Ludovico Abenavoli
email: l.abenavoli@unicz.it

Raffaele Capasso

Natasa Milic

Francesco Capasso

PAST, PRESENT, FUTURE

Keywords: Milk thistle, silybin, silipide, silymarin, liver diseases, reactive oxygen species Milk Thistle, silybin, silipide, silymarin, liver diseases, reactive oxygen species

come

One of the earliest records of MT is found in the Bible (Genesis 3:18). In this verse, God told Adam and Eve when they were banished from the Garden of Eden that "thorns also and thistles shall it bring forth to thee". Some of the earliest people to use and write about MT were ancient Greek and Roman physicians and herbalists, each of whom seemed to have their own name for the herb. Dioscorides called it "sillybon", Pliny the Elder called it "sillybum", and Theophrastus called it "pternix". Dioscorides' use of MT is one of the oldest known references of the medicinal use of this plant. He suggested preparing it in a tea "for those that be bitten of serpents". Another famous ancient herbalist, Pliny the Elder, wrote that mixing the juice of the plant with honey was good for "carrying off bile" [START_REF] Ross | Milk thistle (Silybum marianum): an ancient botanical medicine for modern times[END_REF].

More recently it has been discovered that Indian and Chinese medicines used MT in clinical practice [START_REF] Abenavoli | Complementary therapies for treating alcoholism First Annual meeting by Complementary Medicine Research Group of the Italian Society for Alcohol Studies-May 5, 2006[END_REF]. Available records do not seem to offer us the insight how (i.e., by what anecdotal or empirical evidence) MT came to be advised for liver and gallbladder problems. Although MT is the most often associated with treating liver disorders, physicians have tried to apply its curative properties to other ailments, including the stimulation of breast-milk production and bile secretion, treatment of depression, the protection against the poisonous mushroom Amanita phalloides and other environmental toxins [START_REF] Pradhan | Hepatoprotective herbal drug, silymarin from experimental pharmacology to clinical medicine[END_REF] The Present use of Milk Thistle

Biochemistry

The crude drug contains 15-30% lipids, in the form of triglycerides [linoleic (about 60%), oleic (about 30%) and palmitic (about 9%) acid]; about 30% proteins, sugars (arabinose, rhamnose, xylose, glucose); tocopherol, (0.038%) sterols (0.063%) with cholesterol, compesterol and stigmasterol, and flavonoids including quercetin, taxifolin, eriodictyol and chrysoeriol (Table 1). However, the constituents responsible for the activity are flavanolignans (flavanone derivatives) initially isolated as a mixture of addition products of a coniferyl alcohol, phenylproponoid alcohol, and a 2,3-dihydroflavonol, taxifolin [START_REF] Wu | Drug-drug interactions of silymarin on the perspective of pharmacokinetics[END_REF]. This mixture, known as silymarin, represents 1.5-3 % of the dry drug weight and consists of (Figure 2): silybin (=silybinin, silibinin) (approximately 50 to 60%), isosilibyn (about 5%), silychristin (about 20%) and silydianin (about 10%), as well as silimonin, isosilychristin, isosilibinin, etc. The drug can be identified for its microscopic characteristics by Thin Layer Chromatography (TLC) or High Performance Liquid Chromatography (HPLC); spectrophotometry can also be used.

Pharmacokinetics

About 20-50% of silymarin is absorbed following an oral administration in humans and about 80% of the dose is excreted in the bile while about 10% enters the enterohepatic circulation. However, the pharmacokinetic studies have principally been performed on silibinin, the main component of silymarin.

The bioavailability of silibinin is low and seems to depend on several factors such as (i) the content of accompanying substances with a solubilising character such as other flavonoids, phenol derivates, aminoacids, proteins, tocopherol, fat, cholesterol and other substances found in the preparation and (ii) the concentration of the preparation itself [START_REF] Voinovich | Solid state mechanochemical activation of Silybum marianum dry extract with betacyclodextrins: Characterization and bioavailability of the coground systems[END_REF]. The systemic bioavailability can be enhanced by adding solubilising substances to the extract [START_REF] Saller | The use of silymarin in the treatment of liver diseases[END_REF]. The bioavailability of silybinin can also be enhanced by the complexation with phosphatidylcholine or β-cyclodextrin and possibly by the choice of the capsule material [START_REF] Morazzoni | Comparative bioavailability of Silipide, a new flavanolignan complex, in rats[END_REF][START_REF] Arcari | A new inclusion complex of silibinin and betacyclodextrins: in vitro dissolution kinetics and in vivo absorption in comparison with traditional formulations[END_REF].

The variations in the content, dissolution and (oral) bioavailability of silybinin between different commercially available silymarin products -despite the same declaration of content -are significant [START_REF] Schulz | The solubility and bioequivalence of silymarin preparations[END_REF]. Therefore, the comparisons between studies should be carried out with the caution, considering the differences between the analytical methods used (TLC vs HPLC) and whether free, conjugated or total silybinin is the object of measurement. Systemic plasma concentrations are usually measured, even though the site of action of silymarin is the liver, as they provide an estimate on the quantity of the drug being absorbed from the gastrointestinal tract. The adequate bioavailability accounts for the dose-related oral activity of silymarin in the liver [START_REF] Saller | The use of silymarin in the treatment of liver diseases[END_REF].

In male volunteers, after single oral administration of a standardised dose of silibinin 100 to 360mg, plasma silibinin Cmax was reached after approximately two hours and ranges between 200 and 1400 µg/L, of which approximately 75% was present in the conjugated form [START_REF] Weyhenmeyer | Study on dose-linearity of the pharmacokinetics of silibinin diastereomers using a new stereospecific assay[END_REF][START_REF] Gatti | Plasma concentrations of free and conjugated silybin after oral intake of a silybin-phosphatidyl complex (silipide) in healthy volunteers[END_REF]. For total silibinin, an elimination half-life of approximately six hours was estimated [START_REF] Saller | An updated systematic review with meta-analysis for the clinical evidence of silymarin[END_REF].

Between 3% and 8% of an oral dose was excreted in the urine, while 20 to 40% was recovered from the bile as glucuronide and sulfate conjugates. The remaining part was excreted via faeces (unchanged, not absorbed). Silibinin concentrations in bile reached approximately 100 times those found in serum (10-5 to 10-4 mol/L of silibinin in bile) with peak concentrations reached within two to nine hours [START_REF] Saller | An updated systematic review with meta-analysis for the clinical evidence of silymarin[END_REF].

Barzaghi and co-workers investigated the pharmacokinetics of silybin phosphatidylcholine complex (Id B1016) and detected an increase in the oral bioavailability of silybin in healthy human subjects, probably by a facilitatory role of the drug complex on the passage of the drug across the gastrointestinal tract [START_REF] Barzaghi | Pharmacokinetic studies on IdB 1016, silybin phosphatidylcholine complex, in healthy human subjects[END_REF]. The bioavailability of Id B1016 was shown to be several times higher than that of silymarin in the patients with hepatic cirrhosis [START_REF] Orlando | Silybin kinetics in patients with liver cirrhosis: a comparative study of a silybin-phospatidylcholine complex (Siliphos ® ) And silymarin[END_REF].

Pharmacological studies

Several pharmacological studies have been carried out on the active components of MT, silymarin and silybinin. It has been found that these substances exert hepatoprotective, anti-oxidant, anti-inflammatory and anti-fibrotic properties; in addition, they

Anti-inflammatory and immuno-modulation activity

Silymarin shows anti-inflammatory and immuno-modulation activity in various structures and pathways of the cell. The tumour necrosis factor-α receptor (TNFα-R) super-family, located in the cell membranes, contains several members with homologous cytoplasmic domains known as death domains (DD), important in initiating apoptosis and other signalling pathways following ligand binding by the receptors [START_REF] Saller | An updated systematic review with meta-analysis for the clinical evidence of silymarin[END_REF].

