USE OF TRICYCLIC SESQUITERPENE LACTONES IN THE TREATMENT OF OBESITY AND RELATED DISEASES AND NON-THERAPEUTIC TREATABLE CONDITIONS

Abstract

A compound of the formula I, wherein the symbols are as defined in the remaining specification, or a mixture of two or more compounds of the formula I, for use as active ingredient in the therapeuticincluding prophylactictreatment of a warm-blooded animal for the regulation of body weight (preferred) and/or fat loss (preferred) and/or for the management of obesity and/or for improving the total cholesterol HDL/LDL ratio; where the compound(s) of the formula I may be present in free form, in the form of a pharmaceutically and/or nutraceutically acceptable salt, in the form of tautomers, in the form of esters and/or in the form of solvates, as well as related invention embodiments.

##STR00001##

Claims

1-14. (canceled)

15. A method for improving total cholesterol HDL/LDL ratio, the method comprising administering to a subject a compound of formula (I): ##STR00016## wherein: A.sub.1, A.sub.2 and A.sub.3 are each, independently from each other, CH.sub.2, CHOR.sub.1, or CO; R.sub.1 is hydrogen, a straight-chain or branched-chain alkyl group having 1 to 12 carbon atoms, a cycloalkyl group having 3 to 10 carbon atoms and optionally comprising one or more ring heteroatoms, a carbohydrate having 5 to 12 carbon atoms, a substituted or unsubstituted aryl group with 6 to 12 ring atoms which can comprise one or more ring heteroatoms, a substituted or unsubstituted C.sub.6-C.sub.12-aryl-C.sub.1-C.sub.6-alkyl group which can comprise one or more heteroatoms, a substituted or unsubstituted aryloxy group wherein aryl has 6 to 12 ring atoms and can comprise one or more ring heteroatoms, a (C(O)R.sub.a) group, a (C(S)R.sub.a) group, a (C(O)OR.sub.a) group, a ((CH.sub.2).sub.nCR.sub.aCR.sub.aR.sub.b) group, or a (C(O)(CH.sub.2).sub.nCR.sub.aCR.sub.aR.sub.b) group; n is zero or an integer from 1 to 12; R.sub.a and R.sub.b are each, independently from each other, selected from a group Z, consisting of hydrogen, a straight-chain or branched-chain alkyl group having 1 to 6 carbon atoms that is unsubstituted or substituted by hydroxy, a cycloalkyl group having 3 to 10 carbon atoms and optionally comprising one or more ring heteroatoms, and a substituted or unsubstituted aryl group with 6 to 12 ring atoms which can comprise one or more ring heteroatoms, and a substituted or unsubstituted C.sub.6-C.sub.12-aryl-C.sub.1-C.sub.6-alkyl group which can comprise one or more heteroatoms; B.sub.1, B.sub.2 and B.sub.3 are each, independently from each other, CCH.sub.2, CHCH.sub.2R.sub.1*, COHCH.sub.2X with X selected from the group F, Cl, and Br, COHCH.sub.2OR.sub.1 with R.sub.1 as defined above and R.sub.1* being R.sub.1 as defined above or a straight-chain or branched-chain alkoxy group having 1 to 12 carbon atoms, or C(R.sub.x)(R.sub.y) wherein R.sub.x, R.sub.y and the binding carbon atom together form a cycloalkyl group having 3 to 10 carbon atoms and optionally comprising one or more ring heteroatoms; wherein if one or more heteroatoms mentioned above are present they are present instead of one or more carbon atoms and are selected from the group consisting of S, N, NH, O, P and Se; wherein the compound of the formula (I) may be present in free form, in the form of a pharmaceutically and/or nutraceutically acceptable salt, in the form of tautomers, in the form of esters and/or in the form of solvates; and alternatively, or in addition, in the form of an extract of the plant or plant parts of a plant of the family of Asteraceae, each comprising one or more compounds of the formula (I) in free form, in the form of a pharmaceutically and/or nutraceutically acceptable salt, in the form of tautomers, in the form of esters and/or in the form of solvates.

