarin treatment completely prevented all the changes observed in CCl4-cirrhotic rats (namely, lipid peroxidation, Na+, K+ and Ca(2+)-ATPases), except for liver collagen content which was reduced only 55% as compared with CCl4-treated rats and for alkaline phosphatase and glutamic pyruvic transaminase which still remained above controls. In the CCl4 + silymarin group, the loss of glycogen content was completely prevented. However, when rats were treated with CCl4 + colchicine, liver glycogen content could not be restored. The hepatoprotective effects of colchicine or silymarin were very similar in regard to the prevention of chronic liver damage.

47. De La Puerta R, Martinez E, Bravo L. Effect of silymarin on different acute inflammation models and on leukocyte migration. J Pharm Pharmacol 1996;48:968-970.

In-vivo anti-inflammatory activity of silymarin was tested in different acute inflammation experimental models. In carrageenan-induced paw oedema in rats, silymarin given orally reduced in a dose-dependent manner the food-pad abscesses (ED50 = 62.42 mg kg-1). In xylene-induced ear mouse inflammation, silymarin applied topically was more effective than administered intraperitoneally, with effects comparable with those of indomethacin. Silymarin also produced a dose-dependent inhibition of leukocyte accumulation in inflammatory exudates following intraperitoneal injection of carrageenan in mice; silymarin significantly reduced the number of neutrophils. Silymarin was unable to inhibit phospholipase A2 in an in-vitro assay. Besides its known anti-oxidative properties and its ability to act as a radical scavenger, these results suggest that silymarin exerts an important anti-inflammatory action in-vivo by reducing oedema with the effect markedly influenced by the inhibition of neutrophil migration into the inflamed site.

48. Amdur MO, Doull J, Klaassen CD. Casarett and Doull's Toxicology: The Basic Science of Poisons, 4th ed. New York, NY: McGraw-Hill; 1991.

49. Awang D. Milk thistle . Can Pharm J 1993;October:403-404.

50. Rumyantseva A. No title available.Vrach Delo 1991;5:15-19.

51. Brown D. Drug Store News for the Pharmacist 1994;4:58,60.

52. Lirussi F,et al. Silybin-beta-cyclodextrin in the treatment of patients with diabetes mellitus and alcoholic liver disease. Efficacy study of a new preparation of an anti-oxidant agent. Diabetes Nutr Metab. 2002 Aug;15(4):222-31.

Department of Medical and Surgical Sciences, University of Padova, Italy.

BACKGROUND AND AIMS: In patients with non-insulin dependent diabetes mellitus (T2DM) and associated chronic liver disease, plasma levels of glucose, insulin and triglycerides are high, lipid peroxidation is increased and natural antioxidant reserves are reduced. Thus, we hypothesised that the re-balancing of cell redox levels and amelioration of liver function could result in a better glucose and lipid metabolism. To study this, we assessed the effect of a new oral formulation of an antioxidant agent - silybin-beta-cyclodextrin (named IBI/S) - in patients with chronic alcoholic liver disease and concomitant T2DM. METHODS: Sixty outpatients were enrolled in a three-centre, double blind, randomised, IBI/S vs placebo study. Forty-two (21 in the group IBI/S - 135 mg/d silybin per os - and 21 in the placebo group) concluded the 6-month treatment period. The efficacy parameters included fasting and mean daily plasma glucose levels, glycosylated hemoglobin (HbA1c), basal, stimulated C-peptide and insulin levels, total-, HDL-cholesterol and triglycerides levels in addition to conventional liver function tests. Insulin sensitivity was estimated by HOMA-IR. Malondialdehyde (MDA) was also measured before and after treatment as an index of oxidative stress. RESULTS: Fasting blood glucose levels, which were similar at baseline in IBI/S group and in the placebo group (173.9 mg/dl and 177.1 mg/dl, respectively), decreased to 148.4 mg/dl (-14.7% vs baseline; p = 0.03) in the IBI/S group while they were virtually unchanged in the placebo group. The comparison between the groups at mo 6 (T6) also showed a significant reduction of glucose levels in the IBI/S group (p = 0.03). The same trend was observed in mean daily blood glucose levels, HbA1c and HOMA-IR, although differences were not significant. Basal and stimulated C-peptide values showed that only a few changes had occured in both groups. Such results indicate that insulin secretion was virtually unaffected, as confirmed also by the insulinemia data. Plasma triglycerides concentrations dropped from a baseline value of 186 mg/dl to 111 mg/dl (T6) in the IBI/S group, with significant differences at all instances with respect to baseline values. By contrast, triglycerides increased from 159 mg/dl at entry to 185 mg/dl (T6) in the placebo group. The difference between the groups at T6 was highly significant (p < 0.01). Total and HDL cholesterol as well as liver function tests did not change significantly during the study in both groups. MDA decreased significantly only in the group receiving IBI/S. No clinically relevant side effects were observed in either group. CONCLUSIONS: Oral administration silybin-beta-cyclodextrin in patients with T2DM and compensated chronic alcoholic liver disease causes a significant decrease in both glucose and triglyceride plasma levels. These effects may be due to the recovery of energy substrates, consistent with a reduced lipid peroxidation and an improved insulin activity.