A pre-clinical study showed that silibinin prevented the effect of TNF-α induced with α-amanitin in hepatocytes [START_REF] El-Bahay | Influence of tumor necrosis factor-alpha and silibin on the cytotoxic action of alpha-amanitin in rat hepatocyte culture[END_REF], possibly by reactive oxygen species (ROS)-dependent mechanisms [START_REF] Chovolou | Resistance to Tumor Necrosis Factor-α (TNFα)-induced apoptosis in rat hepatoma cells expressing TNFα is linked to low antioxidant enzyme expression[END_REF]. In mice treated with 750 mg/kg/day silymarin by gavage and 2.25 mg/kg Fumonisin B1 (inhibitor of ceramide synthase), silymarin prevented the Fumonisin B1-induced increases in the TNF receptor 1 (TNF-R1) expression, TNF-R1-associated apoptosis (by caspases pathways) and the induction of lymphotoxin ß and interferon (IFN)-γ [START_REF] He | Silymarin protects against liver damage in BALB/c miceexposed to Fumonisin B1 despite increasing accumulation of free sphingoid bases[END_REF]. Some evidence suggested that silymarin inhibited the expression of adhesion molecules, such as E-selectin [START_REF] Kang | Silymarin inhibits TNF-alpha-induced expression of adhesion molecules in human umbilical vein endothelial cells[END_REF], another family of transmembrane molecules, expressed particularly on the surface of leukocytes, involved in inflammatory pathways. In human cells, silymarin caused "in vitro" a reduction of lectin-dependent and natural killer (NK) cell mediated cytotoxicity but not of antibody dependent cell-mediated cytotoxicity (ADCC) [START_REF] Deák | Effects of two bioflavonoids on certain cellular immune reactions in vitro[END_REF].

The inhibition of the 5-lipoxygenase pathway in the cytoplasm, in particular leukotriene B4 (LTB4), at the silibinin concentrations achieved "in vivo", could well represent a pivotal pharmacological property of silymarin. The study which evaluated the action of silibin in isolated Kupferr cells, indicated a strong inhibitory effect on LTB4 (Dehmlow et al., 1996a). Similar results were found in porcine basilar arteries (IC50 = 100 µmol/L), human blood cells and human omentum endothelial cells (Dehmlow et al., 1996b) This selective inhibition of leukotriene formation by Kupffer and possibly other cells could at least partly account for the hepatoprotective properties of silibinin.

Silymarin anti-inflammatory nuclear DNA/RNA mediated effects involve nuclear factor kappa B (NF-κB), a ubiquitous rapid response transcription factor in inflammatory cells. Also, many genes encoding the proteins of the hepatic acute phase response are under the control of the transcription factor NF-κB [START_REF] Saliou | Selective inhibition of NF-kappa B activation by the flavonoid hepatoprotector silymarin in HepG2. Evidence for different activating pathways[END_REF]. Inactive NF-κB is present in the cytoplasm complexed with an inhibitory protein I-κB. NF-κB is activated by a number of incoming signals from the cell surface. Released from I-κB inhibition, NF-κB translocates into the nucleus and binds to the kappa B motif of the target gene. The NF-κB activation process can be inhibited by pharmacologic agents at each activation step; that is glucocorticoids, cyclosporine, tacrolimus, and antioxidants [START_REF] Lee | Nuclear factor kappa B: important transcription factor and therapeutic target[END_REF].

In vitro silymarin was found to suppress both the kappa B motif of NF-κB DNA binding activity and its dependent gene expression in hepatoma cells. In addition, silymarin blocked the translocation of NF-κB p65 protein (through phosphorylation) to the nucleus without affecting its ability to bind the DNA (Manna et al., 1999). Silymarin also inhibited the TNF-α-induced activation of mitogen-activated protein kinase and c-Jun N-terminal kinase and abrogated TNF-α-induced cytotoxicity and caspase activation [START_REF] Saller | An updated systematic review with meta-analysis for the clinical evidence of silymarin[END_REF]. Kang et al. reported that silymarin had exerted an inhibitory effect on nitric oxide (NO) production and inductible nitric oxide synthase (iNOS) gene expression in macrophages, although at relatively high concentrations of 12.5-25 µg/ml [START_REF] Kang | Inhibition of inducible nitric-oxide synthase expression by silymarin in lipopolysaccharide-stimulated macrophages[END_REF]. The oral administration at the dosage of 100 mg/kg attenuated NO production by peritoneal macrophages in lipopolysaccharide (LPS)-treated mice. Silymarin, also concentration-dependent, suppressed the LPS-induced production of NO in isolated mouse peritoneal macrophages. Moreover, iNOS mRNA and its protein expression were completely abrogated by silymarin in LPS-stimulated murine RAW 264.7 cells. These results suggest that silymarin inhibits NO production and iNOS gene expression by inhibiting NF-κB/Rel activation. This conclusion was subsequently confirmed by Schümann and co-workers [START_REF] Schümann | Silibinin protects mice from T celldependent liver injury[END_REF]. The authors tested silibinin in the mouse model of concanavalin A (ConA)-induced, T cell-dependent hepatitis. Silibinin significantly inhibited ConA-induced liver disease. Silibinin proved to be an immune-response modifier in vivo, inhibiting intrahepatic expression of tumor necrosis factor, interferon-g, interleukin (IL)-4, IL-2, and iNOS, and augmenting synthesis of IL-10. In addition, silibinin inhibited intrahepatic activation of NF-kB. The major mechanism of this hepatoprotective activity appears to be the inhibition of intrahepatic NF-kB activation, which prevents the subsequent synthesis of TNF, IFN-g, IL-2, and iNOS. Furthermore, while the synthesis of IL-10 was augmented, the production of IL-4 was inhibited within the liver.

Enhanced protein synthesis

Regeneration of liver cells is necessary for hepatic recovery from acute or chronic conditions. In chronic disease, fibrosis occurs simultaneously with cellular regeneration.

The ultimate outcome is determined by the type of the process which dominates.

Sonnenbichler and Zetl reported that the intra-peritoneal administration of silibinin had caused a marked increase in the synthesis of ribosomal RNA (increased polymerase I) in rat liver. The same authors found that silibinin had stimulated the synthesis of DNA in partially hepatectomised rats, but neither in healthy controls nor in hepatoma or other neoplastic cells [START_REF] Sonnenbichler | Biochemical effects of the flavonolignan silibinin on RNA, protein, and DNA synthesis in rat liver[END_REF][START_REF] Pradhan | Hepatoprotective herbal drug, silymarin from experimental pharmacology to clinical medicine[END_REF]. The exact mechanism of the action, probably involving polymerase I activation, is unknown. Silybin appeared to stimulate ribonucleic acid (RNA) polymerase-I and ribosomal RNA as it was found in several preclinical studies. This effect leads to more rapid formation of ribosomes,

Antifibrotic activity

Liver fibrosis can result in remodelling of liver architecture leading to hepatic insufficiency, portal hypertension and hepatic encephalopathy. These processes involve complex interplay of cells and mediators [START_REF] Gebhardt | Oxidative stress, plant-derived antioxidants and liver fibrosis[END_REF]. In the initial phase the proliferation of hepatic parenchymal cells is developed. The conversion of hepatic stellate cells (HSC) into myofibroblast is considered as the central event in fibrogenesis. Silymarin inhibits NF-kB and also retards HSC activation. It also inhibits protein kinases and other kinases involved in signal transduction and may interact with the intracellular signalling pathways. The evidence for anti-fibrotic activity comes largely from animal studies. Favari and Perez-Alvez reported that orally administered silymarin (50 mg/kg) to the rats with chronic carbon tetrachloride (CCl4) liver damage could reduce the collagen content in the liver of these animals up to 55% (increased with CCl4 approximately 4-to 6-fold as compared to controls) [START_REF] Favari | Comparative effects of colchicine and silymarin on CCl4chronic liver damage in rats[END_REF]. In similar experiments, there was a reduction of collagen and pro-collagen III contents after biliary obstruction in the rat by 30% with 50 mg/kg/day although not at the level of 25 mg/kg/day of silymarin [START_REF] Mourelle | Prevention of CCL4-induced liver cirrhosis by silymarin[END_REF][START_REF] Boigk | Silymarin retards collagen accumulation in early and advanced biliary fibrosis secondary to complete bile duct obliteration in rats[END_REF]. Silymarin suppressed the expression of pro-fibrogenic procollagen-α1(I) and TIMP-1 most likely via down-regulation of TGF-β1 mRNA in the rats with biliary fibrosis [START_REF] Jia | Antifibrotic effect of silymarin in rat secondary biliary fibrosis is mediated by downregulation of procollagen alpha 1(I) and TIMP-1[END_REF]. Silymarin, administered during three years, retarded the development of alcohol-induced hepatic fibrosis in baboons [START_REF] Lieber | Silymarin retards the progression of alcoholinduced hepatic fibrosis in baboons[END_REF].