16. The method of claim 15, wherein A.sub.1 is CH.sub.2 and B.sub.3 is CH.sub.2.

17. The method of claim 15, wherein the method comprises administering to a subject a compound of the formula (I): ##STR00017## wherein A.sub.1 is CH.sub.2; A.sub.2 and A.sub.3 are each, independently from each other, CH.sub.2, CHOR.sub.1, or CO; B.sub.3 is CCH.sub.2; B.sub.1 and B.sub.2 are each, independently from each other, CCH.sub.2, CHCH.sub.2R.sub.1*, C(OH)CH.sub.2X or C(OH)CH.sub.2OR.sub.1; wherein X is selected from F, Cl and Br, R.sub.1 is hydrogen, a straight-chain or branched-chain alkyl group having 1 to 12 carbon atoms, a cycloalkyl group having 3 to 10 carbon atoms and optionally comprising one or more ring heteroatoms, a carbohydrate having 5 to 12 carbon atoms, a substituted or unsubstituted aryl group with 6 to 12 carbon atoms which can comprise one or more ring heteroatoms, a substituted or unsubstituted C.sub.6-C.sub.12-aryl-C.sub.1-6-alkyl group which can comprise one or more heteroatoms, a substituted or unsubstituted aryloxy group where aryl has 6 to 12 ring atoms and can comprise one or more ring heteroatoms, a C(O)R.sub.a group, a C(S)R.sup.a group, a C(O)OR.sub.a group, a (CH.sub.2).sub.nCR.sub.aCR.sub.aR.sub.b group, or a C(O)(CH.sub.2).sub.nCR.sub.aCR.sub.aR.sub.b group; n is zero or an integer from 1 to 12; R.sub.a and R.sub.b are each, independently from each other, selected from a group Z, consisting of hydrogen, a straight-chain or branched-chain alkyl group having 1 to 6 carbon atoms that is unsubstituted or substituted by hydroxyl, a cycloalkyl group having 3 to 10 carbon atoms and optionally comprising one or more ring heteroatoms, and a substituted or unsubstituted C.sub.6-C.sub.12-aryl-(C.sub.1-C.sub.6)-alkyl group which can comprise one or more heteroatoms; R.sub.1* is R.sub.1 as defined above or a straight-chain or branched-chain alkoxy group having 1 to 12 carbon atoms, or C(Rx)(Ry) where Rx, Ry and the binding carbon atom together form a cycloalkyl group having 3 to 10 carbon atoms and optionally comprising one or more ring heteroatoms; wherein if one or more heteroatoms are present they selected from S, N, NH and O; wherein the compound of the formula (I) is present in free form, in the form of a pharmaceutically and/or nutraceutically acceptable salt, in the form of tautomers and/or in the form of solvates; and wherein the optional substituents of the aryl group with 6 to 12 carbon atoms which can comprise one or more ring heteroatoms, C.sub.6-C.sub.12-aryl-C.sub.1-6-alkyl group which can comprise one or more heteroatoms and aryloxy group where aryl has 6 to 12 ring atoms and can comprise one or more ring heteroatoms are selected from the group consisting of C.sub.1-C.sub.7-alkyl, hydroxy, C.sub.1-C.sub.7-alkoxy, C.sub.1-C.sub.7-alkanoyloxy, C.sub.1-C.sub.7-alkoxycarbooxy, C.sub.1-C.sub.7-alkanesulfonyloxy, phenyl-C.sub.1-C.sub.7-alkoxy, amino, N-mono- or N,N-di-(C.sub.1-C.sub.7-alkyl, C.sub.1-C.sub.7-alkanoyl, C.sub.1-C.sub.7-alkoxycarbonyl, C.sub.1-C.sub.7-alkanesulfonyl and/or phenyl-C.sub.1-C.sub.7-alkyl)-amino, carboxy, C.sub.1-C.sub.7-alkoxycarbonyl, carbamoyl, N-mono- or N,N-di-(C.sub.1-C.sub.7-alkyl)-carbamoyl, sulfamoyl, N-mono- or N,N-di-(C.sub.1-C.sub.7-alkyl)-sulfamoyl and cyano.

18. The method of claim 15, wherein the compound of formula (I) is selected from the group consisting of 3-Epi-11,13-dihydrodeacylcynaropicrin, Subexpinnatin, 11,13-Dihydrodeacylcynaropicrin, 11-beta, 13-Dihydrocynaropicrin, Isoamberboin, 3,11,13-Trihydroxy-10(14)-guaien-12,6-olide, Dehydrocyanaropicrin, Sibthorpin, 8-Deoxy-11,13-dihydroxygrosheimin, Isolipidiol, 8-Hydroxy-3-oxo-4(15), 10(14)-guaiadien-12,6-olide, 3,8-Dihydroxy-10(14),11(13)-guaiadien-12,6-olide, Grossheimin, Integrifolin, 8beta-Hydroxydehydrozaluzanin C, Cynaropicrin, 13-Chloro-3,11-dihydroxy-4(15),10(14)-guaiadien-12,6-olide, 3-Acetyl-13-chloro-13-deoxysolstitialin, 8-Deoxy-11-hydroxy-13-chlorogrosheimin, Cynarascoloside A, Cynarascoloside B, Cynarascoloside C, Cynarinin A, and Cynarinin B, where the compound of the formula (I) may be present in free form, in the form of a pharmaceutically and/or nutraceutically acceptable salt, in the form of tautomers, and/or in the form of solvates.

