EP2303252A1

EP2303252A1 - Compounds for the treatment of metabolic syndrome and insulin resistance

Info

Publication number: EP2303252A1
Application number: EP09745281A
Authority: EP
Inventors: Alois Jungbauer
Original assignee: Individual
Current assignee: Apomedica Pharmazeutische Produkte Gesellschaft M; Universitaet fuer Bodenkultur Wien BOKU
Priority date: 2008-05-15
Filing date: 2009-05-15
Publication date: 2011-04-06
Also published as: WO2009137859A1

Abstract

The present invention provides a compound comprising an ellagic acid moiety, such as ellagic acid itself, ellagitannins and ellagic acid glycosides - as well as plant extracts comprising the same, for the treatment or prevention of Metabolic Syndrome or non-diabetic insulin resistance.

Description

Compounds for the treatment of metabolic syndrome and insulin resistance

The present invention relates to natural compounds suitable for the treatment of insulin related metabolic diseases.

Metabolic Syndrome (also known as the Insuline Resistance Syndrome) is a combination of medical disorders that affect a large number of people in a clustered fashion, the end result of which is to increase one's risk for cardiovascular disease and diabetes. In most cases Metabolic Syndrome culminates in type 2 diabetes. The symptoms of Metabolic Syndrome are related to lipid and carbohydrate metabolism and include obesity, elevated triglycerides, low levels of high density lipoproteins, increased blood pressure or hypertension and increased glucose levels, but also symptoms of inflammation. Generally, the individual symptoms associated with the Metabolic Syndrome are treated separately (e.g. diuretics and ACE inhibitors for hypertension, statins to decrease cholesterol levels or glitazones to treat diabetes) . The problem is compounded when multiple drugs are necessary to control multiple risk factors. For example, once type 2 diabetes develops in patients with the Metabolic Syndrome, patients often require 10 or more drugs for treatment. A new and potentially efficacious class of drugs for the Metabolic Syndrome as a whole, as well as for patients with type 2 diabetes, is the thiazolidinediones (TZDs) . These drugs act by agonizing the nuclear receptor PPAR-gamma, which is predominantly expressed in adipose tissue, but also occurs in other tissues (WO 2005/027661; CA 2 526 589) . TZDs reduce the secretion of unesterified fatty acids and adipokines such as tumour- necrosis factor-alpha (TNF-alpha) , other inflammatory cytokines, resistin and plasminogen-activator inhibitor 1 (PAIl) ; they also enhance adipose-tissue release of adiponectin. The net result of these changes, apparently, is to reduce insulin resistance in muscle and liver and to mitigate prothrombotic and proinflammatory states. These findings therefore suggest that TZDs are hitting at the heart of the Metabolic .Syndrome by improving insulin resistance in adipose tissue. Still, in type 2 diabetes, they have only a modest effect on plasma lipoproteins and blood pressure. So, although they improve the Metabolic Syndrome, they by no means cure it once type 2 diabetes develops. Insulin resistance is a key factor in development of the Metabolic Syndrome. This syndrome is the coexistence of hy- perglycaemia, hypertension, dyslipidemia and obesity. Therefore cardiovascular diseases such as coronary heart diseases and stroke are more prevalent among patients with Metabolic Syndrome. A classification of insulin resistance and the Insulin Resistance Syndrome (Metabolic Syndrome) is present in the ACE- Position-Statements (Endocr. Pract. 2003; 9 (No. 3) 240-252) which shall be used herein. Although these diseases share a common origin, yet should be strictly kept apart as different symptoms and disease progression occur.

The PPAR-gamma is a class II nuclear receptor that forms a heterodimer with the retinoid X-receptor (RXR) and binds to specific regions on the DNA of target genes. Expression of target genes is increased or decreased, depending on the gene. PPAR- gamma is composed of 6 structural regions in 4 functional domains. The A/B region forms the ligand-independent transactiva- tion domain which can be covalently modified by phosphorylation. By the C region, the DNA-binding domain of the receptor can be targeted to the PPAR-gamma response element (PPRE) , a specific sequence of nucleotides within the regulatory region of responsive genes. The E/F region contains the ligand binding domain and the co-activator/co-repressor-binding surface. Binding of agonists leads to conformational changes of the receptor and to its activation. The activated receptor heterodimerizes with RXR and this heterodimer binds to PPRE through the DNA-binding domain.

Natural ligands of the PPAR-gamma receptor are fatty acids such as lauric acid, petroselenic acid, linolenic acid, linoleic acid and arachidonic acid, fatty acid metabolites like 15-deoxy- delta 12, 14-prostaglandin J2. The synthetic ligands comprise the group of thiazolidinedions (Troglitazone, Rosiglitazone, Piogli- tazone) , the non-thiazolidinediones (e.g. GW1929, GW7845) and the non-steroidal anti-inflammatory drugs (e.g. flufenamic acid, fenoprofen) . Thus, natural ligands for the PPAR-gamma are polyunsaturated fatty acids and eicosanoids . This receptor is expressed in many tissues like adipose tissue, heart, muscle, colon, kidney, liver and is mainly responsible for adipocyte differentiation and energy storage. PPAR-gamma may also play a critical role in the pathogenesis of atherosclerosis through in- fluencing the circulating lipid and glucose level or directly- through modulating of macrophage functions. It was recently discovered, that platelets also contain the transcription factor PPAR-gamma and the role of PPAR ligands on prevention of unwanted platelet activation and chronic inflammatory diseases has to be proven.