Antioxidant effects

Free radicals, including the superoxide radical, hydroxyl radical (OH), hydrogen peroxide (H 2 O 2 ), and lipid peroxide radicals were implicated in liver diseases [START_REF] Urtasun | Oxidative and nitrosative stress and fibrogenic response[END_REF]. The mechanism of free radical damage included ROS-induced peroxidation of the polyunsaturated fatty acid in the bilayer cell membrane, which caused the chain reaction of lipid peroxidation, thus damaging the cellular membrane and causing further oxidation of membrane, lipids and proteins [START_REF] Nagata | Common pathogenic mechanism in development progression of liver injury caused by non-alcoholic or alcoholic steatohepatitis[END_REF]. The antioxidant properties of silibinin were evaluated by studying the ability to react with relevant biological ROS or oxidants such as superoxide anion radical (O 2 -), hydrogen peroxide (H 2 O 2 ), hydroxyl radical (HO -) and hypochlorous acid (HOCl) [START_REF] Detaille | Interrelation between the inhibition of glycolytic flux by silibinin and the lowering of mitochondrial ROS production in perifused rat hepatocytes[END_REF]. Several preclinical studies reported that MT was not a good scavenger of O 2 -and no reaction with H 2 O 2 was detected (IC50% >200 µmol/L). However, it reacted rapidly with HO -radicals in free solution at approximately diffusion-controlled rate (IC50% = 1.2-7 µmol/L). It was reported that in vitro incubation with silymarin in the concentration equivalent to the usual therapeutic dosage markedly increased the expression of superoxide dismutase (SOD) in lymphocytes in the patients with alcoholic cirrhosis (Feher et al., 1988;[START_REF] Pradhan | Hepatoprotective herbal drug, silymarin from experimental pharmacology to clinical medicine[END_REF]. Silymarin had no direct effect on ethanol metabolism and had no role in reducing ethanol levels or the rate at which ethanol was removed from the body. In fact, there is no evidence of the interaction of silymarin or silibinin with cytochrome P450-2E1, thus suggesting that these antitoxic effects are due to its antioxidant and free radical scavenging properties [START_REF] Miguez | Hepatoprotective mechanism of silymarin: no evidence for involvement of cytochrome P450 2E1[END_REF].

The phenolic conformation of silymarin is thought to permit the formation of stable compounds from hydroxylic and oxygen radicals [START_REF] Das | Protective effects of silymarin, a milk thistle (Silybium marianum) derivative on ethanol-induced oxidative stress in liver[END_REF]. The in vivo studies in rats indicate that silymarin can reduce the free radical load. The rats exposed to acetaminophen at toxic doses, had increased the levels of reduced glutathione and superoxide dismutase when treated with silymarin compared with the levels in controls [START_REF] Singh | Effects of Embelin on Lipid Peroxidation and Free Radical Scavenging Activity against Liver Damage in Rats[END_REF]. Furthermore, the other studies demonstrated that silymarin could inhibit cell lysis as measured by changes in alanine aminotransferase levels when exposing isolated hepatocytes to carbon tetrachloride and galactosamine [START_REF] Tsai | Effects of silymarin on the resolution of liver fibrosis induced by carbon tetrachloride in rats[END_REF]. The in vitro experiments with simian kidney cells damaged by paracetamol, cisplatin and vincristin demonstrated that administration of silibinin before or after the drug-induced injury could lessen or avoid the toxic effects. In the recently published study, the rats received cisplatin (3 mg/kg) intraperitoneally and silymarin (50 mg/kg) two hrs before or after cisplatin injection, during five days [START_REF] Karimi | Cisplatin nephrotoxicity and protection by milk thistle extract in rats[END_REF]. The pre-treatment with silymarin prevented the nephrotoxic effects of cisplatin while silymarin administered after the chemo-therapeutic agent reduced them to some extent.

Toxin blockade

Silymarin is a suitable candidate to treat iatrogenic and toxic liver diseases. It has a regulatory action on cellular and mitochondrial membrane permeability in association to an increase in membrane stability against xenobiotic injury. It can prevent the absorption of toxins into the hepatocytes by occupying the binding sites as well as inhibiting many transport proteins at the membrane. The phalloidin-transporting system, belonging to the hepatocyte-specific organic anion uptake transporters OATP2 [START_REF] Fehrenbach | Characterization of the transport of the bicyclic peptide phalloidin by human hepatic transport proteins[END_REF], is inhibited in a competitive way by silymarin with no influence on membrane fluidity (Sebastian et al., 2004). The OATP2 may represent the part of a hepatic equipment which clears portal blood from bile acids, lipophilic hormones or xenobiotics. The transporting system incorporates phallotoxins into the hepatocytes leading to the death of these cells.

Phalloidin (a bicyclic heptapeptide) and antamanide (a monocyclic decapeptide from Amanita phalloides) interact with bile-salt-binding polypeptides of the hepatocyte membrane. Similar or possibly identical transport mechanism inhibited by silibinin was described for amanitin [START_REF] Wellington | Silymarin: a review of its clinical properties in the management of hepatic disorders[END_REF]. Therefore, silymarin is able to reduce the cellular absorption of noxious xenobiotics other than mushroom poisons, thereby exerting cell-protection.

MT and lactation

Traditionally MT has been used by nursing mothers for stimulating milk production and it was recently reported that MT increased the lactation in cows [START_REF] Tedesco | Silymarin, a possible hepatoprotector in dairy cows: biochemical and histological observations[END_REF] and women [START_REF] Pierro | Clinical efficacy, safety and tolerability of BIO-C (micronized Silymarin) as a galactagogue[END_REF]. However, the MT mode of action has not been established yet. Prolactin is the principal lactogenic hormone and it was recently shown that MT significantly increased circulating prolactin levels in female rats [START_REF] Capasso | Silymarin BIO-C  , an extract from Silybum marianum fruits, induces hyperprolactinemia in intact female rats[END_REF]. This effect seems to involve, at least partly, dopamine D 2 receptors. In the light of these results MT (or silymarin) could be considered a good candidate for the treatment of lactation insufficiency.

The Present Use of Milk Thistle: Overview of Evidence

The available clinical studies have generally investigated the effects of proprietary products containing silymarin/silybin in patients with hepatic disorders like cirrhosis, acute and chronic hepatitis and liver disease. However, there is also an interest in the use of A systematic meta-analysis, published in the last years, evaluated nineteen studies, eleven double blind and eight single blind [START_REF] Saller | An updated systematic review with meta-analysis for the clinical evidence of silymarin[END_REF]. The authors concluded that it was reasonable to employ silymarin in the treatment scheme in the case of Amanita phalloides poisoning. Also, the available evidence supported the therapeutic use of silymarin, in view of its excellent safety profile, in alcohol-induced liver diseases, including liver cirrhosis (Child-Pugh classification grade A).