19. The method of claim 15, wherein the compound of the formula (I) is obtained from an extract of a plant or plant parts of a plant of the genus selected from the group consisting of Cynara, Centaurea, Saussurea, Amberboa, Grossheimia, Tricholepsis, Cheirolophus, Macroclinidium, Vernonia, Ixeris, Jurinea, Ainsliaea, Pseudostifftia, Crepis, Cartolepsis, Andryala and Volutarella, where the compound may be present in free form, in the form of a pharmaceutically and/or nutraceutically acceptable salt, in the form of tautomers and/or in the form of solvates.

20. The method of claim 15, wherein the compound of the formula (I) is Cynaropicrin, or a pharmaceutically and/or nutraceutically acceptable salt and/or solvate thereof, or Grossheimin, or a pharmaceutically and/or nutraceutically acceptable salt and/or solvate thereof.

21. The method of claim 20, wherein the compound of the formula (I) is obtained from an extract of a plant or plant parts of a plant of the genus selected from the group consisting of Cynara, Centaurea, Saussurea, Amberboa, Grossheimia, Tricholepsis, Cheirolophus, Macroclinidium, Vernonia, Ixeris, Jurinea, Ainsliaea, Pseudostifftia, Crepis, Cartolepsis, Andryala and Volutarella, where the compound may be present in free form, in the form of a pharmaceutically and/or nutraceutically acceptable salt, in the form of tautomers and/or in the form of solvates.

22. The method of claim 15, wherein the compound is administered to the subject in the form of a pharmaceutical and/or nutraceutical formulation and/or dietary supplement and/or functional food, where the compound of the formula (I) may be in free form, in the form of a pharmaceutically and/or nutraceutically acceptable salt, in the form of tautomers, and/or in the form of solvates.

23. The method of claim 15, wherein a mixture of two or more compounds of the formula (I) is administered to the subject for improving the total cholesterol HDL/LDL ratio in the subject.

Description

DESCRIPTION OF THE FIGURES

[0112] FIG. 1: Graphic representation of the body weight gain in correlation to the control group, body weights of the control groups were normalized to 1.00 (weight loss in rats by administration of cynaropicrin).

[0113] FIG. 2: Graphic Representation of the body fat gain (determined by DEXA) in correlation to the control group, body fat of the control groups were normalized to 1.00 (reduction of body fat in rats by cynaropicrin)

[0114] FIG. 3: HPLC analysis of the enriched extract according example 4 A; ELSD upper line, UV (210 nm) lower line.

[0115] FIG. 4: HPLC analysis of the final product according example 5 identified as Cynaropicrin (IMD-009047); ELSD upper line, ret time 9.372 min, UV (210 nm) lower line, ret time 9.235 min.

[0116] FIG. 5: FIG. 3: HPLC analysis of an extract according example 4 B; UV (210 nm).

PREFERRED EMBODIMENTS OF THE INVENTION

[0117] In one preferred embodiment, the invention relates to a USE of a compound of the formula I or a mixture of two or more such compounds, wherein [0118] A1, A2 and A3 independently from each other represent the diradicals **CH.sub.2, **CHOR1, or **CO, [0119] with R1 denoting hydrogen, a straight-chain or branched-chain alkyl group having 1 to 12 carbon atoms, or a (C(O)(CH.sub.2).sub.nCR.sub.aCR.sub.aR.sub.b) group wherein n is zero or an integer from 1 to 12 and [0120] R.sub.a and R.sub.b independently from each other are selected from a group Z, consisting of hydrogen and a straight-chain or branched-chain alkyl group having 1 to 6 carbon atoms that is unsubstituted or substituted by hydroxy, and [0121] B1, B2 and B3 independently from each other represent the diradicals **CCH.sub.2, **CHCH.sub.2R1*, and **COHCH.sub.2OR1 with R1 as defined above and R1* being R1 as defined above or a straight-chain or branched-chain alkoxy group having 1 to 12 carbon atoms, [0122] where if one or more heteroatoms mentioned above are present they are present instead of one or more carbon atoms and are selected from the group consisting of S, N, NH, O, P and Se, preferably S, N, NH and O, where the compound(s) of the formula I may be present in free form, in the form of a pharmaceutically and/or nutraceutically acceptable salt, in the form of tautomers, in the form of esters and/or in the form of solvates.