PPAR-gamma downregulates TNF-alpha, leptin, IL-6, plasminogen activator inhibitor-1 (PAI-I) , resistin, 11-beta- hydroxysteroid dehydrogenase type-1 (11-beta-HSD-l) . Those proteins are responsible for insulin resistance. Thus activation of PPAR-gamma leads to reduced insulin resistance because they regulate the glucose-transporter protein GLUT-4 in the cell membrane .

WO 2005/053724 mentions non-aqueous extracts of Astragalus membranaceus which influence PPAR-gamma. Compounds found in these extracts are for example calycosin, formononetin, gen- istein, afromorsin, biochanin A, coumestrol, odoratin and daidzein. PPAR-gamma mediated diseases which can be treated with the extracts are dyslipidaemia, atherosclerosis, coronary heart disease, obesity and colon cancer.

WO 2005/027661 describes catechines, which are obtainable from green tea, as activating ligands of PPAR-gamma. The ligands mentioned therein, in particular glitazone, rosiglitazone, ciglitazone and pioglitazone, fall under the group of TZDs. Further TZDs, for example troglitazone are mentioned in the US 2006/0030597 Al.

CA 2 526 589 Al describes ligands of PPAR-gamma, in particular glabrene, glabridine, glabrol and their derivatives, and glitazones. These compounds are mentioned in connection with the multiple risk factor syndrome, another name of the Metabolic Syndrome, which is related to insulin resistance and can be treated with PPAR-gamma ligands. Also described is a licorice extract for the treatment of Metabolic Syndrome.

EP 1 350 516 Bl claims a hydrophobic licorice extract, and extracts from turmeric, clove and cinnamon for the use of treating Metabolic Syndrome as well as associated diseases like visceral obesity and diabetes mellitus . The activity of the extracts is measured in reference to troglitazone and pioglitazone.

JP 2005/097216 mentions dehydrodieugenol A and B, magnolol, oleanic acid and betulic acid as PPAR-gamma ligands that are useful for preventing or ameliorating Metabolic Syndrome.

The EP 1481669 Al describes polyhydroxy phenol and polyphenols such as ellagic acid for the inhibition of P-selectin.

The WO 2006/022502 describes compounds with diphenoyl structure for a treatment of immune diseases.

The EP 1312374 describes the use of an extract from evening primrose to relieve symptoms of diabetes mellitus. The extract may contain ellagic acid.

The WO 2007/038768 describes a method for preventing or treating metabolic syndrome using a grape extract as dietary supplement. According to this document the absence of epicate- chin-gallate or only a small amount thereof in addition to a high amount of low molecular weight compounds is responsible for increased vasodilatation which is believed to be responsible for a drop in blood pressure in individuals with metabolic syndrome.

It is the goal of the present invention to provide compounds with particular exceptional potential to treat or prevent Metabolic Syndrome and diseases associated with insulin resistance.

Therefore, the present invention provides a compound comprising an ellagic acid moiety, such as ellagic acid itself, el- lagitannins and ellagic acid glycosides - as well as plant extracts comprising the same, for the treatment or prevention of Metabolic Syndrome or non-diabetic insulin resistance. Also described is the method of treating or preventing Metabolic Syndrome or non-diabetic insulin resistance with the compound comprising the ellagic acid moiety or the extract. In addition, the invention also relates to the use of the invention and extracts for the manufacture of a pharmaceutical preparation for the treatment or prevention of said diseases or conditions.

The Metabolic Syndrome, also termed "Insulin Resistance Syndrome" is a non-diabetic accumulation of risk factors, which can lead to the development of diabetes but it is not identical with diabetes. As defined by the American Association of Clinical Endocrinology the Metabolic Syndrome (i.e. the Insulin Resistance Syndrome) is defined by four factors:

Metabolic Syndrome

1. Triglycerides > 150 mg/dL

2. HDL cholesterol Men < 40 mg/dL

. i Women < 50 mg/dL

3. Blood pressure > 130/85 mm Hg

4. Glucose

Fasting 110-125 mg/dL

120 min post-glucose challenge 140-200 mg/dL (ACE Position Statement, Endocr Pract. 9(3), 2003: 240-252)

In comparison to this definition the World Health Organization (WHO) defines diabetes by the following criteria:

Diabetes mellitus ^' Venous plasma glucose concentration mπιol/1 mg/dL fasting or ≥7.0 ≥126

2-hour post-75 g glucose load ≥ll.l ≥200 (Khatib, Guidelines for the prevention, management and care of diabetes mellitus, EMRO Technical Publications Series 32, chapter 2 p. 13-19; 2006)

Insulin resistance is by itself characterized by an insufficient function of insulin, it can give rise to compensatuary hy- perinsulinemia (as a precursor to the Metabolic Syndrome) or result in an inadequate insulin response (as a precursor to diabetes) . However, insulin resistance, the Metabolic Syndrome and diabetes type 2 are conditions which are defined to be mutually exclusive (ACE Position Statement, Endocr Pract. 9(3), 2003: 240-252) . The present invention provides the use of the inventive compounds and pharmaceutical compositions for the treatment or prevention of the Metabolic Syndrome and insulin resistance - in particular non-diabetic - but also contemplates their use for the treatment of diabetes, in particular type 2 diabetes.