MT in alcohol-induced liver disease

Randomized, blinded, placebo-controlled studies assessed the effectiveness of MT in chronic alcoholic liver disease. The results of these trials might be conflicting and confounded because of heterogeneity of the degree of disease severity and alcoholic intake or abstinence. In a double-blind controlled study by Salmi and Sarna, one hundred and six patients with mild acute and sub-acute liver disease, of which ninety with confirmed histological diagnosis, were randomly allocated to either silymarin or placebo treatment, for four weeks [START_REF] Salmi | Effect of silymarin on chemical, functional, and morphological alterations of the liver. A double-blind controlled study[END_REF]. Alcohol was forbidden during the trial.

The difference between the two groups was significant in an important decrease in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the silymarin group. No difference, instead, in reduction of serum bilirubin in both. Among the subgroup with the second histological work-up, the normalization of histological changes occurred more often in the silymarin group (eleven out of fifteen) than in controls (four out of fourteen).

Feher and co-workers, studied thirty six patients suffering from chronic alcoholic liver disease, treating them with an oral dose of 420 mg/day of silymarin or placebo for six 15 activity were normalized in the silymarin group, with an improvement in histology of the liver and a decrease of pro-collagen III peptides (a marker of fibrotic progression in liver disease), but only a few patients had control histology (Feher et al., 1989).

In a double blind comparative study of 106 patients with histological proven alcoholic hepatitis, MT did not significantly influence the liver biopsy findings (Trichet et al., 1989).

MT in viral hepatitis

Even though silymarin does not affect viral replication, it might play a beneficial role in viral hepatitis by its inhibitory action on inflammatory cascade induced by viral infection.

Lirussi and Okolicsanyi compared silymarin and ursodeoxicholic acid in a rather heterogeneous population of patients with active cirrhosis, the majority were HCV positive [START_REF] Lirussi | Cytoprotection in the nineties: experience with ursodeoxycholic acid and silymarin in chronic liver disease[END_REF]. No efficacy was detected in this study. More recently, a new silybin-phosphatidylcholine complex was studied in a short pilot study on twenty patients with chronic active hepatitis [START_REF] Buzzelli | A pilot study on the liver protective effect of silybin-phosphatidylcholine complex (IdB1016) in chronic active hepatitis[END_REF]. Transaminase levels were reduced in silymarin group, but without consistent differences in other liver function tests (bilirubin, alkaline phosphatase, albumin).

Rambaldi et al. in the meta-analyses of randomized clinical trials valuated 915

patients with alcoholic hepatitis and hepatitis B or C [START_REF] Rambaldi | Milk thistle for alcoholic and/or hepatitis B or C liver diseases -a systematic Cochrane Hepato-Biliary Group Review with metaanalyses of randomized clinical trials[END_REF]. The primary outcome measure was the number of patients dying. The secondary outcome measures were the development of clinical symptoms and complications analyzed separately and combined with liver biochemistry, liver biopsy findings, as well as the number and type of adverse events. The authors found no significant effect of MT on all-cause mortality and observed a potential beneficial effect of MT on mortality in patients with alcoholic liver disease, but this effect could not be confirmed in two high-quality trials. A potential 16 beneficial effect of MT on liver-related mortality was observed, but again, this effect could not be demonstrated in three high quality trials.

Subsequently, Federico et al. evaluated the antioxidant and antifibrotic activity of Realsil® (complex silybin -vitamin E -phospholipids) to improve insulin resistance and liver damage in the patients with non-alcoholic fatty liver disease (NAFLD) and HCV chronic infection [START_REF] Federico | A new silybin-vitamin E-phospholipid complex improves insulin resistance and liver damage in patients with non-alcoholic fatty liver disease: preliminary observations[END_REF]. This study enrolled eighty five patients; fifty nine were affected by primitive NAFLD (group A) and twenty six by HCV related chronic hepatitis C in combination with NAFLD, all HCV genotype-1b, and non-responders to previous antiviral treatment (group B). All the patients with a diagnosed liver disease two years prior to the study, according to histological criteria, were enrolled over six consecutive months and further divided into two subgroups using a systematic random sampling procedure: fifty three [thirty nine NAFLD and fourteen HCV) were treated with four tablets/day of Realsil® (one tablet contained 94 mg of silybin, 194 mg of phosphatidylcholine, and 90 mg of vitamin E) for six months followed by another six months of follow up, while the other thirty two patients (twenty NAFLD and twelve HCV) served as a control group (no treatment). For 0, 6, and 12 months, the outcomes were:

body mass index, bright liver by ultrasonography, transaminase and GGT levels, blood glucose and insulin plasma levels with a contemporaneous determination of insulin resistance by Homeostasis Model Assessment (HOMA) test and, as indices of liver fibrosis, plasma levels of transforming growth factor β, hyaluronic acid, and metalloproteinase.

The results showed the improvement of liver enzyme levels in the treated group, but this only persisted in the group A. Hyperinsulinaemia, present in both groups, was significantly reduced only in the treated patients. The treatment with Realsil® significantly reduced all indices of liver fibrosis in both treated groups, with a persistent effect only in group B. was performed to determine the effect of silymarin in patients with cirrhosis [START_REF] Ferenci | Randomized controlled trial of silymarin treatment in patients with cirrhosis of the liver[END_REF]. It was a randomized controlled study comprising 170 patients with cirrhosis. Eighty seven patients (alcoholic forty, non-alcoholic forty-one) were treated with silymarin 140 mg oral dose, three times daily; eighty three patients (alcoholic forty five, non-alcoholic thirty eight) received a placebo. The mean observation period was forty one months. The survival rate, to four years of the treatment, was 58% in the first group and 39% in the controls. The analysis of subgroups indicated that the treatment was effective in the patients with alcoholic cirrhosis (p=0.01) and in the patients initially rated Child-Pugh A (p=0.03).

A French study group, conducted a randomized double-blind trial of silymarin versus placebo in 116 patients with histologically proven alcoholic hepatitis, fifty eight of them with cirrhosis (Trichet et al., 1989). Fifty seven patients received oral silymarin 420 mg/day and fifty nine received placebo for three months. The biological parameters were assessed in the serum, and the percutaneous liver biopsy was obtained both at the start of the trial and after three months (control histology available in thirty two silymarin and in thirty five placebo patients). The 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 but clearly correlated with abstention from alcohol.

However, this study is relatively short and half the population was not classified as having cirrhosis; since the deaths were not allocated to a diagnostic group, the entire group of the patients was considered "at risk". Bunout et al. conducted a placebo-controlled trial with silymarin in the patients with alcoholic liver cirrhosis. Seventy-one patients were randomly assigned to oral silymarin (n=34) at a relatively low dose of 280 mg/day or an equal number of placebo tablets (n=37) [START_REF] Bunout | Controlled study of the effect of silymarin on alcoholic liver disease[END_REF]. Both groups did not differ in their initial laboratory assessments and were followed up for an average of fifteen months. Ten patients died during the follow up (five in placebo and five in silymarin; liver-related death in nine). No details concerning distribution by the therapeutic group were provided. No significant differences were observed between these two groups. It was concluded that silymarin did not change the evolution in this trial. A subsequent Spanish study was specifically aimed to determine the effect of silymarin in the patients with alcoholic liver cirrhosis, respect to survival, clinical and laboratory changes [START_REF] Parés | Effects of silymarin in alcoholic patients with cirrhosis of the liver: results of a controlled, double blind, randomized and multi-center trial[END_REF]. From

February 1986 to June 1989, this randomized double-blind multicentre trial, comparing silymarin 450 mg/day in three divided doses (n=103) with placebo (n=97) enrolled 200 alcoholics with histologically-(n=191) or laparoscopically-proven (silymarin six, placebo three) liver cirrhosis. The primary outcome was the time of death and the secondary outcome was the progression of liver failure. The survival rate was similar in both groups and was not influenced by gender, persistence of alcoholic intake, severity of disease or by the presence of alcoholic hepatitis in the liver biopsies. Silymarin did not produced any significant effect on the progression of disease, but the frequency of complications was lower in the treated patients (p=0.06). The study by Bunout had the same end-point,

Toxic and iatrogenic liver disease

In spite of the large number of pre-clinical studies, the clinical trials in toxic liver disease are scarce and of poor quality. Only one study was set up to assess the ability of silymarin to antagonize or to prevent the hepatotoxic effects of tacrine [START_REF] Allain | Aminotransferase levels and silymarin in de novo tacrine-treated patients with Alzheimer's disease[END_REF].