Publication Types:

•  Clinical Trial
•  Randomized Controlled Trial

53. Piscitelli SC, Formentini E, Burstein AH, Alfaro R, Jagannatha S, Falloon J. Effect of milk thistle on the pharmacokinetics of indinavir in healthy volunteers. Pharmacotherapy. 2002 May;22(5):551-6.

STUDY OBJECTIVE: To characterize the pharmacokinetics of indinavir in the presence and absence of milk thistle and to determine the offset of any effect of milk thistle on indinavir disposition. DESIGN: Prospective open-label drug interaction study. SETTING: Outpatient clinic. SUBJECTS: Ten healthy volunteers. Intervention. Blood samples were collected over 8 hours after the volunteers took four doses of indinavir 800 mg every 8 hours on an empty stomach for baseline pharmacokinetics. This dosing and sampling were repeated after the subjects took milk thistle 175 mg (confirmed to contain silymarin 153 mg, the active ingredient) 3 times/day for 3 weeks. After an 11-day washout, indinavir dosing and blood sampling were repeated to evaluate the offset of any potential interaction. MEASUREMENTS AND MAIN RESULTS: Indinavir concentrations were measured by using a validated high-performance liquid chromatography method. The following pharmacokinetic parameters were determined: highest concentration (Cmax), hour-0 concentration, hour-8 concentration (C8), time to reach Cmax, and area under the plasma concentration-time curve over the 8-hour dosing interval (AUC8). Milk thistle did not alter significantly the overall exposure of indinavir, as evidenced by a 9% reduction in the indinavir AUC8 after 3 weeks of dosing with milk thistle, although the least squares mean trough level (C8) was significantly decreased by 25%. CONCLUSION: Milk thistle in commonly administered dosages should not interfere with indinavir therapy in patients infected with the human immunodeficiency virus.

Publication Types:

•  Clinical Trial

54 . Allain H, et al.. Aminotransferase levels and silymarin in de novo tacrine-treated patients with Alzheimer's disease. Dement Geriatr Cogn Disord. 1999 May-Jun;10(3):181-5.

BACKGROUND: Silymarin is a well-known hepatoprotective agent. Tacrine, the first drug marketed for Alzheimer's disease (AD), induces an elevation of serum liver transaminase prohibiting an effective dosage in many patients. This 12-week randomised, double-blind, placebo-controlled study was undertaken to evaluate the ability of silymarin to antagonise or prevent the hepatotoxic effects of tacrine and to analyse its action on tacrine efficacy and tolerability. METHODS: Outpatients suffering from mild-to-moderate dementia of the Alzheimer type were randomly assigned to two treatment groups: tacrine + silymarin and tacrine + placebo. The study was double-blind for silymarin and open for tacrine and was conducted in 22 French neurology and geriatric centres. Silymarin (420 mg/day) was given first (1 week) and tacrine was added at 40 mg/day for 6 weeks, then increased to 80 mg/day (6 weeks). Serum ALAT was the main evaluation criterion (> upper limit of normal, ULN). Serum ASAT as well as adverse side effects and cognitive performance assessed by MMSE and the Syndrome Kurtz test (SKT) were secondary evaluation criteria. Null hypotheses were evaluated with Fisher's exact test. FINDINGS: 222 patients were recruited and received silymarin and tacrine (110 patients) or placebo and tacrine (112 patients). 28 patients dropped out; 217 were included in the intent-to-treat analysis. No statistical difference was observed between the two groups for serum ALAT (p = 0.39). Fewer patients had ALAT levels >5 ULN in the silymarin group (-33.3%). Side effects and notably gastrointestinal disorders were much less frequent in the silymarin group. Cognitive performance remained unchanged in both groups. INTERPRETATION: Silymarin does not prevent tacrine-induced ALAT elevation but does reduce the rate of gastrointestinal and cholinergic side effects without any impact on cognitive status. As a consequence, silymarin (420 mg/day) could be co-administered with tacrine to improve tolerability in the initial phases of AD treatment.