The abnormalities related to elevated triglycerides, HDL cholesterol values and increased blood pressure are characteris^¬ tics of the Metabolic Syndrome. Drugs for the treatment of Metabolic Syndrome are required to enhance insulin sensitivity, as well as the other manifestations of the insulin resistance syn^¬ drome. This action was surprisingly found in the plants and com^¬ pounds of the present invention which interact with the PPAR- gamma. PPAR-gamma interaction has a beneficial effect on insulin sensitivity but also on the lipid profile, blood pressure, haemostasis and can significantly reduce the risk of cardiovascular disease associated with Metabolic Syndrome (Walcher et al., Diabetes and Vascular Res. 2004 (2): 76-81) .

In contrast, impaired transactivation, which has been described in the frequent genetic polymorphism Alal2Pro of the PPAR-gamma gene, leads to higher insulin sensitivity (but lower postprandial hypertriglyceridemia) , and a haplotype for which higher transcriptional activity is postulated has an increased risk for Metabolic Syndrome. Therefore, a non-linear activity- effect curve has been proposed for PPAR-gamma activity, with small increases in activity having opposite effects than stronger activation by ligands .

The known antidiabetic effect of grape seed extracts and the lower cardiovascular disease (CVD) incidence due to red wine consumption is well documented. The present invention provides the identification of ellagic acid (2, 3, 7, 8-tetrahydroxy- [1] Benzopyrano [5, 4, 3-cde] [1] benzopyrano-5, 10-dione) and its associated ellagitannins found in plants with potential health benefit through PPAR-gamma binding and therefore responsible for transactivation and/or repression.

"Prevention" in the sense of the present invention should not be construed in an absolute sense in that the target diseases and conditions are with 100% certainty prevented but in a relative sense, i.e. as decreasing the risk of developing said diseases or conditions. Accordingly, the compounds are used for prophylactic administration.

As used herein a "ellagic acid moiety" is to be understood as the molecular backbone of ellagic acid not counting its hydrogen atoms. A compound with said moiety is thus ellagic acid itself or a derivative wherein hydrogens are substituted. These derivatives include esters, ethers or amides of ellagic acid, in particular glycosides, glyco esters of ellagic acid such as ellagic acid-galactoside or -glucoside. Ellagic acid naturally forms intermolecular esters (lactone) that can be hydrolized, in particular upon derivatisation, such as during ellagitannin formation. The ellagic acid moiety may therefore be one of the following:

(formula 1 - lactone (formula 2) [formula 3) form of ellagic acid) or one of its deprotonized salts, preferably a pharmaceutically acceptable salt.

Ellagic acid is found in plants in its free form (cyclic intramolecular ester form, i.e. lactone form) but predominantly as complex ester (ellagitannins) or non-tannic glycosidic forms

(Koponen et al . , J. Agric. Food Chem. 2007, 55, 1612-1619) . An ellagitannin may contain one, two, three or more "ellagic acid moieties", in particular hexahydroxydiphenic acid of formula 3 or the intermediate of formula 2. In particular the carboxylic acid groups may be subject of derivatisation or substitution forming esters, or amines. An ellagitannin may further be an ester of tannic or gallic acid, sugar units, preferably glucose, and/or further hydroxydipehnic acids. Preferably the ellagitannin derivative of ellagic acid is of the following structure

(formula 4 ) :

(formula 4)

Preferably the inventive compound is hydrolyzable to ellagic acid (or convertable to ellagic acid by hydrolysis) , such as ellagitannins which contain one or more hexahydroxydiphenic acid moieties (such as of formula 3) esterified to a polyol, usually glucose. Hydrolysis of ellagitannins with acids or bases yields hexahydroxydiphenic acid which spontaneosly lactonizes to el- lagic acid of formula 1. In further embodiments the phenolic hy- droxygroups may be substituted by ether, ester or amide groups.

Hydrolyis may be effected by strong or weak acids or bases, e.g. at a pH of below 4, 3, 2, or 1 or above 10, 11, 12 or 13. In particular hydrolysis can occur in human gastric conditions.

In another aspect the present invention provides a plant extract comprising the inventive compounds for the treatment or prevention of metabolic syndrome or non-diabetic insulin resistance, preferably the compound is enriched in said extract.

In yet another aspect a plant extract is provided comprising the compound as described above, in particular ellagic acid, wherein the compound is specifically enriched by at least 50%, preferably by at least 75% or even more preferred by a factor of at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, 60, 70, 80, 100, 120, 140, 160, 180, 200, 250, 300, 400, or at least 500. An extract of the present invention may be a solid or fluid extract. The "specific enrichment" refers to the increased concentration of the inventive compounds, ellagitannins, ellagic acid and/or ellagic acid glycosides, with reference to all solid plant constituents of an untreated plant or plant portion (such as fruits) from which the extract is derived. Preferably the extract comprises at least 1%, 2%, 3%, 4%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or at least 50% (w/w of the solid mass) of the inventive compounds.