Tacrine is an anticholinesterase drug used for the treatment of Alzheimer's disease. In this randomized double-blind placebo controlled study on twenty three patients suffering from mild to moderate dementia of the Alzheimer type, the silymarin oral dose (420 mg/day) was given for one week, and then tacrine was added, first at 40 mg/day for six weeks, then at 80 mg for six additional weeks. The statistical difference was not observed for serum ALT, but the side effects, especially gastrointestinal disorders, were much less frequent in the silymarin group. Therefore, the co-administration in the initial phases of the treatment may improve tolerability.

In the study of prophylactic MT during the treatment for tuberculosis, twenty nine subjects with normal liver function tests received anti-tuberculosis drugs plus Hepabene ® , a mixture of silymarin and Fumaria officinalis alkaloids. The differences in the AST and ALT reductions were statistically significant. Therefore, the combination of silymarin with such potentially hepatotoxic drugs may prevent such adverse reactions [START_REF] Comelli | Toward the definition of the mechanism of action of silymarin: activities related to cellular protection from toxic damage induced by chemotherapy[END_REF].

Mushroom poisoning

The Amanita phalloides mushroom ("death cap") has been known and feared for at least two millennia and continues to cause serious illness and death. The most important, cytotoxin, out of many cytotoxins produced by mushrooms, is the potent amanitin found in Phalloidin, a cyclic heptapeptide that accompanies amanitin and may interfere with actin polymerization, is probably responsible for the initial gastrointestinal symptoms. Amanita phalloides intoxications are not very frequent. Unfortunately, there are no controlled studies available and so case-control studies collected over several decades, individual cases reports and "expert opinion" were reviewed [START_REF] Saller | An updated systematic review with meta-analysis for the clinical evidence of silymarin[END_REF].

Diabetic patients with chronic liver disease

Glucose intolerance in the patients with cirrhosis results from two abnormalities that occur simultaneously: insulin resistance in muscle and inadequate response of β-cells to secrete insulin appropriately in order to overcome the defect in insulin action [START_REF] Petrides | Pathogenesis of glucose intolerance and diabetes mellitus in cirrhosis[END_REF]. Diabetes mellitus in the insulin-resistant patients with cirrhosis is the result of the progressive impairment in insulin secretion together with the development of hepatic insulin resistance leading to fasting hyperglycaemia and a diabetic glucose tolerance profile (Younossi and McCullough, 2009).

The prevalence of diabetes mellitus was reported to be higher in the patients with HCV-related cirrhosis (23.6%) than in those with HBV-related cirrhosis (9.4%) [START_REF] Negro | Hepatitis C virus and type 2 diabetes[END_REF]. The prevalence of diabetes mellitus is also associated closely with the Child-Pugh score and increasing age. In a study by Velussi et al., the authors showed the effectiveness of a long-term treatment with MT to reduce lipoperoxidation and insulin resistance in the diabetic patients with cirrhosis [START_REF] Velussi | 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[END_REF]. A group of patients was enrolled to receive an oral dose of 600 mg/day of silymarin plus standard therapies, while the control group standard therapies alone. The trials outcomes were: fasting blood glucose levels, daily glucosuria, glycosylated haemoglobin (HbA1c) and malondialdehyde levels. There was a significant decrease in fasting blood glucose levels and in fasting 40%) already after four months of the treatment in the silymarin group, also seen for the mean daily blood glucose levels (-14.6%) and daily glucosuria (-32%).

However, the HbA1c levels at the end of twelve months were lowered by 8.8%. In addition, there was a significant decrease in the mean exogenous insulin requirements in the treated group from 55 ± 5 IU/day to 45 ± 3 IU/day after six months and 42 ± 2 IU/day after twelve months, while the untreated group showed the significant increase in fasting insulin levels and a stabilized insulin requirement. The authors concluded that these results

showed that silymarin might reduce the lipoperoxidation of cell membranes and insulin resistance, significantly decreasing endogenous insulin overproduction and the need for exogenous insulin administration, thus reducing the daily insulin dose required by almost 25% patients.

Preparations of Milk Thistle for humans

A water-soluble derivative of silybinin (silybinin dihemisccinate disodium) is available in Europe for Amanita phalloides poisoning. Madaus Legalon ® is produced in tablets containing 70 or 140 mg silymarin and is given in a dose of one to two tablets up to three times daily, with a maximum dosage of 420 mg. The formulation includes extraction with alcohol, filtration and evaporation and may also include pressing, heat drying, and blending with other compounds. Some brands may add choline, inositol, tumeric extract, artichoke extract, whole herb powder, dandelion, licorice, curcuma, boldo, iron, or vtamins A and C.

One formulation is combined with kutkin, the roots and rhizome of Picrorhiza kurroa, a perennial herb found only in the higher mountains of the north-western Himalayas. Other concentrated oral formulations include tablets and softgel capsules. Silipide ® is a complex of one part of silybin and two parts of phosphatidycholine from soybean phopholipids (lecithin), for which the standardization is expressed as silybin equivalents. It was more 22 bio-available than standardized silymarin after oral ingestion in the normal volunteers, cirrhotics, and patients after cholecystectomy [START_REF] Barzaghi | Pharmacokinetic studies on IdB 1016, silybin phosphatidylcholine complex, in healthy human subjects[END_REF][START_REF] Federico | A new silybin-vitamin E-phospholipid complex improves insulin resistance and liver damage in patients with non-alcoholic fatty liver disease: preliminary observations[END_REF].

The bioavailability of silybin in Silipide ® is approximately tenfold greater than the silybin content of standard MT preparations [START_REF] Morazzoni | Silybum marianum (Carduus marianus)[END_REF]. Other preparations containing MT standardized to silymarin or to silybin are also found in commerce in different forms (granulates, tablets or coated tablets and drops).

Complications and adverse effects

Silymarin is reported to have a very good safety profile. No side-effects were observed in volunteers following a single oral dose of silymarin corresponding to 254 mg silybin [START_REF] Weyhenmeyer | Study on dose-linearity of the pharmacokinetics of silibinin diastereomers using a new stereospecific assay[END_REF]. The patients with liver disorders of various origins who received oral silymarin (600-800 mg/day) for six months did not manifest the adverse effects [START_REF] Velussi | Silymarin reduces hyperinsulinemia, malondialdehyde levels, and daily insulin need in cirrhotic diabetic patients[END_REF][START_REF] Palasciano | The effect of silymarin on plasma levels of malon-dialdehyde in patients receiving long-term treatment with psychotropic drugs[END_REF]. The clinical trials involving about 3500 patients, of whom 2637 with liver disease, treated with Legalon (560 mg/day) for eight weeks reported that the frequency of adverse effects with silymarin was about 1%.

The pooled adverse effects were transient gastrointestinal complaints like bloating, nausea, dyspepsia and diarrhea. (Leng-Pschow, 1996).

It was also shown that MT was generally non toxic and without side effects when administered to the adults in an oral dose range of 240-900 mg/day in two or three divided doses. At higher doses of more than 1500 mg/day silymarin may produce a laxative effect with an increased bile flow and secretion. Among the randomized controlled studies neither optimal nor maximal therapeutic doses were defined. In most of the trials the reportedly affective daily doses of silymarin were 420-600 mg [START_REF] Saller | An updated systematic review with meta-analysis for the clinical evidence of silymarin[END_REF].

However the case reported described an adverse reaction of the MT herbal medication. The symptoms included severe sweating, abdominal cramping, nausea, vomiting, diarrhea and weakness (Adverse Reaction Advisory Committee, 1999). Another report described an anaphylactic shock in a 54-year-old man with immediate-type allergy to kiwi fruit [START_REF] Geier | Anaphylactic shock due to an extract of Silybum marianum in a patient with immediate-type allergy to kiwi fruit[END_REF].

