SYNERGISTIC COMPOSITIONS CONTAINING LYCOPENE AND SILYMARIN FOR TREATMENT OF LIVER DISEASE

Abstract: The present invention provides a novel synergistic composition comprising lycopene and sylimarin and its use in treating liver diseases. The composition of the invention is further effective as a free oxygen radical scavenger, thus the composition of the invention is effective in treating liver diseases induced by free oxygen radical formation.

Description: SYNERGISTIC COMPOS ITIONS CONTAIN ING LYCOP ENE AND SI LYMARIN FOR TREATMENT OF LIVER DISEASE Field of the Invention The present invention concerns a composition containing lycopene and sUymarin. The present invention more particularly concerns the synergistic mixture of both lycopene and sUymarin and its use in the treatment of acute, subacute and chronic liver diseases, Background of the Invention Toxic nutritive liver diseases are the most frequent metabolic diseases of the modem world, in particular the alcohol-induced liver diseases like alcohol hepatitis, alcohol-induced fatter liver hepatitis and alcohol-induced liver cirrhosis. Free oxygen radical formation is found to be involved in ethanol-induced hepatotoxicity in experimental models and in man (Younes and Strubelt, 1987). Lycopene is a β-carotinoid with radical-scavenging properties. Among other radical-scavengers like thiols or flavonoids, lycopene is specialized to scavenge singlet-oxygen (Gerster, 1997). This action results in a protection of hepatocytes against carbon tetrachloride induced injury and lipid peroxidation (Kirn, 1995). SUymarin is an extract of mild thistle fruits containing three main flavonoids, silibinin, silidianin and silicristin (Wagner et al., 1968), a class of flavonolignanes. Three main mechanisms of hepatoprotection are discussed: 1. Stimulation of RNA-polymerase 1 in the cell nucleus and thereby stimulation of liver cell regeneration (Sonnenbichler and Zetl, 1984) 2. Stabilization of lipid cell membranes 3. Antiperoxidative properties by scavenging free radicals (Feher and co workers, 1987) Clinical experiences with sUymarin-containing drugs have proven efficacy in the treatment of toxic liver diseases (Ferenci et al., 1989; Fintelmann and Albert, 1980; Floersheim et al., 1980). Even, adjuvant treatment of chronic viral hepatitis and liver cirrhosis were reported (Kiescwetter et al., 1977; Ferenci et al., 1989). Objectives It is an objective of the present invention to provide a novel synergetic pharmaceutical composition. It is a further objective of the present invention to provide a novel synergetic dietary composition. Yet an additional objective of the present invention is to provide a composition effective in treating liver disorders. It is a further objective of the present invention to provide a mixture to scavenge free radicals of different origins in order to counteract the inflammation, toxic cell injury and lipid peroxidation involved in the alcohol-induced liver diseases such as alcohol hepatitis, alcohol-induced fatter liver hepatitis, and alcohol-induced liver cirrhosis. Summary of the Invention The present invention provides a synergetic mixture comprising of lycopene and sUymarin. Furthermore, the present invention provides a method for inhibiting or preventing free oxygen radical formation in a subject, wherein said method comprises adrriinistering to a subject a free oxygen radical scavenging effective dose of the synergistic mixture of the present invention. Furthermore, the present invention provides a use of a novel synergistic mixture comprising lycopene and sylimarin in the