Preferably the extract is an extract of berry, fruit and nut species or of oak, including oak wood. The extract may be of plants rich in ellagic acid or ellagitannins such as berry, fruit, and nut species. They have been detected in berries of the genus Rubus (raspberry, blackberry, cloudberry, arctic bramble) and the genus Fragaria (strawberry) , pomegranate, walnuts, and some other nuts, and oak-aged wines (Koponen et al., J. Ag- ric. Food Chem. 2007, 55, 1612-1619) . In addition to polymeric ellagitannins, minor amounts of ellagic acid are usually present as free and glycosylated forms. The extract may also benefit from more than one plant, e.g. extraction of more than one berry species or a berry and oak extraction.

In preferred embodiments the extract is of grapes, preferably of grape seeds and/or skin. Grape constituents including ellagic acid and its derivatives are also found in red wine, which can also be used for obtaining the extract. In addition oak aged wine is also rich in tannins found in oak providing a further increase of the ellagitannin and ellagic acid concentration. Previously, red wine consumption has been made responsible for the beneficial effect on cardiovascular diseases and inflammation. This has been mainly referred to resveratrol in the past. Due to the low abundance of resveratrol in red wine and medium potency to many hormone receptors the beneficial effects was surprisingly found due to the PPAR-gamma activity. As is shown in the examples ligand binding of different wines was studied and neat wine compounds and extracts assayed for peroxisome pro- liferators activated receptor gamma binding in a competitive ligand binding assay. Grape skin derived compounds like resveratrol, apigenin, piceatannol, kaempferol and myricetin showed only medium binding affinity in the μM range. With the help of GCMS ellagic acid was identified and assessed in the ligand binding assay as strong binder with a IC50 of 5.7xlO^"7 M.

In especially preferred embodiments the extract is a tannin extract. Tannin fraction of diverse plants are especially rich in ellagitannins and yield a high amount of ellagic acid.

In particular embodiments the extract is a red wine or a red wine concentrate, preferably of oak containers aged wine. Red wines are natural extracts of grapes, including grape skin or seeds which are rich in ellagic acid containing moieties. A further enrichment occurs due to contact with oak, such as by storage in oak containers, barrels or caskets.

In a further aspect the present invention provides a pharmaceutical composition of the extract or compound. The composition may be in solid form, preferably a powder, tablet or capsule, or fluid. In a preferred embodiment, the pharmaceutical preparation according to the invention comprises a pharmaceutical carrier or additive. A pharmaceutical carrier includes wetting, emulsifying, or pH buffering agents or vehicles with which the pharmaceutical preparation can be contained in or administered. Examples are oils, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Preferably, the agents of the pharmaceutical preparation are formulated in solid form, including salt forms, or fluid form. Pharmaceutically ac- ceptable salts include those formed with anions ^; such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric, butyric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

Preferably the preparation is an oral preparation, in particular preferred in form of a juice or tablet. The extracts, juices or compounds can be dried and formulated into tablets and administered orally. For example, the preparations can be formulated in the form of tablets, capsules, cachets, gelcaps, solutions, suspensions, and the like. Tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose) ; fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch glycolate) ; or wetting agents (e.g., sodium lauryl sulphate) . The tablets may be coated by methods well-known in the art. Liquid preparations for oral administration may take the form of, but are not limited to, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); nonaqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl- p-hydroxybenzoates or sorbic acid) . The preparations may also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated for slow release, controlled release, or sustained release of a prophylactic or therapeutic agent (s) .

The present invention is further illustrated by the following figures and examples without being limited thereto.

F i g u r e s :

Fig. 1: Binding analysis of twelve different wines. The relative binding affinity (RBA) was calculated by relating the binding affinities with the maximum binding affinity of rosiglitazone. Fig. 2: Fractionation wine W6: (A) HPLC run and (B) ligand binding assay from the fractions. 4xl00μl were injected, 1 ml fractions were collected (B: x-axis: fraction number), solvent was evaporated and the residues were dissolved in 100 μl DMSO and used for the ligand binding assay.

Fig. 3: Logistic dose response curves of oak tannin (tannin Q) and grape tannin extracts.

Fig. 4: Logistic dose response curves from ellagic acid, epigal- locatechingallate and epicatechingallate and the synthetic PPAR- gamma ligand rosiglitazone for the determination of the inhibitory concentrations (IC₅₀ values) .

Fig. 5: GCMS analysis of wine Wβ (A), oak tannin extract (B) and grape tannin extract (C) .

E x a m p l e s :

Epidemiological studies have suggested the health benefit of moderate red wine consumption. This was first attributed to the alcohol contents at the beginning of the 20th century. A correlation between dietary habits and red wine consumption and the risk of cardiovascular diseases in France lead to the so called French paradox. Recent epidemiological studies on alcohol intake, type of beverage and risk of Type 2 diabetes performed in Australia showed a strong correlation for moderate wine consumption and lower diabetes risk for men and women.

But also animal studies suggest an impact of wine exercising a protective role in chronic pathologies. Such advantages are described for cardiovascular heart diseases and atherosclerosis, hypertension, diabetes, neurodegenerative diseases as well as cancer. But the molecular mechanism of wine compounds and the different pathways are not fully understood. One well known mechanism is the activation of the estrogen receptors through isoflavones. This can lead to activation of e-NOS and further to release of NO. A similar effect of e-NOS activation and relaxation of the artery vessels with a decrease in blood pressure is also described for wine polyphenols.