Preclinical data has not documented the acute toxicity for sylimarin and silybin.

Silymarin, in particular, given orally to mice and dogs at doses of 20 or 1 g/kg did not cause adverse effects or mortality. However, a long-term oral administration of silymarin (100 mg/kg/day) to rats for 22 weeks did not also cause the adverse effects [START_REF] El-Bahay | Influence of tumor necrosis factor-alpha and silibin on the cytotoxic action of alpha-amanitin in rat hepatocyte culture[END_REF][START_REF] Saller | An updated systematic review with meta-analysis for the clinical evidence of silymarin[END_REF]. MT preparations are contra-indicated for the individuals with hypersensitivity to Asteraceae. None of the studies on cirrhosis, carried out with silymarin, considered clinical outcomes other than death as the primary end-point. Based on the available data, several issues could be partially examined in the above mentioned studies.

Different issues were usually addressed in different trials; although these issues are interrelated, they have to be regarded individually and cannot be consolidated into one global picture.

Bleeding oesophageal varices constitute one of the most serious complications of cirrhosis. The total incidence of upper gastrointestinal bleedings (UGBs), both as a cofactor of death and as "last data", were reported in the two largest trials: 4.6% with silymarin vs. 9.6% with placebo, and 6.3% vs. 13.5% 79, respectively, and showed the differences in favour of the active treatment [p=0.042; Odds Ratio (OR) 0.44 (95% CI 0.20, 0.97)] [START_REF] Ferenci | Randomized controlled trial of silymarin treatment in patients with cirrhosis of the liver[END_REF]. These findings suggest that the decreased rate of UGBs reflects an overall improvement in the patients, also evidenced by the lower liver-related mortality rate, rather than being a direct effect of silymarin.

The Future: New Prospective uses of Milk Thistle

The cancer chemopreventive role of MT flavonolignans has been reported in recent literature [START_REF] Mazzio | In vitro screening for the tumoricidal properties of international medicinal herbs[END_REF]. Silymarin modulates imbalance between cell survival and apoptosis through interference with the expressions of cell cycle regulators and proteins involved in apoptosis. In addition, silymarin also showed anti-inflammatory, anti-angiogenic and anti-metastatic effects [START_REF] Ramasamy | Multitargeted therapy of cancer by silymarin[END_REF]. Particularly, the protective effects of silymarin and its major active constituent, silibinin, studied in a variety of in vitro and in vivo cancer models, including liver cancer, suggest that they should be established in therapies as adjuncts in the clinical application in these patients to prevent or reduce chemotherapy as well as radiotherapy-induced toxicity. The molecular mechanisms of silibinin-mediated antiproliferative effects are mainly via receptor tyrosine kinases, androgen receptor, NF-κB, cell cycle regulatory and apoptotic signalling pathways in various cancer cells [START_REF] Li | Targeting silibinin in the antiproliferative pathway[END_REF]

  and liver regeneration, increase lactation and possess immuno-modulation activity. The following is the synthesis of the selected studies on this subject.

  IC50 of 15 µmol/L silibinin

  increases protein synthesis. This action has important therapeutic implications in repairing the damaged hepatocytes and restoring the normal liver functions.

  silymarin in toxin-(mushroom A. phalloides) induced hepatitis.The Agency for Healthcare Research and Quality data, published in 2002, analyzed sixteen prospective placebo-controlled trials. Twelve studies used Legalon®; eight of which used a dosage of 240 to 800 mg/day for the treatment period varying from seven days to six years. No information about doses or duration was reported in four studies. In remaining four of placebo-controlled studies, two used silymarin (no further characterization regarding preparation) and two studies used Silipide®, a silybin phosphatidyl cholin complex (240 mg/day). Seventeen additional trials did not include controls. Silymarin, silybin, silipide and silimarol were used with no standardized dose in nine studies. In the remaining two trials, silymarin was administered to the subjects with no liver disease diagnosed. These trials were probably aimed to determine the prophylactic in the subjects treated with concomitant anti-tuberculosis drugs and tacrine, but the results were contradictory, for the absence of hepatic illness.

  months. Serum bilirubin, transaminase values and gamma glutamil transferase (GGT)

  More recently El-Kamary et al. conducted a randomized controlled trial of silymarin vs placebo with 105 patients with symptoms compatible with acute clinical hepatitis and serum alanine aminotransferase levels > 2.5 times the upper limit of normal[START_REF] El-Kamary | A randomized controlled trial to assess the safety and efficacy of silymarin on symptoms, signs and biomarkers of acute hepatitis[END_REF]. The patients received 420 mg/day of silymarin (Legalon  ) or vitamin placebo for four weeks. The treatment with silymarin significantly reduced the symptoms related to biliary retention (dark urine, jaundice, scleral icterus), while the biomarkers considered (transaminase and direct bilirubin) were not significantly modified.Liver cirrhosisClinical trials in liver cirrhosis are complicated by a series of confounding factors, such as concomitant therapies and complications of cirrhosis. The study byFerenci et al.

  and Parés, however a low dose was used, and it did not state whether it was properly blinded.

  to the genera Amanita and Galerina. Amanitin is a cyclic octapeptide which inhibits RNA polymerase II, thus interfering with protein synthesis.

Figure 1 :

 1 Figure 1: Distribution of Milk thistle in European countries (in black).

Figure 2 :

 2 Figure 2: Chemical formulas of Silybin, Silydianin, Isosilibyn and Silychristin.

Table 1 :

 1 . Additional studies are necessary to evaluate chemotherapeutic effects of silymarin. Younossi ZM, McCullough AJ. 2009. Metabolic syndrome, non-alcoholic fatty liver disease and hepatitis C virus: impact on disease progression and treatment response. Liver Int. 29: Main constituents of Milk Thistle.

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sterols (cholesterol, campesterol, stigmasterol and sitosterol) 25-30% protein, some mucilage THE FRUITS flavonoids (apigenin and its 7-O-glucoside, 7-O-glucuronide; 4,7diglucoside, kaempferol and its 7-glucoside and 3-sulphate) luteolin and its 7-glucoside sitosterol and its glucoside triterpene acetate fumaric acid THE HERB polyacetylenes

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http://mc.manuscriptcentral.com/ptr Phytotherapy Research

http://mc.manuscriptcentral.com/ptr


Kádár

Ferenc Budán
email: ferencbudánbudfer2@gmail.com

István Szabó

Tímea Varjas

Ghodratollah Nowrasteh

Tamás Dávid

Péter Gergely

Zsuzsa Varga

Kornélia Molnár

Balázs Kádár

Zsuzsa Orsós

István Kiss

István Ember

Mixture of Uncaria and Tabebuia Extracts are Potentially Chemopreventive in CBA/Ca Mice -A Long-term Experiment

Keywords: Gene expression, carcinogenesis, chemoprevention, DMBA, Uncaria, Tabebuia Gene expression, carcinogenesis, chemoprevention, DMBA, Uncaria, Tabebuia

A long-term experimental animal model was developed by our research group for the evaluation of potential chemopreventive effects. The inhibitory effects of agents on carcinogen (7,anthracene (DMBA)) induced molecular epidemiological biomarkers, in this case the expression of key onco/suppressor genes were investigated.

Expression pattern of c-myc, Ha-ras, Bcl-2, K-ras protoonco and p53 tumoursuppressor genes were studied to elucidate early carcinogenic and potential chemopreventive effects.