Publication Types:

•  Clinical Trial
•  Randomized Controlled Trial

55. Velussi M, et al. Long-term (12 months) treatment with an anti-oxidant drug (silymarin) is effective on hyperinsulinemia, exogenous insulin need and malondialdehyde levels in cirrhotic diabetic patients. J Hepatol. 1997 Apr;26(4):871-9.

BACKGROUND/AIMS: Several studies have demonstrated that diabetic patients with cirrhosis require insulin treatment because of insulin resistance. As chronic alcoholic liver damage is partly due to the lipoperoxidation of hepatic cell membranes, anti-oxidizing agents may be useful in treating or preventing damage due to free radicals. The aim of this study was to ascertain whether long-term treatment with silymarin is effective in reducing lipoperoxidation and insulin resistance in diabetic patients with cirrhosis. METHODS: A 12-month open, controlled study was conducted in two well-matched groups of insulin-treated diabetics with alcoholic cirrhosis. One group (n=30) received 600 mg silymarin per day plus standard therapy, while the control group (n=30) received standard therapy alone. The efficacy parameters, measured regularly during the study, included fasting blood glucose levels, mean daily blood glucose levels, daily glucosuria levels, glycosylated hemoglobin (HbA1c) and malondialdehyde levels. RESULTS: There was a significant decrease (p<0.01) in fasting blood glucose levels, mean daily blood glucose levels, daily glucosuria and HbA1c levels already after 4 months of treatment in the silymarin group. In addition, there was a significant decrease (p<0.01) in fasting insulin levels and mean exogenous insulin requirements in the treated group, while the untreated group showed a significant increase (p<0.05) in fasting insulin levels and a stabilized insulin need. These findings are consistent with the significant decrease (p<0.01) in basal and glucagon-stimulated C-peptide levels in the treated group and the significant increase in both parameters in the control group. Another interesting finding was the significant decrease (p<0.01) in malondialdehyde/levels observed in the treated group. CONCLUSIONS: These results show that treatment with silymarin may reduce the lipoperoxidation of cell membranes and insulin resistance, significantly decreasing endogenous insulin overproduction and the need for exogenous insulin administration.

Publication Types:

•  Clinical Trial
•  Controlled Clinical Trial
•  Randomized Controlled Trial

56. Nassuato G, et al. Effect of Silibinin on biliary lipid composition. Experimental and clinical study. J Hepatol. 1991 May;12(3):290-5.

The effect of Silymarin, a natural flavonoid, on biliary lipid composition, was studied in rats and humans. Bile flow, biliary cholesterol, phospholipid and total bile salt concentrations were measured in 23 control rats and in 27 rats treated with Silibinin, the active component of Silymarin, at the dose of 100 mg/kg body weight i.p. (n = 21) or 50 mg/kg body weight i.p. (n = 6) for 7 days. Biliary cholesterol and phospholipid concentrations were significantly reduced after the higher Silibinin dose (60.9 and 72.9% of the control values), whereas bile flow and biliary total bile salt concentration were unchanged. After the lower Silibinin dose all parameters remained unchanged. Total liver cholesterol content was not affected by Silibinin. On the other hand, in vitro determination of rat liver microsomal 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity showed a significant dose-dependent inhibition by Silibinin (0.5-8 mg/kg). Biliary lipid composition was also assayed in four gallstone and in 15 cholecystectomized patients before and after Silymarin (420 mg per day for 30 days) or placebo administration. In both groups, biliary cholesterol concentrations were reduced after Silymarin treatment and the bile saturation index significantly decreased accordingly. These data suggest that Silibinin-induced reduction of biliary cholesterol concentration both in humans and in rats might be, at least in part, due to a decreased synthesis of liver cholesterol.