Another nuclear receptor which is a target for the dietary prevention of metabolic diseases is the peroxisome prolifera- tors-activated receptor-gamma (PPAR-gamma) . The different bio- logical^, actions Of ellagic acid and ellagitanins, in particular as wine active compounds, and the molecular mechanism by acting on the PPAR are studied herein.

Example 1: Materials

For ligand binding assay and transactivation assay the wines were used without further purification. Standard compounds purchased from Sigma, Indofine and Extrasynthese . In a pre- screening eight international wines were tested for the ability to bind on the PPAR-gamma. For detailed analysis twelve wine varieties were tested: two Austrian white wines (Neuburger and Rotgipfler) and ten Austrian red wines (Pinot noir, St. Laurent, Zweigelt and Blaufrankisch) . One very potent red wine was used for further investigation and isolation of active compounds. A chemical analysis of all twelve wines was done with an HPLC system. The grape extract Tannin Grape (Erbslδh, Geisenheim, Germany) and the oak extract Tannin Q (Sulfometa, Krems, Austria) were purchased in a local wine supplement shop. The extract were made using a 20% ethanolic solution.

Example 2: HPLC system:

Analytical -HPLC system for sample fractionation: All HPLC runs were performed with an Agilent 1100 HPLC system (Waldbronn, Germany) . The fractionation was achieved by RP-HPLC using a C 18 (2) Luna 3μm column (Phenomenex, Torrance CA, USA) . Eluent A was de- ionized and 0.22 μm filtered water, supplemented with 5% ace- tonitrile and 0.1% TFA, eluant B was acetonitrile supplemented with 0.1% TFA. Different compounds were eluted with a linear gradient from 0 to 17.5% eluant B in 20 min, 17.5 to 50% eluant B in 25 min, and a 10 min hold at 50 % eluant B, regeneration of the column was effected by a step gradient to 90% B. The flow rate was 0.5 mL/min.

Preparative HPLC system for ligand identification: Isolation and identification of the single compounds was achieved with a preparative Agilent 1200 HPLC system. A reversed phase column C18 (2), 250x20.21mm column volume and 15μm particle diameter (Phenomenex, Torrance CA, USA) was used. Buffer and gradient conditions were the same as for the analytical separation. The flow rate was 26.5 mL/min and the fractions were collected every 2 min. They were evaporated and .the residues dissolved in etha- nol and used for ligand binding assay and GCMS analysis, respectively.

Example 3: Competitive ligand binding assay:

The commercial available polarScreen PPAR competitor assay green was used to measure binding on the PPAR-gamma ligand binding domain (Invitrogen PV3355) . Briefly, PPAR-LBD is added to a fluorescent PPAR ligand (Fluormone™ PPAR Green) to form a PPAR - LBD/Fluormone™ PPAR Green complex resulting in a high polarization value. Competitors displace the fluorescent Fluormone™ PPAR Green ligand from the ligand binding domain, resulting in a low polarization value. Noncompetitors will not displace the fluorescent ligand from the complex, so the polarization value remains high. The shift in polarization value is used to determine relative affinity of test compounds for the PPAR-LBD.

Example 4: GC-MS-Analysis

GC-MS was performed using a GC 6890N / MSD 5973B instrument (Agilent Technologies) . For silylation, all samples were treated with Bistrimethylsilyltri-fluoroacetamide (BSTFA) at 70⁰C for one hour. Separation of the compounds was achieved on fused silica HP-5ms (30m, 0.25mm, 25μm) column using helium as carrier gas, a helium column flow of 0.9ml /min, an oven programme starting with 100⁰C (5 min), then 10°C/min to 280°C (20 min), and an auxiliary temperature programme starting at 24O⁰C (18min), then 10°C/min to 280⁰C (14min) . Inlet was operated in split mode (25:1) at 280°C. Ionization was performed in EI mode at 70 eV, 230⁰C, and 1.5*10^~5 Torr. Data acquisition and processing were performed using the MSDChem software package (Agilent Technologies) . Furthermore, the mass spectral library NIST 2002 (National Institute of Standards and Technology, USA) and its implemented search routine were employed for identifying the multitude of peaks in the obtained total ion chromatograms .

Example 5: Data analysis

Data of the competitive ligand binding assay were fitted using a logistic dose response model, described as (equationl) : Equation 1 _y . b i+{c/_xy Parameter a equals the baseline, b is the efficiency or plateau of the curve, c the ligand potency and d gives the transition width. Calculation was performed with Table Curve 2D software (Jandel) .

Example 6: Calculation of the equivalent rosiglita- zone . concentration (EC) :

Rosiglitazone is a known synthetic strong PPAR-gamma binder and is used as a reference substance to compare extracts and wines. The equivalent concentration of the wine samples is calculated by dividing the potency of rosiglitazone in μmol/L by the potency of the wine samples in L/L (equation.2) :

Equation 2

One grape tannin extract and one oak tannin extract were tested (Fig. 3) . Very high binding affinities were found for the grape and oak tannin extracts with potencies of 350 and 270 ng/ml, respectively. Similar high binding affinities are found for pomegranate extracts with 300 ng/ml.