Consumption of so called "CoD TM tea" (abbreviation of Claw of Dragon tea) containing bark of Uncaria guianensis, Cat's Claw (Uncaria sp. U. tomentosa) and Palmer trumpet-tree (Tabebuia sp. T. avellanedae) was able to decrease the DMBA-induced onco/suppressor gene overexpression in a short-term animal experiment. In following study CBA/Ca mice were treated with 20 mg/bwkg DMBA intraperitoneally (ip.) and expression patterns of onco/suppressor genes were examined at several time intervals. According to the examined gene expression patterns in this long-term experiment the chemopreventive effect of CoD TM tea consumption could be confirmed. In our previous studies a unique animal test model was developed through the investigation of onco/suppressor genes expression patterns, as molecular epidemiological biomarkers (Ember et al., 1998). 7,12-dimetilbenz[a]anthracene (DMBA) is a pluripotent and complete carcinogen, therefore it is applied to induce carcinomas in animal experiments. DMBA increases the expression of c-myc, Ha-ras, Bcl-2, K-ras protoonco (Gyöngyi and Somlyai, 2002) and consequently p53 tumoursuppressor genes in the lungs and in the liver, as well [START_REF] Sándor | The function of the p53 gene suppressor in carcinogenesis[END_REF] in several animal species and also in both sex, indicating both carcinogenic exposure and early steps of tumour formation [START_REF] Ember | Effect of 7,12-dimethylbenz[a]anthracene on onco/suppressor gene action in vivo: a short-term experiment[END_REF]. The purpose was to study the early biological effect of DMBA and the protective effect of some potential chemopreventive agents in so called "CoD TM tea" (abbreviation of Claw of Dragon tea).

Consumption of "CoD TM tea", which contains extract of Uncaria guianensis, Cat's Claw (Uncaria sp. U. tomentosa) and Palmer trumpet-tree (Tabebuia sp. T. avellanedae), was able to decrease the DMBA-induced overexpression of the genes mentioned above, in a short-term animal experiment [START_REF] Zs | Effect of CoD TM tea consumption in a "short-term" test system on the expression of onco-and tumor suppressor genes[END_REF]. According to pharmacological studies Uncaria species have cytotoxic, anti-inflammatory, antiviral, immunostimulative, antioxidant and antibacterial properties [START_REF] Heitzman | Ethnobotany, phytochemistry and pharmacology of Uncaria (Rubiaceae)[END_REF]. Keplinger et al. confirmed that the pentacyclic oxindol alkaloides (POA) are the main biological active components of Uncaria species, which in vitro activate lymphocyte-proliferation regulating factor in endothelial cells [START_REF] Laus | Alkaloids of Peruvian Uncaria tomentosa[END_REF][START_REF] Keplinger | Uncaria tomentosa (Willd.) DC. -Ethnomedicinal use and new pharmacological, toxicological and botanical results[END_REF]. The bark of Palmer trumpet-tree is abundant in lapachol (2hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone) (2-7%), lapachone and xyloidone [START_REF] De Santana | Antitumoral and toxicological properties of extracts of bark and various wood components of Pau d'arco (Tabebuia avellanedae)[END_REF], which are anticarcinogenic [START_REF] Ueda | Production of anti-tumour-promoting furanonaphthoquinones in Tabebuia avellanedae cell cultures[END_REF] and inhibit the growth of the human keratinocyte cell line HaCaT culture in vitro [START_REF] Müller | Potential antipsoriatic agents: lapacho compounds as potent inhibitors of HaCaT cell growth[END_REF]. Our research group supposed that CoD TM tea consumption was able to decrease the overexpression of certain key onco/suppressor genes. In this long term study the effect of CoD TM tea was investigated on the expression of c-myc, Ha-ras, Bcl-2, K-ras and p53 onco/suppressor genes in CBA/Ca mice test model [START_REF] Cs | Comparative studies on genotoxic and carcinogenic effects of different cytostatic protocols. I. In vivo cytogenetic analyses in CBA mice[END_REF][START_REF] Ember | Szekunder tumorok állatkísérletes modellje. In vivo citosztatikus kezelési protokollok karcinogén-leukemogén hatása[END_REF][START_REF] Gyöngyi | Deuterium depletion can decrease the expression of C-myc Ha-ras and p53 genes in carcinogen-treated mice[END_REF].

Materials and methods

Animals and Treatments.

Infuse of CoD TM tea (D/Eu reg. numb.: PZN Deutschland: 4226037) was prepared according to the instruction of the manufacturer. 10g of the powder was suspended in 750 ml tap water and left to soak for twelve hours, and then it was heated to 80 ºC for thirty minutes. After filtration and water refill, the infuse was kept in a closed bottle avoiding light at 4 ºC. 250 ml extract is advised to be consumed three times a day, therefore the human dose is 0.167 g powder/kg per os (calculated for 60 kg mean body weight). Calculated equivalent dose for mice is 0.0033 g powder in 3 ml tap water.

Quantity of total pentacyclic oxindol alkaloides (POA) is supposed to be 0.1-1.12%, based on the results of Csapi et al. [START_REF] Csapi | Phythochemical analysis of Uncaria products commercially available in Hungary[END_REF].

Six-to eight-week-old (20±4 g) conventionally kept CBA/Ca inbred H-2 K haplotype mice (6 males and 6 females in each group) were used for this experiment according to the following arrangement: control groups, DMBA treated groups, CoD TM consuming groups and DMBA treated groups with CoD TM consumption. These setups were measured at five time intervals after the first DMBA treatment: 1 week, 1 month, 3, 6 and 12 months later.

Control groups were given tap water and treated with ip. corn oil. DMBA treated groups received 20 mg/bwkg DMBA. (Sigma Aldrich Budapest, Hungary) dissolved in corn oil (Teva Pharmaceutical Industries Ltd. Debrecen, Hungary) intraperitoneally. DMBA treatment was applied at the beginning of the experiment and in addition two months later.

Gene expression investigations.

One week, one, three, six and twelve months later the actual four groups were sacrified and their liver, lungs, kidneys and spleen were removed. 100 mg tissue samples from each organ were collected, and samples of one group were pooled.

After homogenization of the organs, total cellular RNA was isolated using TRIZOL reagent (Invitrogen, Paisley, UK). The RNA quality was checked by denaturing gel-electrophoresis, and absorption measurement was performed at 260/280 nm (A260/A280 was over 1.8). After the necessary dilution, 10 µg RNA was dot-blotted onto Hybond N + nitrocellulose membrane (ECL kit, Amersham, Little Chalfont, UK) and hybridized with chemiluminescently labelled specific probes for c-myc, Bcl-2, K-ras, p53 and Ha-ras (Professor J. Szeberényi, University of Pécs, Hungary) genes. Isolation of RNA, hybridization and detection were performed

Results

In this study we investigated the level of certain onco/suppressor gene expressions. We found, that in the control group basal c-myc, Ha-ras, p53, Bcl-2 and K-ras gene expressions vary both in time and in different organs. In the liver of male mice gene expression was low comparing to the beta-actin reference with a slight fluctuation (Figure 1.), in the lungs and kidneys (Figure 3, 5.) key onco/suppressor expression was higher with the same reference.

Compared to the results of male mice (Figure 1, 3, 5, 7.) we found, that in female mice (Figure 2, 4, 6, 8.) gene expressions were slightly lower nearly in every organ. Gene

The effect of DMBA treatment:

DMBA caused the overexpression of the investigated key onco/suppressor genes. Male mice showed major elevations, especially in the lungs, kidneys and spleen (Figure 3, 5, 7.). In the lungs and kidneys these overexpressions decayed after one month but in the spleen key gene expressions remained high.

In the liver the key gene expression elevations were present -they were not as high as in other organs (Figure 1.). Moreover, at the 12 th month control major elevations were present. In the lungs, spleen and kidneys key gene expression elevations were detected.

In male mice highly increased key gene expression levels were measured in the lungs, kidneys and spleen (Figure 3, 5, 7.). In female mice similar -but milder -gene expressions elevations were detected in the organs mentioned above (Figure 4, 6, 8.).

In female mice major gene expression elevations were present at the 12 th month in every organ (Figure 2,4,6,8.).

The effect of CoD TM on DMBA treatment:

We compared the results of CoD TM tea consumption on DMBA treated mice to the DMBA induced overexpressed cases (positive control). In case of the DMBA we arbitrary chose more than 100% gene expression increase as overexpression, and in case of the CoD TM treatment we chose more than 100% key onco/suppressor gene expression decrease as major decrease in comparison to the positive control.