Publication Types:

•  Clinical Trial
•  Controlled Clinical Trial

57. Lang I, Nekam K, Gonzalez-Cabello R, Muzes G, Gergely P, Feher J. Hepatoprotective and immunological effects of antioxidant drugs. Tokai J Exp Clin Med. 1990 May;15(2-3):123-7.

Second Department of Medicine, Semmelweis University Medical School, Budapest, Hungary.

The hepatoprotective and immunomodulatory effects of silymarin and amino-imidazole-carboxamide-phosphate were studied in 40 patients with alcoholic cirrhosis of the liver in a one-month double-blind clinical trial. Treatment with either of the drugs normalized the elevated levels of aspartate aminotransferase, alanine aminotransferase and serum bilirubin, markedly reduced the high level of gamma-glutamyl transferase, increased lectin-induced lymphoblast transformation, decreased the percentage of OKT8+ cells and suppressed lymphocytotoxicity. None of these changes occurred in the placebo-treated group. Thus, the hepatoprotective effects of silymarin and amino-imidazole-carboxamide-phosphate in alcoholic cirrhosis can partly be attributed to the immunomodulatory activity of the drugs.

Publication Types:

•  Clinical Trial
•  Controlled Clinical Trial

58. Muzes G, et al. [Effect of silimarin (Legalon) therapy on the antioxidant defense mechanism and lipid peroxidation in alcoholic liver disease (double blind protocol)] Orv Hetil. 1990 Apr 22;131(16):863-6.

Semmelweis Orvostudomanyi Egyetem, II. Belgyogyaszati Klinika, Budapest.

A double blind study. Antioxidant and antiperoxidative effects of the free radical scavenger agent silymarin (Legalon) were investigated in patients with chronic alcoholic liver disease in a double blind clinical trial. Six month treatment (at a daily dose of 420 mg) with silymarin significantly enhanced the originally low superoxide dismutase activity of erythrocytes and lymphocytes and also restored the diminished superoxide dismutase expression on lymphocytes as measured by flow-cytofluorimetry. In addition, silymarin therapy markedly increased the serum level of ree--SH groups and the activity of glutathione peroxidase. In contrast, a considerable fall in serum malondialdehyde concentration was detected in patients having received silymarin. However, in case of placebo-treated patients the above mentioned parameters of antioxidant defense system and lipid peroxidation failed to change significantly. These data indirectly suggest that antioxidant, antiperoxidative effects might be important factors in the mechanism of hepatoprotective action of silymarin.

Publication Types:

•  Clinical Trial
•  Controlled Clinical Trial

59. Somogyi A, Ecsedi GG, Blazovics A, Miskolczi K, Gergely P, Feher J. Short term treatment of type II hyperlipoproteinaemia with silymarin. Acta Med Hung. 1989;46(4):289-95.

Second Department of Medicine, Semmelweis University, Chinoin Pharmaceutical and Chemical Works, Ltd., Budapest, Hungary.

In a seven-month open clinical study on 14 type-II hyperlipidaemic outpatients, the effects of silymarin (Legalon), an antioxidant and hepatoprotective agent, were investigated. Blood lipid, lipoprotein and apolipoprotein concentrations, as well as liver and renal function parameters were measured. After determining baseline values, patients were treated with 420 mg Legalon daily for three months. After a two-month placebo period, the treatment was repeated with Legalon for a further month. In respect to the serum lipid and lipoprotein concentrations, there were no remarkable changes except that the total cholesterol and HDL-cholesterol levels slightly decreased. At the 12th week, in all cases, the apolipoprotein levels were somewhat decreased compared to the baseline values. By the significant decrease of both apo A-I and A-II values, a decrease of the total structural protein amount of HDL, and thus a relative increase in the proportion of cholesterol in HDL fraction was suggested. There were minor changes in serum protein concentration and liver function tests, but all values remained within the normal range. All of the renal function parameters remained unchanged during both treatments and the placebo periods. An additive role of Legalon in the therapy of secondary hyperlipoproteinaemia resulting from different liver diseases is discussed.

Publication Types:

•  Clinical Trial

60. Tanasescu C, Petrea S, Baldescu R, Macarie E, Chiriloiu C, Purice S. Use of the Romanian product Silimarina in the treatment of chronic liver diseases. Med Interne. 1988 Oct-Dec;26(4):311-22.