preparation of a medicament for treating liver diseases. Additionally, the present invention provides a use of a novel synergistic mixture comprising lycopene and sylimarin in the preparation of a dietary composition. Further provided by the present invention is a pharmaceutical composition comprising lycopene and sylimarin. Description of the Drawings Fig. 1 is a graph of GPT U I perfusate levels over time and is a comparison of ethanol; ethanol + dimethyl sulfoxide; and ethanol + silymarine. Fig. 2 is a graph of GPT U/I perfusate levels over time and is a comparison of ethanol; ethanol + tetrahydrofuran + butylated hydroxytoluene; ethanol + lycopene; and ethanol + Lyc-O-mato®. Fig. 3 is a graph of GPT U/I perfusate levels over time and is a comparison of ethanol; ethanol + dimethyl sulfoxide + tetrahydrofuran + butylated hydroxytoluene; ethanol + lycopene + silymarine; and ethanol + Lyc-O-Mato® + silymarine. Fig. 4 is a graph of LDH U/I perfusate levels over time and is a comparison of ethanol; ethanol + dimethyl sulfoxide; and ethanol + silymarine. Fig. 5 is a graph of LDH U/I perfusate levels over time and is a comparison of ethanol; ethanol + tetrahydrofuran + butylated hydroxytoluene; ethanol + lycopene; and ethanol + Lyc-O-mato®. Fig. 6 is a graph of LDH U/I perfusate levels over time and is a comparison of ethanol; ethanol + dimethyl sulfoxide + tetrahydrofuran + butylated hydroxytoluene; ethanol + lycopene + silymarine; and ethanol + Lyc-O-Mato® + silymarine. Fig. 7 is block diagram of GSH levels in the liver and is a comparison of ethanol; ethanol + dimethyl sulfoxide; and ethanol + silymarine. Fig. 8 is block diagram of GSH levels in the liver and is a comparison of ethanol; ethanol + tetrahydrofuran + butylated hydroxytoluene; ethanol + lycopene; and ethanol + Lyc-O-mato®. Fig. 9 is block diagram of GSH levels in the liver and is a comparison of ethanol; ethanol + dimethyl sulfoxide + tetrahydrofuran + butylated hydroxytoluene; ethanol + lycopene + silymarine; and ethanol + Lyc-O-Mato® + silymarine. Fig. 10 is block diagram of MDA levels in the liver and is a comparison of ethanol; ethanol + dimethyl sulfoxide + tetrahydrofuran + butylated hydroxytoluene; ethanol + lycopene + silymarine; and ethanol + Lyc-O-Mato® + silymarine. Fig. 11 is block diagram of MDA levels in the liver and is a comparison of ethanol; ethanol + tetrahydrofuran + butylated hydroxytoluene; ethanol + lycopene; and ethanol + Lyc-O-mato®.. Fig. 12 is block diagram of MDA levels in the liver and is a comparison of ethanol; ethanol + dimethyl sulfoxide; and ethanol + silymarine. Detailed Description of the Invention The following description is illustrative of embodiments of the invention. The following description is not to be construed as limiting, it being understood that the skilled person may carry out many obvious variations. The present invention is based on the unexpected discovery that there is a surprising synergism between lycopene and silymarin in counteracting the free oxygen radical formation found to be involved in alcohol-induced liver diseases. The source of lycopene can be tomato oleoresin, algal, fermented, fungal, genetically modified organism (GMO), synthetic lycopene, and mixtures thereof. Lycopene and Lyc-O-mato®. (6% tomato oleoresin) were obtained from the Lycored Natural Products Industries Ltd. Silymarin extract was delivered by the Paul Muggenburg Company, Germany. The combination of silymarin and Lyc-O-mato® (the latter a 6% tomato oleoresin) was found to be most effective in counteracting the effects of alcohol-induced liver diseases