[0123] A preferred embodiment relates to a USE of one or more compounds selected from the group consisting of 3-Epi-11,13-dihydrodeacylcynaropicrin (99305-01-8), Subexpinnatin (81421-79-6), 11,13-Dihydrodeacylcynaropicrin (66761-12-4), 11 beta, 13-Dihydrocynaropicrin (160661-30-3), Isoamberboin (30825-69-5), 3,11,13-Trihydroxy-10(14)-guaien-12,6-olide (70894-20-1), Dehydrocynaropicrin (35821-02-4), Sibthorpin (94410-23-8), 8-Deoxy-11,13-dihydroxygrosheimin (83551-03-5), Isolipidiol (30825-68-4), 8-Hydroxy-3-oxo-4(15), 10(14)-guaiadien-12,6-olide (38142-62-0), 3,8-Dihydroxy-10(14), 11(13)-guaiadien-12,6-olide (84305-03-3), Grossheimin (22489-66-3), Integrifolin (89647-87-0), 8beta-Hydroxydehydrozaluzanin C (83991-71-3), Muricatin (52597-25-8), Cynaropicrin (35730-78-0), 13-Chloro-3,11-dihydroxy-4(15), 10(14)-guaiadien-12,6-olide (142563-68-6), 3-Acetyl-13-chloro-13-deoxysolstitialin (142546-23-3), Cynaroside A (117804-06-5), 8-Deoxy-11-hydroxy-13-chlorogrosheimin (83551-02-4), Cynarascoloside A (518034-52-1), Cynarascoloside B (518034-53-2), Cynarascoloside C (518034-54-3), Cynarinin A (849146-43-6), Cynarinin B (849146-44-7), where the compound(s) of the formula I may be present in free form, in the form of a pharmaceutically and/or nutraceutically acceptable salt, in the form of tautomers, in the form of esters and/or in the form of solvates, where the given CAS numbers represent only preferred stereoisomeric forms, so that the given names can also represent other stereoisomeric forms, or mixtures of two or more such forms.

[0124] One or more of the structures of the invention presented in the following list are especially preferred for USE according to the invention:

TABLE-US-00001 Names CAS-RN Isolipidiol 3,8-Dihydroxy-10(14)-guaien-12,6-olide; (1alpha,3beta,4alpha,5alpha,6alpha,8alpha,11alpha) Dentalactone 3,8-Dihydroxy-10(14)-guaien-12,6-olide; (1alpha,3beta,4beta,5alpha,6alpha,8beta,11beta) [00003] 30825-68-4 155662-30-9 Isoamberboin 3,8-Dihydroxy-10(14)-guaien-12,6-olide; (1alpha,3beta,4alpha,5alpha,6alpha,8alpha,11alpha); 3-Ketone 8-Hydroxy-3-oxo-10(14)-guaien-12,6-olide [00004] 30825-69-5 22339-28-2 3-Epi-11,13-dihydrodeacylcynaropicrin 3,8-Dihydroxy-4(15),10(14)-guaiadien-12,6-olide; (1alpha,3alpha,5alpha,6alpha,8alpha,11betaH) 11,13-Dihydrodeacylcynaropicrin 3,8-Dihydroxy-4(15),10(14)-guaiadien-12,6-olide; (1alpha,3beta,5alpha,6alpha,8alpha,11betaH) 3,8-Dihydroxy-4(15),10(14)-guaiadien-12,6-olide 8alpha-Hydroxy-11alpha,13-dihydrozaluzanin C 3,8-Dihydroxy-4(15),10(14)-gualadien-12,6-olide; (1alpha,3beta,5alpha,6alpha,8alpha,11alphaH) [00005] 99305-01-8 66761-12-4 35932-54-8 84813-81-0 3,8-Dihydroxy-4(15),10(14)-guaiadien-12,6-olide; (1alpha,3beta,5alpha,6alpha,8beta,11alphaH)-form 8beta-Hydroxy-11beta,13-dihydrozaluzanin C 153753-47-0 3,8-Dihydroxy-4(15),10(14)-guaiadien-12,6-olide; (1alpha,3beta,5alpha,6alpha,8beta,11betaH) 8-Hydroxy-3-oxo-4(15),10(14)-guaiadien-12,6-olide 3,8-Dihydroxy-4(15),10(14)-guaiadien-12,6-olide; (1alpha,3beta,5alpha,6alpha,8alpha,11betaH); 3-Ketone; 8-Hydroxy-3-oxo-4(15),10(14)- guaiadien-12,6-olide [00006] 38142-62-0 3,8-Dihydroxy-10(14),11(13)-guaiadien-12,6-olide 3,8-Dihydroxy-10(14)-guaien-12,6-olide; (1alpha,3beta,4alpha,5alpha,6alpha,8beta,11alpha)- form; 11,13-Didehydro [00007] 84305-03-3 405283-65-0 8-Epigrosheimin 3,8-Dihydroxy-10(14)-guaien-12,6-olide; (1alpha,4alpha,5alpha,6alpha,8beta); 11,13- Didehydro, 3-ketone Grossheimin 8-Hydroxy-3-oxo-10(14),11(13)-guaiadien-12,6- olide [00008] 22489-66-3 3,8-Dihydroxy-4(15),10(14),11(13)-guaiatrien-12,6- olide; (1xi,3xi,5xi,6xi,8xi)-form Integrifolin 3,8-Dihydroxy-4(15),10(14),11(13)-guaiatrien-12,6- olide; (1alpha,3beta,5alpha,6alpha,8beta) 8-Hydroxyzaluzanin C 3,8-Dihydroxy-4(15),10(14),11(13)-guaiatrien-12,6- olide; (1alpha,3beta,5alpha,6alpha,8alpha) [00009] 89647-87-0 31565-50-1 8beta-Hydroxydehydrozaluzanin C 3,8-Dihydroxy-4(15),10(14),11(13)-guaiatrien-12,6- olide; (1alpha,3beta,5alpha,6alpha,8beta); 3-Ketone 8-Hydroxy-3-oxo-4(15),10(14),11(13)-guaiatrien- 12,6-olide [00010] 83991-71-3 67667-77-0 Cynaratriol 3,11,13-Trihydroxy-4(15),10(14)-guaiadien-12,6- olide; (1alpha,3beta,5alpha,6alpha,11alpha); 4beta, 15- Dihydro [00011] 70894-20-1 Sibthorpin 3,11,13-Trihydroxy-4(15),10(14)-guaiadien-12,6- olide; (1alpha,3alpha,5alpha,6alpha,11alpha) Solstitialin 3,11,13-Trihydroxy-4(15),10(14)-guaiadien-12,6- olide; (1alpha,3beta,5alpha,6alpha,11alpha) [00012] 94410-23-8 22738-70-1 8-Deoxy-11,13-dihydroxygrosheimin 11,13-Dihydroxy-3-oxo-10(14)-guaien-12,6-olide; (1alpha,4alpha,5alpha,6alpha,11xi) 3,11,13-Trihydroxy-4(15),10(14)-guaiadien-12,6- olide; (1alpha,3beta,5alpha,6alpha,11alpha)-form; 4,15- Dihydro, 3-ketone 11,13-Dihydroxy-3-oxo-10(14)-guaien-12,6-olide; (1alpha,4alpha,5alpha,6alpha,11alpha)-form [00013] 83551-03-5 255722-99-7 Muricatin 3,8-Dihydroxy-10(14)-guaien-12,6-olide; (1alpha,3beta,4alpha,5alpha,6alpha,8alpha,11alpha); 11,13-Didehydro, 8-O-(2-hydroxymethylpropenoyl) [00014] 52597-25-8 Cynaropicrin 3,8-Dihydroxy-4(15),10(14),11(13)-guaiatrien-12,6- olide; (1alpha,3beta,5alpha,6alpha,8alpha); 8-O-(2- Hydroxymethyl-2-propenoyl) Saupirin 3,8-Dihydroxy-4(15),10(14),11(13)-guaiatrien-12,6- olide; (1xi,3xi,5xi,6xi,8xi); 8-O-(2-Hydroxymethylpropenoyl) [00015] 35730-78-0 35932-39-9

[0125] Also preferred are the esters (meaning e.g. C.sub.1-C.sub.6alkanoyl, C.sub.3-C.sub.6alkenoyl, hydroxy-C.sub.1-C.sub.6alkanoyl or C.sub.3-C.sub.6alkenoyl esters) of the above shown non-acylated structures, especially but not limited to their ester presented as last compound in the above list.

[0126] The geometries of the double bonds within the structures shall not be assigned as been drawn. So they can be present either as E or Z configuration isomers, or as mixture of such isomers.

[0127] Especially preferred for USE according to the invention are Cynaropicrin (IUPAC name: (8-hydroxy-3,6,9-trimethylidene-2-oxo-3a,4,5,6a,7,8,9a, 9b-octahydroazuleno[4,5-b]furan-4-yl) 2-(hydroxymethyl)prop-2-enoate), or Grossheimin IUPAC name: 4-hydroxy-9-methyl-3,6-dimethylene-3a,4,5,6a,7,9,9a,9b-octahydroazuleno[5,4-d]furan-2,8-dione), or a mixture of these two compounds.