N. Gh. Lupu Institute of Internal Medicine, Bucharest, Romania.

The Romanian product Silimarina (synonym Legalon) was administered in a randomized double-blind trial, to a group of 180 patients with chronic persistent hepatitis (CPH), chronic active hepatitis (CAH) and hepatic cirrhosis (HC). The trial lasted for 40 days. The results showed favourable effects similar with those obtained with other preparations produced by foreign drug industries. The Romanian product proved to have no toxic effect. The authors discuss the present possibilities of estimating the evolution of chronic liver disease.

Publication Types:

•  Clinical Trial
•  Randomized Controlled Trial

61. Szilard S, Szentgyorgyi D, Demeter I. Protective effect of Legalon in workers exposed to organic solvents. Acta Med Hung. 1988;45(2):249-56.

Tisza Chemical Works Leninvaros, Occupational Health Care Service, Hungary.

Abnormal result of liver function tests (elevated levels of AST, ALT activity) and/or abnormal haematological values (low platelet counts, leucocytosis, relative lymphocytosis) were observed in 49 of 200 workers exposed to toluene and/or xylene vapours for 5-20 years. Thirty of the affected workers were treated per os with Legalon (MADAUS, FRG) t.i.d. for 30 days. The remaining 19 were left without treatment. Under the influence of Legalon the liver function tests and the platelet counts significantly improved. The leukocytosis and relative lymphocytosis showed a nonsignificant tendency of improvement.

Publication Types:

•  Clinical Trial
•  Controlled Clinical Trial

62. Benda L, Dittrich H, Ferenzi P, Frank H, Wewalka F. [The influence of therapy with silymarin on the survival rate of patients with liver cirrhosis]. Wien Klin Wochenschr. 1980 Oct 10;92(19):678-83.

A randomized double-blind study about the therapy of the cirrhosis of the liver shows a significant higher surviving rate of the alcoholic cirrhosis in the group treated with Silymarin. This result can be well explained by the protective influence of this substance against toxic injuries. The etiology of many chronic liver diseases is uncertain and therefore it is advisable to try the therapy also in other cases. The conditions of the study are exactly reported. The influence on the clinic of the disease and on the laboratory data obtained in many controls will be published later, because the statistical analysis needs some more time.

Publication Types:

•  Clinical Trial
•  Randomized Controlled Trial

63. Magliulo E, Gagliardi B, Fiori GP. [Results of a double blind study on the effect of silymarin in the treatment of acute viral hepatitis, carried out at two medical centres] Med Klin. 1978 Jul 14;73(28-29):1060-5.

In a double blind study carried out under standard conditions at two treatment centers silymarin, 2 sugar-coated tablets 70 mg three times daily, showed a definite therapeutic influence on the characteristic increased serum levels of bilirubin, GOT and GPT associated with acute viral hepatitis. The above mentioned values in 28 patients treated with silymarin were compared with those in 29 patients treated with placebo. The laboratory parameters in the silymarin group regressed more than in the placebo group after the 5th day of treatment. The number of patients having attained normal values after 3 weeks' treatment was higher in the silymarin group than in the placebo group. A statistical comparison revealed a difference between bilirubin and GOT values in the placebo and silymarin groups and a definite trend in the regression of GPT values in favour of silymarin. The course of the immune reaction in HBS Ag patients was not influenced by silymarin. As already proved by other investigators, the use of silymarin in acute viral hepatitis can lead to an accelerated regression in pathological values, thus indicating its use in the treatment of this liver disease.

Publication Types:

•  Clinical Trial
•  Controlled Clinical Trial

64. Kreeman V, et al. Silymarin inhibits the development of diet-induced hypercholesterolemia in rats. Planta Med 1998;64:138-142.