such as alcohol-induced fatter liver hepatitis, and alcohol-induced liver cirrhosis. The relative parts by weight of silymarin to lycopene is from 50: 1 to 1 :250, preferably 30: 1 to 1:30. Ethanol hepatotoxicity could be tested in an ex-vivo model because of high ADH-activities which are necessary for the ethanol metabolism and oxygen radical formation (Younes and Strubelt, 1987). As a measure of hepatotoxic response to ethanol (3%o) the release of enzymes (LDH, GLDH, GPT) into the perfusate are determined in a time dependent manner and also functional parameters are looked at like perfusion flow, bile flow, oxygen consumption, glucose, lactate and pyruvate release into the perfusate. Biochemical parameters at the end of the perfusion in the livers give information on mechanistic aspects like lipid peroxidation (MDA), ATP-depletion or glutathione depletion (GSH/GSSG). Alterations in the liver weight are indicative of general toxicity (hydropic swelling of injure cells). The composition of the present invention can be used straight or contain dietary components, additives, excipients, binding agents, coatings, preservatives, and mixtures thereof. The composition of the present invention can be contained in a variety of dosage forms such as tablets, caplets, vegecaps, and hard shell gelatin capsules. Examples General HepG2, a human hepatocellular tumor cell line (ATCC-Nr. HB 8065) was cultured in RPM1/1640 /Boehringer, Mannheim, Germany) medium supplemented with 5% fetal calf serum and l% L-glutamine. The neutral-red assay was performed as a measure of cell growth according to Borenfreund and Puerrier (1984). Enzyme releases into the medium were a measure of cytotoxicity; LDH, GPT, GPT and GLDH were determined by using commercially available test kits (Boehringer and Sigma, Germany). Perfusion of isolated rat livers with ethanol was performed according to published procedures from our lab (Younes and Strubelt, 1987; Deters et al., 1998). Arithmetic means and their standard errors are given, IC50 value were calculated by graphic interpolation. Until now, checks for statistical significance of differences between means were not performed because of the problems of multiple comparisons. Males Wistar rats (conventional animals, 320-380 gr breeder Winkelrnann, Borchen) were used throughout. They had free access to a standard diet (AJtromin pellets) and tap water. Unless otherwise stated, reagents used for liver perfusion and biochemical determinations were of analytical grade and obtained from either Sigma Aldrich (Deisenhofen, FRG) or Merck (Darmstadt, FRG). Removal of the liver and its connection to a recirculating perfusion system was performed as previously described (Strubelt et al., 1986). After removal of the livers, rats died by exsanguination. The albumin- and serum-free perfusion medium consisted of 259 ml Krebs Henseleit buffer, pH 7.4 (118 mmoVl NaCl, 6 mmol/1 KC1, 1.1 mmol/1 MGSO4 24 mmol/1 CaC-5). Sodium taurocholate (36.7 g/1) was infused into the perfusate at a rate of 1.2 ml/h to stimulate bile secretion. The perfusion medium was continuously gassed with carbogen (95% O 2 , 5% CO 2 ) yielding an oxygen partial pressure of about 600 rnmHg. Perfusion was performed under conditions of constant pressure (240 mmiπo) throughout the experiment and the perfusion flow rate was initially regulated at 60 ml/min using a tube clamp. The experiments were started after a 30-min equilibration period (time 0) by adding ethanol (130.2 mmol/1) to the perfusate before they were finished 120 rnin later. Oxygen consumption of the isolated perfused livers