Subsequently several wines were tested. Table 1 lists an overview of their types and production qualities. Table 2B gives theis PPAR-gamma binding activity as rosiglitazone equivalents.

Table IA. Description of the wines - technological aspects

Wine l Wine 2 Wine 3 Wine 4 Wine 5 Wine 6 Wine 7 Wine 8 Wine 9 Wine 10 Wine 11 Wine 12

Variety Neuburger Rotgipflei St. Laurent Pinot noir St. Laurent Zweigelt BF BF Zweigelt BF BF Zweigelt

Vintage 2005 2005 2003 2004 2003 2004 2003 2004 2005 2004 2005 2004

Sun exposure - - + + + -H- + + + ++ -B- -H-

Skin contact 1 hour 3 hours 21 days 18 days 10 days 7 days >14 days >14 days 12-14 days ? days 10 days 10 days

Fining agent bentonite bentonite gelatin - PVPP PVPP - - - - - -

New oak

Oak contact - - barrique barrique barrique cask Oak cask Oak cask Oak cask barrique - -

Table IB: Description of the wines - chemical aspects Compound/Analysis Wl W2 W3 W4 W5 W6 W7 W8 W9 WlO WIl W12

Density 0.9916 0.9930 0.9936 0.9921 0.9927 0.9919 0.9927 0.9916 0.9924 0.9915 0.9924 0.9915

Ethanol (%vol) 13.2 13.8 13.1 14.1 13.2 13.5 13.4 13.6 13.5 13.3 13.5 13.3

Sugar (g/L) 3.3 5.3 2.0 1.8 0.3 1.4 0.8 1.7 1.8 1.3 1.8 1.3

Fructose (g/L) 4.2 6.1 0.5 0.5 n.d. 0.1 n.d. 0.1 0.1 n.d. 0.1 n.d.

Glucose (g/L) 1.3 1.4 1.3 1.0 n.d. 0.9 0.2 0.8 0.7 1.0 0.7 1.0

Acidity (g/L) 5.3 5.83 4.74 4.75 4.74 4.05 5.05 4.89 5.28 4.99 5.28 4.99 pH-value 3.7 3.8 3.6 3.7 3.6 3.6 3.6 3.6 3.5 3.5 3.5 3.5

Volatile acid (g/L) 0.5 0.7 0.7 0.8 0.8 0.5 0.8 0.6 0.6 0.4 0.6 0.4

Tartaric acid (g/L) 1.2 1.1 0.8 1.0 0.5 1.1 1.1 1.7 2.2 2.5 2.2 2.5

Malic acid (g/L) 2.2 3.2 0.2 n.d. n.d. n.d. n.d. 0.3 0.1 0.2 0.1 0.2

Lactic acid (g/L) 1.0 1.1 1.9 2.5 3.1 1.8 2.7 1.9 1.8 1.5 1.8 1.5

Citric acid (g/L) 0.2 0.2 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.

Glycerol (g/L) 6.5 6.9 10.8 11.3 11.0 10.7 10.7 10.2 10.5 10.7 10.5 10.7

Gallic acid (mg/L) 2.5 3.9 50.1 40.8 22.5 71.9 46.7 40.7 25.7 49.8 35.7 25.5

Caftaric acid (mg/L) 32.1 48.1 61.5 81.6 13.7 31.4 127.0 109.3 91.9 87.1 135.4 57.5

Tyrosol (mg/L) 7.1 7.0 34.0 19.0 31.5 22 32.3 45.2 28.1 20.5 24.9 23.1 c-Cutaric acid

3.2 4.4 2.7 5.4 1.2 1.3 2.1 3.2 2.9 3.0 3.3 1.7 (mg/L) ϊ -Cutaric acid 6 6.8 15.7 23.2 3.9 6.3 16.8 16.7 18.5 14.8 20.5 12.3 (mg/L) 4.

Catechin (mg/L) 22.0 25.4 94.5 141.7 85.0 85.3 111 68.6 79.9 78.6 61.2 26.7

Caffeic acid (mg/L) 4.5 4.9 3.6 5.1 9.8 15.4 5.7 7.7 15.8 5.7 4.6 3.5

Fertaric acid (mg/L) 4.2 5.7 2.5 2.6 1.6 2.1 4.8 3.7 3.4 3.7 4.5 2.0 p-Cumaric acid

1.3 1.3 2.9 1.5 6.5 3.7 1.4 2.9 4.5 3.5 1.2 6.6 (mg/L)

Epicatechin (mg/L) 8.3 7.5 43.7 94.5 32.6 77.9 42.5 45.7 52.6 61.1 40.9 17.0

Ferulic acid (mg/L) 0.3 0.9 0.9 0.9 1.0 0.8 0.5 0.4 0.8 0.5 0.6 0.6

Mono. Anthocyanins n.d. n.d. 107 88 56 127 33 62 215 83 146 95 (mg/L)

Total Polyphenols

0.08 0.09 1.46 1.33 1.51 1.65 1.71 1.05 1.55 1.2 1.26 0.80 (g/L)

AOP (mMol/L) 2.0 2.3 19.8 24.9 30.5 34.8 47.0 21.8 26.8 22.8 29.7 19.4

Table 2. Equivalent concentrations of twelve wines from the detailed analysis.