To study the ability of silymarin, a standardized mixture of antioxidant flavonolignans from the medicinal plant Silybum marianum, and of silybin, the main flavonolignan of silymarin, to inhibit the development of diet-induced hypercholesterolemia the rats were fed high cholesterol diet (HCD). Silymarin or silybin were given as dietary supplements, and their influences on serum cholesterol levels were compared to those of probucol, an antioxidant hypocholesterolemic drug. Anticholesterolemic effect of silymarin was parallel to that of probucol, and dose-dependent at dietary drug concentrations of 0.1-0.5-1.0% (w/w). However, in contradistinction to probucol, silymarin caused an increase in high density lipoprotein (HDL)-cholesterol and a decrease in liver cholesterol content, changes considered to be of benefit. In addition to its anticholesterolemic effect silymarin partially prevented the HCD-induced decrease in liver reduced glutathione, an endogenous antioxidant. Silybin was not so effective as silymarin suggesting that either other constituent(s) of silymarin may be responsible for its anticholesterolemic effect or the bioavailability of silybin alone might be lower than that of silybin as a compound of silymarin.

65. Kock HP, et al. Silymarin: Potent inhibitor of cyclic AMP phosphodiesterase. Meth Find Expel Clin Pharmacol 1985;7:409-413.

Silymarin, the active principle of the Milk Thistle (Silybum marianum Gaertner), is a very potent inhibitor of cyclic AMP breakdown in vitro by a commercial beef heart phosphodiesterase preparation. Its main constituents, silybin, silydianin and silychristin, are 12.66 to 52.06 times more active than theophylline and 0.77 to 3.17 times more active than papaverine in this respect. Using a novel HPLC technique, the enzyme kinetical analysis can be performed much faster than by the classical methods.

66. Singh RP, Sharma G, Dhanalakshmi S, Agarwal C, Agarwal R. Suppression of advanced human prostate tumor growth in athymic mice by silibinin feeding is associated with reduced cell proliferation, increased apoptosis, and inhibition of angiogenesis. Cancer Epidemiol Biomarkers Prev. 2003 Sep;12(9):933-9.

68. Dhanalakshmi S, et al. Silibinin sensitizes human prostate carcinoma DU145 cells to cisplatin- and carboplatin-induced growth inhibition and apoptotic death. Int J Cancer. 2003 Sep 20;106(5):699-705.

69. Tyagi A, Agarwal C, Agarwal R. Inhibition of retinoblastoma protein (Rb) phosphorylation at serine sites and an increase in Rb-E2F complex formation by silibinin in androgen-dependent human prostate carcinoma LNCaP cells: role in prostate cancer prevention. Mol Cancer Ther. 2002 May;1(7):525-32.

Several studies have identified silibinin as an anticarcinogenic agent. Recently, we showed that silibinin inhibits cell growth via G1 arrest, leading to differentiation of androgen-dependent human prostate carcinoma LNCaP cells (X. Zi and R. Agarwal, Proc. Natl. Acad. Sci. USA, 96: 7490-7495,1999). Here, we extend this study to assess the effect of silibinin on total retinoblastoma protein (Rb) levels and its phosphorylation status, levels of E2F family members, and Rb-E2F binding in LNCaP cells. Compared with controls, silibinin resulted in an increase in total Rb levels that was largely attributable to an increase in unphosphorylated Rb (up to 4.1-fold). This effect of silibinin was mainly attributable to a large decrease (70-97%) in the amount of Rb phosphorylated at specific serine sites. In other studies, silibinin showed a moderate effect on E2F1 but up to 98 and 90% decreases in E2F2 and E2F3 protein levels, respectively. Silibinin treatments also resulted in an increase in the amount of Rb binding to E2F1 (3.8-fold), E2F2 (2.2-fold), and E2F3 (2.2-fold). Cyclin-dependent kinases (CDKs), together with their catalytic subunit cyclins, phosphorylate Rb, which makes transcription factor E2Fs free from Rb-E2F complexes, resulting in cell growth and proliferation. Conversely, CDK inhibitors inhibit this phosphorylation, maintaining E2Fs bound to Rb, which causes growth inhibition. On the basis of our data showing that silibinin induces both unphosphorylated Rb levels and Rb-E2F binding, we also assessed its effect on upstream cell cycle regulators. Silibinin-treated cells showed up to 2.4- and 3.6-fold increases in Cip1/p21 and Kip1/p27 levels, respectively, and a decrease in CDK2 (80%), CDK4 (98%), and cyclin D1 (60%). Consistent with these results, silibinin showed both G1 arrest and growth inhibition. Together, these findings identify modulation of Rb levels and its phosphorylation status as a molecular mechanism of silibinin-induced neuroendocrine differentiation of human prostate carcinoma LNCaP cells and suggest that this could be a novel approach for prostate cancer prevention by silibinin.