was calculated by measuring the differences in oxygen concentrations between the influent and affluent perfusate using a micro pH/blood gas analyzer 1306 (Instrumentation Laboratory). Perfusion flow was determined every 30 rnin by damming up the effluent perfusate in a special vial without imp-airing the perfusion flow and measuring the volume after 20 s. Bile was sampled every 30 rnin and the rate of bile secretion was calculated in g Uver/min. For biochemical determinations, samples of 2 ml were also taken from the perfusate every 30 rnin. Livers were weighed before connecting them to the perfusion system. At the end of the experiments they were frozen in liquid nitrogen until further analysis. The activities of GPT, LDH and GLDH were assayed using commercial kits from Bochringer Mannheim (Mannheim, FRG). Perfusate enzyme concentrations were distributed normally as checked by the method of Sachs (1978). Malondialdehyde (MDA) was measured both in the perfusate and livers by coupling to thiobarbituric acid (Buege and Aust, 1978). Total glutathione was determined in liver and perfusate samples according to Brehe and Burch (1976). Oxidized glutathione (GSSG) was estimated by the same procedure after blocking GS11 with 2-vinylpyridine (Griffith, 1980). For ATP determination, hepatic tissue was frozen immediately in liquid nitrogen and extracts were prepared according to Williamson and Corkey (1969). Adenosine triphosphate (ATP) was assayed enzymatically using a reagent kit from Sigma (Munich, FRG). Example 1 GPT enzyme released into the perfusate is markedly depressed by the combination of silymarin and lycopene: GPT U/I Perfusate (a) Control 45 (See Fig.1,2,3) (b) 130.2 mmol/1 ethanol 310 (c) 10 mg/1 Silymarin + (b) 90 (See Fig.1) (d) 300 mg/1 Lyc-O-mato® + (b) 300 (See Fig.2) (e) 10 mg/1 Lycopene + (b) 300 (See Fig.2) 10 mg/1 Silymarin + 300 mg/1 Lyc-O-mato® 100 (See Fig.3) + (b) (g) 10 mg/1 Silymarin + 10 mg/1 Lycopene + (b) 80 (See Fig.3) Example 2 LDH enzyme released into the perfusate is markedly depressed by the combination of silymarin and Lyc-O-mato® LDH U/I Perfusate (a) Control 500 (See Fig.4, 5, 6) (b) 1130.2 mmol/1 ethanol + (b) 3100 (c) 10 mg/1 Silymarin + (b) 1085 (See Fig.4) (d) 300 mg/1 Lyc-O-mato® + (b) 3000 (See Fig.5) (e) 10 mg/1 Lycopene + (b) 3000 (See Fig.5) 10 mg/1 Silymarin + 300 mg/1 Lyc-O-mato® 750 (See Fig.6) + (b) (g) 10 mg/1 Silymarin + 10 mg/1 Lycopene + (b) 2600 (See Fig.6) Exa ple 3 MDA is markedly decreased by the combination of silymarin and lycopene or Lyc-O-mato®: MDA nmol/g liver (a) Control 16.5 (See Fig. lo,: (b) 130.2 mmol/1 ethanol 22.5 (c) 10 mg/1 Silymarin + (b) 17.5 (See Fig. 10) (d) 300 mg/1 Lyc-O-mato® + (b) 14 (See Fig. 11) (e) 10 mg/1 Lycopene + (b) 20 (See Fig. 11) 10 mg/1 Silymarin + 300 mg/1 13 (See Fig. 12) Lyc-O-mato® + (b) (g) 10 mg/1 Silymarin + 10 mg/1 Lycopene + (b) 14 (See Fig. 12) Example 4 GSH depletion is markedly inhibited by the combination of silymarin and lycopene or Lyc-O-mato®: GSH μmol/g liver (a) Control 3.5 (See Fig. 7, 8, 9) (b) 130.2 mmol/1 ethanol 2.3 (c) 10 mg/1 Silymarin + (b) 3.6 (See Fig. 7) (d) 300 mg 1 Lyc-O-mato® + (b) 4.2 (See Fig. 8) (e) 10 mg/1 Lycopene + (b) 3.3 (See Fig. 8) 10 mg/1 Silymarin + 300 mg/1 Lyc-O-mato® 4.75 (See Fig. 9) + (b) (g) 10 mg/1 Silymarin + 10 mg/1 Lycopene + (b) 4.2 (See Fig. 9) While embodiments of the invention have been described by way of illustration, it will be apparent that the invention may be carried out with many modifications, variations and adaptations, without departing from its spirit or exceeding the scope of the claims.