Potency ■^^rosiglitazone

Wine [L/L] [μM]

Wl n.d. n.d.

W2 n.d. n.d.

W3 5.0*10-4 240.0

W4 8.6*10-4 139.5

W5 3.8*10-4 315.8

Wβ 2.4*10-4 500.0

W7 2.3*10-4 521.7

W8 6.0*10-4 200.0

W9 2.9*10-4 413.8

WlO 8.5*10-4 141.2

WIl 4.8*10-4 250.0

W12 2.3*10-3 active

Two white wines were also tested but showed no binding on the receptor, due to the low phenol content of white wines. All red wines had remarkable equivalent concentrations of rosiglita- zone ranging from 52 to 521 μmol/L (Table 2) . Wines which were ^■ stored in oak barrels always showed a higher binding affinity, than wines produced without oak contact (wine 12) - fig. 1.

Example 7 : Fractionation

Wine 6 with an equivalent concentration of 500 μmol/L was used for further analysis. RP-HPLC on a C-18 (2) column was performed for fractionation of the most potent ligands. Fractions were collected every 2 min, the solvent was evaporated, the residues dissolved in DMSO and used for binding analysis. Compounds eluting up to 20% acetonitrile in the solvent seem to be the most active ones, they are low to medium hydrophobic. Especially fraction 15 and 16 showed high binding response (Fig. 2) .

Fraction 15 is also the most coloured part of the linear gradient. Anthocyanins, especially cyanidin were mentioned to regulate adipocyte function. Therefore, malvidin, cyanidin and the glucosides were evaluated as the most abundant wine anthocyanins for PPAR-gamma binding. Malvidin and especially cyanidin aglycon showed high binding affinity (Table 3) . Malvidin-O- glucoside neat compound has the same retention time as the dominant peak in fraction 15, but with no binding affinity in the ligand binding assay. One possible reason could be a structure change due to oxidation or cleavage and also the presence of other potent ligands in this fraction.

Since the amount of phenolic compounds in the fractions was too small a semi-preparative RP-HPLC was performed. Therefore 10 mL wine sample were injected and separated on an 80 mL RP-HPLC column C18. GCMS analysis leads to the identification of the most abundant compounds of each fraction. They are listed in table 3.

Table 3: Identified compounds in fraction 11-20 with GCMS analysis

Fraction 11 Vanillic acid, syringic acid, p-coumaric acid, cis-caffeic acid, trans-caffeic acid, epicatechin, catechin

Fraction 12 Protocatechuic acid, syringic acid, gallic acid, ferulic acid, affeic acid, epi- We could identify ellagic acid in fraction 15 and quercetin in fraction 16 as very potent ligands and therefore responsible for the high activity of these fractions.

Example 8 : GC-MS results

GCMS analysis of wine W6 was performed to determine potential ligands. Beside known grape derived compounds oak polyphenols, in particular ellagic acid, were identified.

Table 4: Compounds identified in the GCMS analysis (Fig. 5A) : retention time and numbering

Compound no . Retention Compound time

1 7.85 Phenylethanol - TMS

2 8.04 g-Hydroxybutyric acid - 2 TMS

4 8.18 2-Hydroxy-3-methylpentanoic acid - 2 TMS

5 8.31 Succinic acid ethyl ester - TMS

6 8.53 Caprylic acid - TMS

7 8.64 a-Hydroxyvaleric acid - 2 TMS

8 8.94 Glycerol - 3 TMS

9 9.61 Succinic acid - 2 TMS

10 10.96 a-Methyl-b-hydroxydicarboxylioc acid

TMS

11 12.11 Dicarboxylic acid - 2 TMS

12 12.32 Malic acid - 3 TMS

13 13.32 4-Hydroxyphenyl ethanol - 2 TMS

14 13.44 2-Hydroxyglutaric acid - 3 TMS

15 13.51 Tricarboxylic acid - 3 TMS

16 13.55 Phenyllactic acid - 2 TMS

17 13.68 Furanose - 4TMS

18 13.87 Ethyl tartrate - 3 TMS

19 13.99 4-Hydroxybenzoic acid - 2 TMS

20 14.25 1, 3, 5-Trihydroxybenzene - 3 TMS

21 14.33 Tartaric acid - 4 TMS

22 14.85 o-Phthalic acid - 2 TMS

23 15.00 4-Hydroxy-3-methoxyphenylethanol - 2 TMS

24 15.55 4-Hydroxy-hydrocoumaric acid - 2 TMS

25 15.62 Vanillic acid - 2 TMS

26 15.67 3, 4-Dihydroxyphenylethanol - 3 TMS 15.78 Gentisic acid - 3 TMS

15.87 4-Coumaric acid --2 TMS

15.95 Citric acid ethyl ester - 3 TMS

16.24 Protocatechuic acid - 3 TMS

17.00 Resorcylic acid - 3 TMS

17.05 Syringic acid - 2 TMS

17,12 4-Hydroxyphenyl lactic acid - 3 TMS

17.42 p-Coumaric acid - 2 TMS

17.52 Ethyl gallate - 3 TMS

17.73 Gallic acid - 4 TMS

18.11 3, 4-Dihydroxymandelic acid - 3 TMS

18.37 Palmitic acid - TMS

18.90 Ferulic acid - 2 TMS

19.34 Caffeic acid - 3 TMS

19.44 Amino acid

19.90 Linoleic acid - TMS

20.15 Stearic acid - TMS

21.56 Resveratrol - 3 TMS

22.24 Pentacosane

22.60 Flavanoide; m/z = 484 [M+, 100%], 427, 233

23.00 Hexacosane

23.80 Heptacosan

24.15 Resveratrol - 3 TMS

25.72 flavanoide compound m/z= 648 [M+], 532, 395

[100%]