70. Tyagi A, Agarwal C, Agarwal R. The cancer preventive flavonoid silibinin causes hypophosphorylation of Rb/p107 and Rb2/p130 via modulation of cell cycle regulators in human prostate carcinoma DU145 cells. Cell Cycle. 2002 Mar-Apr;1(2):137-42.

71. Tyagi AK, et al. Silibinin strongly synergizes human prostate carcinoma DU145 cells to doxorubicin-induced growth Inhibition, G2-M arrest, and apoptosis. Clin Cancer Res. 2002 Nov;8(11):3512-9.

72. Tyagi A, Bhatia N, Condon MS, Bosland MC, Agarwal C, Agarwal R. Antiproliferative and apoptotic effects of silibinin in rat prostate cancer cells. Prostate. 2002 Nov 1;53(3):211-7.

73. Singh RP, Dhanalakshmi S, Tyagi AK, Chan DC, Agarwal C, Agarwal R. Dietary feeding of silibinin inhibits advance human prostate carcinoma growth in athymic nude mice and increases plasma insulin-like growth factor-binding protein-3 levels. Cancer Res. 2002 Jun 1;62(11):3063-9.

74. Dhanalakshmi S, et al. Silymarin inhibits function of the androgen receptor by reducing nuclear localization of the receptor in the human prostate cancer cell line LNCaP.
Carcinogenesis. 2001 Sep;22(9):1399-403.

75. Sharma Y, et al. Inhibitory effect of silibinin on ligand binding to erbB1 and associated mitogenic signaling, growth, and DNA synthesis in advanced human prostate carcinoma cells. Mol Carcinog. 2001 Apr;30(4):224-36.

76. Zi X, Zhang J, Agarwal R, Pollak M. Silibinin up-regulates insulin-like growth factor-binding protein 3 expression and inhibits proliferation of androgen-independent prostate cancer cells. Cancer Res. 2000 Oct 15;60(20):5617-20.

77. Bhatia N, Zhao J, Wolf DM, Agarwal R. Inhibition of human carcinoma cell growth and DNA synthesis by silibinin, an active constituent of milk thistle: comparison with silymarin. Cancer Lett. 1999 Dec 1;147(1-2):77-84.

78. Zhao J, Agarwal R. Tissue distribution of silibinin, the major active constituent of silymarin, in mice and its association with enhancement of phase II enzymes: implications in cancer chemoprevention. Carcinogenesis. 1999 Nov;20(11):2101-8.

79. Zi X, Agarwal R. Silibinin decreases prostate-specific antigen with cell growth inhibition via G1 arrest, leading to differentiation of prostate carcinoma cells: implications for prostate cancer intervention. Proc Natl Acad Sci U S A. 1999 Jun 22;96(13):7490-5.

Reduction in serum prostate-specific antigen (PSA) levels has been proposed as an endpoint biomarker for hormone-refractory human prostate cancer intervention. We examined whether a flavonoid antioxidant silibinin (an active constituent of milk thistle) decreases PSA levels in hormone-refractory human prostate carcinoma LNCaP cells and whether this effect has biological relevance. Silibinin treatment of cells grown in serum resulted in a significant decrease in both intracellular and secreted forms of PSA concomitant with a highly significant to complete inhibition of cell growth via a G1 arrest in cell cycle progression. Treatment of cells grown in charcoal-stripped serum and 5alpha-dihydrotestosterone showed that the observed effects of silibinin are those involving androgen-stimulated PSA expression and cell growth. Silibinin-induced G1 arrest was associated with a marked decrease in the kinase activity of cyclin-dependent kinases (CDKs) and associated cyclins because of a highly significant decrease in cyclin D1, CDK4, and CDK6 levels and an induction of Cip1/p21 and Kip1/p27 followed by their increased binding with CDK2. Silibinin treatment of cells did not result in apoptosis and changes in p53 and bcl2, suggesting that the observed increase in Cip1/p21 is a p53-independent effect that does not lead to an apoptotic cell death pathway. Conversely, silibinin treatment resulted in a significant neuroendocrine differentiation of LNCaP cells as an alternative pathway after Cip1/p21 induction and G1 arrest. Together, these results suggest that silibinin could be a useful agent for the intervention of hormone-refractory human prostate cancer.