[0128] The invention also relates to the USE according to the invention of any one of the compounds of the formula I mentioned in the examples, alone or in combination with one or more other compounds of the formula I mentioned therein.

[0129] Finally, the invention also relates to the embodiments in the claims which are incorporated here by reference, dependent claims representing preferred embodiments of the invention.

EXAMPLES

[0130] The present invention is further explained by the following examples. The specific examples which follow illustrate the methods in which the compositions of the present invention may be prepared, components therein and their use, as well as other embodiments of the invention, but are not to be construed as limiting the invention in scope.

General Experimental Procedures:

[0131] If not mentioned otherwise, chemicals are obtained in analytical grade from Merck (Darmstadt, Germany) or Sigma-Aldrich (Deisenhofen, Germany). LC-MS analyses are performed using an Agilent HP1100 (Agilent, Waldbronn, Germany) liquid chromatograph coupled with a LCT mass spectrometer (Micromass, Manchester, UK) in the positive and negative electrospray ionisation (ESI) mode, based on slight modification of a previously described method [9]. A Waters symmetry column is used as stationary phase. Mobile phase A: 0.1% Formic acid in water, mobile phase B: 0.1% Formic acid in acetonitrile; gradient: 0-1 min. 100% A, from 1-6 min. to 90% B, from 6 to 8 min to 100% B, from 8-10 min 100% B. LC-MS spectra are recorded in the range of molecular weights between 150 and 1.600 U. HPLC-UV/Vis analyses are carried out on a HP 1100 Series analytical HPLC system (Agilent, Waldbronn, Germany) comprising a G 1312A binary pump system, a G 1315A diode array detector, a G 1316A column compartment, a G 1322A degasser and a G 1313A autoinjector. Mobile phase: A=0.1% Trifluoroacetic acid in water, B=0.1% Trifluoroacetic acid in acetonitrile. A Nucleodur (Trademark by Macherey & Nagel) RP 18 column (1254 mm, particle size 5 m) serves as stationary phase. Aliquots of the samples (representing 2-10 g of methanol-soluble materials, according to the concentrations of main metabolites) are analysed at 40 C. with a flow of 1 ml/min in the following gradient: Linear from 0% B to 100% B in 20 min, thereafter isocratic conditions at 100% acetonitrile for 5 min; followed by regeneration of the column for 5 min. HPLC-UV chromatograms are recorded at 210 nm and 254 nm. Diode array detection (DAD) is employed to record HPLC-UV/Vis spectra in the range of 190-600 nm. The HP ChemStation (Trademark by Agilent Technologies, Inc; see Agilent above) software allows for an automated search for calibrated standard compounds in crude extracts.

Example 1: Feeding Experiments

[0132] Feeding experiments can be performed in any mammal and are done in different species.

[0133] For the present experiments, the compound to be tested is added to the feed of the individuals. This is beneficial against enforcement because it allows a validation of the acceptance of the test compounds. The experiments are performed with negative control and are correlated to a commercial slimming product.

[0134] Cynaropicrin is tested using six weeks old, none-adult, male Sprague-Dawley (SD) rats (Charles Rivers, Sulzfeld, Germany). Cynaropicrin is mixed with the chow (ssniff Spezial-diten GmbH, Soest, Germany) to a final concentration of 0.1% by weight and fed for 8 weeks in two different treatment regimes: [0135] Balanced diet (standardised to 4% fat) [0136] High fat diet (standardised to 34% fat)

[0137] Controls obtain the same diet without Cynaropicrin or other anti-obesity additives.

[0138] Body weight is studied and analysed every week, body fat and mineral bone density are analysed by DEXA (scanner, see Example 3) in week 0, in week 4 and at the last day of the study in week 8. Furthermore, the white body fat weight is determined after removal of the tissue at the end of the feeding study in week 8. For the DEXA measurements the same apparatuses and methodology are used as described in example 3.

[0139] The GE Lunar Piximus2 DEXA scanner (GE Healthcare, Munich, Germany) gives accurate information on differences in body composition in mice (see Nagy, T R and Clair, A-L. Precision and accuracy of dual-energy X-ray absorptiometry for determining in vivo body composition in mice. Obesity Res. 2000; 8:392-398; Brommage Am J Physiol Endocrinol Metab 285: E454-E459, 2003). Validation and calibration of DEXA body composition in mice; Sarah L. Johnston, Wendy L. Peacock, Lynn M. Bell, Michel Lonchampt, and John R. Speakman, Obesity Research 2005 13 (9), 1558). One scanning procedure takes up to five minutes per individual and provides data on fat mass, lean mass, bone mineral content and bone mineral density. The method is validated against Soxhlet (SOX) fat extraction in mice and a strong correlation (r 2>0.95) between fatDEXA and fatSOX is determined (published information).