25.83 Epicatechin - 5 TMS

26.20 Naringenin - 3 TMS '

26.11 Catechin - 5 TMS

26.69 Catechingallate

27.22 Catechin - 4 TMS

27.37 flavanoide compound; m/z = 578 [M+], 368

[100%], 283

28.07 Epicatechin - 4 TMS

28.25 flavanoide compound; m/z = 666 [M+], 384,

355, 283 [100%] 29.21 flavanoide compound; m/z = 666 [M+], 384,

355, 283 [100%] 29.44 Kaempferol - 4 TMS 29.84 flavanoide compound; m/z = 652 [M+], 382,

253 [100%], 219 62 30. 92 Quercetin - 5 TMS

63 32. 54 678, 663 (100), 575, Myricetin - 5 TMS

64 33. 84 Ellagic acid - 4 TMS

65 34 . 58 b-Sitosterol - TMS

The neat compounds were tested in the ligand binding assay. Grape polyphenols like myricetin, quercetin, kaempferol and nar- ingenin are medium potent ligands in the μM range. The oak polyphenol ellagic acid, however, was identified as high potent PPAR-gamma ligands with IC50s of 5.7xlO^~7 M. The binding potencies of the most abundant wine phenols were determined and listed (Table 5) .

Table 35. Calculated potencies of some neat wine and oak compounds

Compound CAS -number IC50 [mol /L]

Rosiglitazone 122320-73-4 2 . IxI O^"7 trans- 501-36-0 active

Resveratrol

Cyanidin 528 -58 -5 1 . 4xl O^~6

Malvidin 643-84 -5 active

Ellagic acid 476-66-4 5 . 7xl O^~7

Ellagitannins are extracted form the oak barrels and oak supplements during wine production and undergo an acid hydrolysis. They can also be extracted from the woody grape seeds and are converted during fermentation. Ellagic acid, which is also present in pomegranate in high amounts, was identified as strong PPAR-gamma binder. The antidiabetic effects of pomegranate are well known, but ellagic acid was not identified as PPAR-gamma ligand till now. Ellagitannins and ellagic acid are the most potent ligands for the PPAR-gamma receptor in wine; they are mainly derived from grape seeds and oak wood.

Red wine shows remarkable PPAR-gamma binding affinity up to an equivalent concentration of 500 μmol rosiglitazone / L. Grape derived polyphenols have only a medium potency in the μM range, but oak derived compounds could be identified as strong PPAR- gamma ligands with IC50 values comparable with rosiglitazone, the synthetic compound for treatment of Metabolic Syndrome. El- lagic acid was first reported as strong PPAR-gamma binder with an IC50 of 5.7xlO^~7 M and is therefore a potent compound for treatment of vascular diseases, inflammatory diseases, Metabolic Syndrome and insulin resistance due to PPAR-gamma pathway activity. Recent findings of PPAR-gamma in platelets leads to the hypothesis, that such wine compounds can be responsible for the beneficial health effects of moderate wine consumption.

Claims

Claims :

1. A compound comprising an ellagic acid moiety for the treatment or prevention of metabolic syndrome or non-diabetic insulin resistance .

2. Compound according to claim 1, characterized in that the ellagic acid moiety is selected from:

(formula 1) (formula 2) (formula 3) or one of its pharmaceutically acceptable salts.

3. Compound according to claim 1 or 2, being an ellagitannin, ellagic acid or an ellagic acid glycoside.

4. Compound according to claims 1 to 3, characterized in that it is acidic or basic hydrolysable to yield ellagic acid.

5. A plant extract comprising a compound of claims 1 to 4 for the treatment or prevention of metabolic syndrome or non- diabetic insulin resistance.

6. A plant extract comprising a compound of claims 1 to 4, characterized in that the compound is specifically enriched by at least 50%, preferably by at least 75% or even more preferred by a factor of at least 2.

7. Extract according to claim 5 or 6, being an extract of berry, fruit and nut species, preferably of raspberry, blackberry, cloudberry, arctic bramble, strawberry, pomegranate, walnut, or of oak.

8. Extract according to any one of claims 5 to 7, characterized in that the extract is of grape, preferably of grape seeds and/or skin.

9. Extract according to any one of claims 5 to 8, characterized in that it is a tannin extract.

10. Extract according to claim 8, characterized in that it is red wine or a red wine concentrate, preferably of oak barrel aged wine .

11. Pharmaceutical composition comprising the extract according to any one of claims 5 to 10 in solid form, preferably a powder, tablet or capsule, or fluid.

12. Pharmaceutical composition according to claim 11 furthermore comprising a pharmaceutically acceptable carrier or additive.