In Table 1. (male) and Table 2. (female) we highlighted the organs -and also the overexpressed genes -where major decrease were present.

Comparing to the DMBA treated group, COD TM tea consumption decreased onco/suppressor gene expression in the following cases: checking the one week control in the kidneys all of andp53 expression (Figure 1, 2, 5, 6, 7, 8.). Ha-ras also decreased in the lungs, the kidneys and spleen ( Figure 3,4,5,6,7,8.). There was decrease of Bcl-2 in liver and the kidneys (Figure 1, 2, 5, 6.) and K-ras in liver, the lungs and spleen (Figure 1, 2, 3, 4, 7, 8.).

In female mice we found fewer differences than in male mice, the definite decrease of onco/suppressor gene expressions are shown in Table 2.

We highlight the kidney (Figure 6.) at the one week control, in which c-myc, Ha-ras, p53 and K-ras onco/suppressor gene expression levels were decreased. In the lungs, kidneys and spleen almost every onco/suppressor gene expression was dropped at the 12 th month (Figure 4, 6, 8.).

Discussion

The increase of c-myc, Bcl-2, K-ras, Ha-ras and p53 gene expression indicates early carcinogenic effects (Gyöngyi and Somlyai, 2002) and their overexpression is characteristic in certain manifest tumours. The amplification and overexpression of c-myc gene was found to exert an initiator role in human and in mice breast carcinogenesis as well (Dickinson et al., 1992). The activation and overexpression of Ha-ras protooncogene through point mutations was detected in colorectal carcinomas, as well [START_REF] Csontos | Oncogene and tumor suppressor gene expression changes in the peripheral blood leukocytes of patients with colorectal cancer[END_REF]. Moreover Bcl-2 is an antiapoptotic gene, which is capable of causing immortalisation of tumour cells, especially if it is overexpressed [START_REF] Norton | Coupling of cell growth control and apoptosis functions of Id proteins[END_REF]. The overexpression and activation of K-ras contribute to the formation of metastasis [START_REF] Smakman | Control of colorectal metastasis formation by K-Ras[END_REF] and enhances initiation and progression as well. The carcinogenic effect of DMBA in the lungs is well known [START_REF] Stowers | Formation and persistence of benzo(a)pyrene metabolite-DNA adducts[END_REF], and correlates to our finding, because due to the treatment, all of the examined genes had increased expression in comparison to the control in both sexes. According to the gene expression patterns, CoD TM tea consumption in the lungs of male group seems to exert tumour formation inhibitory effect, usually as the results of several anticancer effects, such as anti-inflammatory, antioxidant [START_REF] Heitzman | Ethnobotany, phytochemistry and pharmacology of Uncaria (Rubiaceae)[END_REF] and DNA topoisomerase II partial inhibitory [START_REF] Esteves-Souza | Cytotoxic and DNA-topoisomerase effects of lapachol amine derivatives and interactions with DNA[END_REF] effects, as well.

Complete (both initiator and promoter) chemical carcinogens activate and overexpress Ha-ras proto-oncogene (Dickinson et al., 1992;[START_REF] Perjési | Effect of the chalcone analog E,E-bis(2-hydroxybenzylidene) acetone on the 7,12dimethylbenz[a]anthracene-induced Ha-ras gene activity in vivo[END_REF], so the Ha-ras overexpression caused by DMBA correlates to these facts. Furthermore, CoD TM tea consumption decreased the overexpression of onco/suppressor genes induced by DMBA in the kidneys of male mice, presumably due to the result of antitumour properties of lapachone [START_REF] Ueda | Production of anti-tumour-promoting furanonaphthoquinones in Tabebuia avellanedae cell cultures[END_REF] and inhibitory effects of POA on cancer cell proliferation [START_REF] Lee | Inhibition of phospholipase cgamma1 and cancer cell proliferation by triterpene esters from Uncaria rhynchophylla[END_REF].

Generally speaking, in male animals CoD TM tea consumption seemed to exert higher chemopreventive effect on carcinogen increased onco/suppressor gene expression, than in female groups. This could be explained with the noncompetitive, concentration-dependent oestrogen receptor binding property, and therefore oestrogen inhibitory effect of U. tomentosa's. But -according to the litarature-in long-term experiment, tamoxifen like paradox oestrogen enhancer effects were found [START_REF] Salazar | Depletion of specific binding sites for estrogen receptor by Uncaria tomentosa[END_REF].

Oestrogen receptor beta (ERbeha) is supposed to be effected [START_REF] Bukulmez | Inflammatory status influences aromatase and steroid receptor expression in endometriosis[END_REF] by the oestrogen receptor activating effect of Uncaria extracts.

Anti-inflammatory activity of Uncaria extracts [START_REF] Heitzman | Ethnobotany, phytochemistry and pharmacology of Uncaria (Rubiaceae)[END_REF] is able to silence COX-2, thereby aromatase expression as well [START_REF] Sirianni | Inhibition of cyclooxygenase-2 down-regulates aromatase activity and decreases proliferation of Leydig tumor cells[END_REF], leading to oestrogen level decreasing. Oestrogen level decreasing might slow the proliferation of certain sensitive cells [START_REF] Sirianni | Inhibition of cyclooxygenase-2 down-regulates aromatase activity and decreases proliferation of Leydig tumor cells[END_REF]. However, oestrogen increased the expression of Ha-ras -and [START_REF] Sándor | The function of the p53 gene suppressor in carcinogenesis[END_REF] -in endometrial cancer cells via increasing tyrosine kinase (TK) activity [START_REF] Fujimoto | Estrogen induces c-Ha-ras expression via activation of tyrosine kinase in uterine endometrial fibroblasts and cancer cells[END_REF]. Aromatase synthetize oestrogen in males dominantly, thus the inhibition of it underpins the higher efficacy of CoD TM tea in male groups, than in observed in female groups.

According to the expression patterns of the examined molecular epidemiological key onco/suppressor biomarker genes, in this long-term experiment the chemopreventive effect of CoD TM tea consumption could be confirmed.

F

  according to the instructions of the manufacturer. The membranes were rehybridized with the constitutively expressed beta-actin gene as a positive control. The chemiluminescent signals were detected on X-ray films, scanned into a computer and evaluated by Quantiscan software (Biosoft, Cambridge, UK). The results were expressed in the percentage of the positive control. (Data are shown in Figure 1-8.)

  the liver were nearly the same (Figure 1, 2.), but in the lungs and in the kidneys expression was lower in female mice (Figure 4, 6.).

  /suppressor gene levels decreased (Figure5, 6.). In the lungs c-myc, Ha-ras, p53, K-ras showed major decrease (Figure3, 4.) and in the spleen c-myc, Ha-ras and K-ras declined at the same time point (Figure7, 8.). In case of liver, the kidneys and spleen at one year, results showed decreasing of c-myc

Figure 4 .Figure 5 .Figure 6 .Figure 7 .Figure 8 .

 45678 Figure 4. The gene expression profile of the lungs of female CBA/Ca mice

Table 1 .

 1 The effect of CoD TM on key onco/suppressor genes in male CBA/Ca mice

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Table 2 .

 2 The effect of CoD TM on key onco/suppressor genes in female CBA/Ca mice

	100
					control	DMBA	COD tea		COD + DMBA
	80
	60
	40
	20
	0	c-myc	Ha-ras	p53	BCL2	F o r K-ras c-myc Ha-ras p53 BCL2	K-ras	c-myc	Ha-ras	p53	BCL2	K-ras	c-myc	Ha-ras	p53	BCL2	K-ras	c-myc	Ha-ras	p53	BCL2	K-ras
				1 week			P 1 month			3 month					6 month				12 month
	e Figure 1. The gene expression profile of liver of male CBA/Ca mice
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Acknowledgements

The authors express their special thanks to Zsuzsanna Bayer and Mónika Herczeg for their valuable technical assistance.