[0140] As a control experiment within the high fat diet, epigallocatechin-3-gallate (EGCG, Chengdu Purification Technology Development Co. Ltd, Chengdu, China) in pure form is used as the standard for comparison of potency and differentiation as well as benchmark against competing compounds or formulations against obesity. Anti-obese and fat reducing effects are demonstrated for EGCG in animal studies and in human trials. EGCG is marketed as a functional food ingredient claiming an anti-obesity effect (Wolfram, S et al. Mol Nutr Food Res 2006; 50(2):176-87; Wolfram, S et al. Ann Nutr Metab 2005; 49(1):54-6, Klaus, S et al. Int J Obes 2005; 29(6):615-23; Hill, A M et al. J Am Coil Nutr. 2007; 26(4):396S-402S). The final concentration of EGCG used is 0.1%, too.

[0141] Result: Feeding of cynaropicrin results under high fat diet conditions in a reduced body weight accumulation and reduced body fat accumulation in comparison to the control groups. No effects are observed for EGCG in this study, as presented in FIGS. 1 and 2.

[0142] Efficiency of assimilation is determined on the last 3 days and the first 3 days of begin to feed cynaropicrin as at the last 3 days of the feeding experiment in week 8.

[0143] Therefore the food intake and faeces are analysed and the energy content are determined by using a bomb calorimeter (IKA C7000). Efficiency of assimilation (in %) is calculated from the ratio between the energy consumption from food intake and the difference between energy intake and energy excretion.

[0144] Feeding of cynaropicrin has no influence on food intake in correlation to the control groups. Further the assimilation efficiency is the same for same types of diet: [0145] LF control group: 87% [0146] LF cynaropicrin: 86% [0147] HF control group: 83% [0148] HF cynaropicrin: 83%

[0149] Based on the same food intake and assimilation efficiencies reduction of body weight and body fat is most presumably caused by effects on energy metabolism. Application of Cynaropicrin results in a reduced body weight and body fat gain by identical energy intake.

Example 2: Clinical Blood Chemistry Findings

[0150] The following parameters are involved in energy metabolism and thereby related to the metabolic syndrome. They are determined from blood samples taken in week 1; 4; and 8 of the rat study: Total cholesterol levels; HDL, LDL, TG (triglycerides), and glucose. Furthermore, the activity of liver enzymes such as ALT (alanin-aminotransferase), AST (aspartat-aminotransferase), and AP (alkaline phosphatase), as well as the concentration of Bilirubin, Albumin, Creatin Kinase, and electrolytes (Na+; K+; Ca++; Cl) are determined as safety markers to exclude any toxic effects such as e.g. liver damage.

[0151] Blood samples are taken from the sublingual vein and collected in Li-Heparin coated vials. The blood samples are stored on ice for a short period of time before it is centrifuged for 15 minutes at 8 C. and 3500 rpm. The supernatant plasma is transferred in a reaction tube and stored at 80 C. until clinical chemistry measurements are performed. All measurements are conducted using an Olympus AU 400 at the gene centre of the LMU Munich, working group of Prof. Wolf. Statistical calculations are performed with Statistika (Statsoft (Europe) GmbH, Hamburg, Germany) und Sigma Stat (Jandel Scientific, San Raphael, Calif., USA).

[0152] Electrolytes, bilirubin and liver enzymes such as e.g. AP are found to be statistically unchanged in all diet groups' indicating absence of any obvious toxic effects or liver damage.

[0153] Most importantly the data enables us to demonstrate an improved total cholesterol:HDL/LDL ratio for Cynaropicrin treated rats under high fat (HF) diet compared to the control group (HF only). Other than the atherogenic LDL, HDL is considered to act cardio-protective. Usually, a high HDL/LDL ratio (More HDL than LDL) is considered as a beneficial blood parameter by physicians.

Example 3: Pathological Parameters, Clinical Findings

[0154] Main organs are analysed macroscopically for safety reasons and to investigate the effects of the different diets on these organs (e.g. induction of cholestatic (fatty) livers etc.). After organ removal, macroscopic inspections of e.g. heart, kidneys; GI tract, spleen, liver are performed.

[0155] Thereby, no significant alterations are found. Additionally, a histo-pathological analysis of the kidneys is done but does not result in any observation of an adverse effect on kidney morphology and function. Therefore it can be concluded that the observed biological activities of Cynaropicrin are most likely due to a real metabolic effect rather than being caused by simple toxic effect such as e.g. a test item related organ damage.