Claims: CLAIMS 1. A synergistic pharmaceutical or dietary composition which comprises lycopene and silymarin. 2. A composition in accordance with Claim 1 wherein the lycopene is selected from the group consisting of lycopene from tomato oleoresin, algal, fermented, fungal, genetically modified organism (GMO), synthetic lycopene, and mixtures thereof. 3. A composition in accordance with Claim 1 wherein the lycopene is selected from the group consisting of lycopene and tomato oleoresin. 4. A composition in accordance with Claim 1 which comprises synthetic lycopene. 5. A composition in accordance with Claim 1 wherein the sπymarin is extracted from the Silybum marianum plant. 6. A composition in accordance with Claim 1 wherein the sUvmarin is extracted from the fruit of the Silybum marianum plant. 7. A composition in accordance with Claim 1 wherein the silymarin is extracted from the Silybum marianum plant and is standardized on a silybin content between 75% and 90%. 8. A composition in accordance with Claim 1 wherein the relative parts by weight of suymarin to lycopene is from 50:1 to 1 :50 preferably 30:1 to 1 :30. 9. A composition in accordance with any of Claims 1 to 8 wherein the composition contains compounds selected from the group consisting of dietary components, additives, excipients, binding agents, coatings, preservatives, and mixtures thereof. 10 A composition in accordance with any of Claims 1 to 9 wherein the composition is contained in dosage forms selected from the group consisting of tablets, caplets, vegecaps and hard shell gelatin capsules. Use of a synergistic composition containing lycopene and silymarin for the preparation of pharmaceutical compositions for treatment of acute, subacute and chronic liver diseases. A use in accordance with Claim 1 1 wherein the lycopene is selected from the group consisting of lycopene from tomato oleoresin, algal, fermented, fungal, genetically modified organism (GMO), synthetic lycopene, and mixtures thereof. A use in accordance with Claim 11 wherein the diseases are selected from the group consisting of alcohol-hepatitis and alcohol-induced fatter liver hepatitis and alcohol induced liver cirrhosis. A use in accordance with Claim 11 wherein the lycopene is tomato oleoresin. A use in accordance with Claim 1 1 which comprises synthetic lycopene. A use in accordance with Claim 1 1 wherein the silymarin is extracted from the Silybum marianum plant. A use in accordance with Claim 11 wherein the silymarin is extracted from the fruit of the Silybum marianum plant. A use in accordance with Claim 1 1 wherein the suymarin is extracted from the Silybum marianum plant and is standardized on a sirybin content between 75% and 90%. A use in accordance with Claim 1 1 wherein the relative parts by weight of silymarin to lycopene is from 50:1 to 1:50 preferably 30:1 to 1 :30. A use in accordance with any of Claims 11 to 19 wherein the composition contains compounds selected from the group consisting of dietary components, additives, excipients, binding agents, coatings, preservatives, and mixtures thereof. A use in accordance with any of Claims 11 to 20 wherein the composition is contained in dosage forms selected from the group consisting of tablets, caplets, vegecaps and hard shell gelatin capsules. A method for combating acute, subacute, and chronic liver diseases which comprises contacting the liver with a synergistic mixture of lycopene and silymarin. A method in accordance with Claim 22 wherein the lycopene is selected from the group consisting of lycopene from tomato oleoresin, algal, fermented, fungal, genetically modified organism (GMO), synthetic lycopene, and mixtures thereof. A method in accordance with Claim 22 wherein the lycopene is selected from the group consisting of lycopene and tomato oleoresin. A method in accordance with Claim 22 which comprises synthetic lycopene. A method in accordance with Claim 22 wherein the silymarin is extracted from the Silybum marianum plant. A method in accordance with Claim 22 wherein the silymarin is extracted from the fruit of the Silybum marianum plant. A method in accordance with Claim 22 wherein the silymarin is extracted from the Silybum marianum plant and is standardized on a silybin content between 75% and 90%. A method in accordance with Claim 22 wherein the relative parts by weight of silymarin to lycopene is from 50: 1 to 1:50 preferably 30: 1 to 1:30. A method in accordance with any of Claims 22 to 29 wherein the composition contains compounds selected from the group consisting of dietary components, additives, excipients, binding agents, coatings, preservatives, and mixtures thereof. A method in accordance with any of Claims 22 to 30 wherein the composition is contained in dosage forms selected from the group consisting of tablets, caplets, vegecaps and hard shell gelatin capsules. Use of a composition claimed in claim 1 for preventing or inhibiting the free oxygen radical formation found to be involved in alcohol-induced liver diseases.

Inventors: GRUENWALD JEORG, NIR ZOHAR, SIEGERS CLAUS PETER, GRUENWALD, JEORG, NIR, ZOHAR, SIEGERS, CLAUS, PETER
Applicants: GRUENWALD JEORG, LYCORED NATURAL PROD IND LTD, NIR ZOHAR, SIEGERS CLAUS PETER, GRUENWALD, JEORG, LYCORED NATURAL PRODUCTS INDUSTRIES LTD., NIR, ZOHAR, SIEGERS, CLAUS, PETER
Application year: 2000
Main classification: A61K35
IPC codes: A23L1, A61K31, A61P5