Example 4: Preparation of an Enriched Extract

[0156] A) 3000 g of dried Cynara scolymus leaves (Alfred Galke GmbH, Gittelde/Harz, Germany) are extracted for 30 minutes at room temperature with 12,000 ml 70% Ethanol twice using ultrasonic irradiation. The resulting supernatant is separated from the remaining material and concentrated under reduced pressure. The remaining aqueous phase is adjusted with water to a final volume of 3,000 ml and is extracted twice with 3,000 ml n-heptane and subsequently twice with 3,000 ml ethyl acetate by liquid/liquid separation. The combined organic n-heptane phases are analysed for the presence of IMD-009047 and are discarded in case of absence of Cynaropicrin. The ethyl acetate phases are dried (Na.sub.2SO.sub.4), the solvents are evaporated under reduced pressure and the amount of the crude dry extract is determined. This process allows for an enrichment of the medium polar organic compounds out of the artichoke leaves. Typical yields are 81 g enriched extract out of the ethyl acetate phase starting with 3,000 g of Cynara scolymus leaves. This enriched extract contains about 20% Cynaropicrin. A typical analytical HPLC profile of this extract is shown in FIG. 3.
B) An alternative extract was prepared using 100 g hackled areal parts of Cynara scolymus which were heated under reflux with 700 g of a mixture of 80% methanol/20% water (w/w) for 90 min. After cooling to room temperature the resulted mixture was filtered over a folded filter. The separated material was washed with an additional amount of 150 g of extraction solvent of the same constitution as above. The volume of the combined solutions was 900 ml. Approximately 10 ml of this solution was evaporated to dryness (80 C., normal pressure) and yielded in 180 mg raw material. A sample of this extract was analysed by HPLC-UV/VIS method (see general experimental methods, gradient: begin 10% eluent B to 20 min 40% eluent B, 25 min 90% B). The chromatogram is presented in FIG. 5. The signal with the retention time of 14.3 min refers to Cynaropicrin (detected by the use of a wavelength of 210 nm) (obtained using the HPLC methods described in the general experimental procedures).

Example 5: Preparation of Cynaropicrin

[0157] Two liquid chromatography steps are optimised for purification of Cynaropicrin. An aliquot of the enriched extract as prepared above, e.g. in this case 81 g, is separated on reverse phase material by low-medium pressure liquid chromatography on a Chromabond (Trademark by Machery-Nagel GmbH & Co. KG, Dren Germany) P300-20 C18 column, 37090 mm, (Macherey & Nagel). The elution scheme as outlined in the following table is used in a batch procedure (defined solvent mix at defined volume used):

TABLE-US-00002 % H.sub.20 % Acetonitril % 2-Propanol Volumen/Fraction 80 20 0 1 2000 mL (Fraction 1) 60 40 0 1 1000 mL (Fraction 2) 60 40 0 4 250 mL (Fraction 3) 40 60 0 4 250 mL (Fraction 4) 40 60 0 2 500 mL (Fraction 4) 0 100 0 1 1000 mL (Fraction 4) 0 0 100 1 4000 mL (Fraction 4)

[0158] Eluent batches are acidified by addition of TFA to a final concentration of 0.1%. The separation is performed with a pressure of 2-3 bar at room temperature. The resulting fractions are analysed by analytical HPLC-UV-ELSD. Fractions containing Cynaropicrin are combined. A typical yield of this first step of purification is 14 g, containing already about 70% Cynaropicrin, starting from 81 g extract.

[0159] The second and final purification step is performed at room temperature on a preparative HPLC system (Gilson Abimed, Ratingen, Germany), comprising Gilson Unipoint software, 305 or 306 binary pump system, 204 fraction collector, 155 UV-Vis detector, 806 manometric module, and 811C dynamic mixer, using gradients and stationary phases as described below. The purification is performed on Chromabond or Nucleodur C18 columns (13040 mmm, Macherey & Nagel) with a flow rate of 20 ml/min whereas the fractions are collected each minute using following gradient and solvents, which were acidified with 0.1% TFA.:

TABLE-US-00003 % H.sub.20 % Acetonitril Time [min] 100 0 0 50 50 50 0 100 60 0 100 130 100 0 135 100 0 145

[0160] Using a UV/VIS-155 detector (Gilson, Langenfeld, Germany) the signals are detected at 210 nm and 254 nm. Cynaropicrin elutes typically in the range of 40-60% acetonitrile. A typical yield of the procedure is 3.0 g cynaoropicrin starting with 4.6 g from the first purification step; this final product contains more than 90% cynaropicrin as shown in FIG. 4.

[0161] The final product of this example is used for the above described examples 1 to 3 for determining biological activities.