Method for testing a substance which is potentially active in the field of lipolysis and its mainly cosmetic use

Abstract

A method of testing a substance potentially active in the field of lipolysis comprising: preparing a substrate containing at least one triacylglycerol; placing this substrate in contact with a substance potentially active in the field of lipolysis, and with a lipoprotein lipase, in the presence of a cofactor of lipoprotein lipase, for a period of time sufficient to release at least in part one fatty acid of the triacylglycerol; and determining the capacity of inhibition of the release of the fatty acid resulting from the activity of the lipoprotein lipase, under the action of said potentially active substance, and evaluating the result of the inhibition which is either compared to the result obtained in the absence of the potentially active substance tested or compared with the result obtained in the presence of a known inhibitor acting as reference. Cosmetic and pharmaceutical uses are described.

1 . A method of testing a substance which is potentially active in the field of lipolysis, comprising the steps: 
 a) preparing a substrate which contains at least one triacylglycerol,    b) placing this substrate in contact with a substance which is potentially active in the field of lipolysis, and with a lipoprotein lipase, in the presence of a cofactor of lipoprotein lipase, for a period of time sufficient for releasing at least in part one fatty acid of the triacylglycerol, and    c) determining the capacity of inhibition of the release of the fatty acid resulting from the activity of the lipoprotein lipase, under the action of said potentially active substance, and evaluating the results of the inhibition which are compared to the result obtained in the absence of the potentially active substance tested or which are compared with the result obtained in the presence of a known inhibitor acting as reference.    
     
     
         2 . The method  claim 1 , wherein the lipoprotein lipase is present with said cofactor which comprises or is constituted of apolipoprotein C-II.  
     
     
         3 . The method of  claim 2 , wherein the cofactor is of human origin.  
     
     
         4 . The method of  claim 1 , wherein the lipoprotein is placed in contact with the substrate in the presence of a fatty acid-acceptor substance or fatty acid-sequestering substance which prevents the blockage of the enzymatic activity of the lipoprotein lipase.  
     
     
         5 . The method of  claim 4 , wherein the fatty acid-acceptor substance or fatty acid-sequestering substance comprises or is essentially constituted by bovine or human albumin.  
     
     
         6 . The method of  claim 1 , wherein the lipoprotein lipase is obtained from bovine milk, or is obtained from bacteria.  
     
     
         7 . The method of  claim 1 , wherein the triacylglycerol comprises an acyl part which is obtained from a long chain fatty acid.  
     
     
         8 . The method of  claim 1 , wherein the triacylglycerol has an acyl part comprising 12 to 30 carbon atoms.  
     
     
         9 . The method of  claim 1 , wherein the acyl part is a straight or branched saturated C12-30 chain.  
     
     
         10 . The method of  claim 1 , wherein the acyl part is a straight or branched unsaturated C12-C30 chain.  
     
     
         11 . The method of  claim 1 , wherein the triacylglycerol comprises or is essentially constituted of triolein.  
     
     
         12 . The method  claim 1 , comprising measuring the capacity of inhibition of the lipoprotein lipase by the potentially active substance in several steps: 
 a) first of all, the lipoprotein lipase is incubated for a determined period of time in the presence of the substance which is potentially active as inhibitor,    b) the substrate which contains the triacylglycerol is incubated in the presence of the lipoprotein lipase cofactor,    c) the mixture of the triacylglycerol/lipoprotein lipase cofactor, is incubated in the presence of the enzyme lipoprotein lipase with or without the potentially active substance tested for its capacity of inhibition of the lipoprotein lipase,    d) upon completion of this incubation, a determination is made upon the reaction medium of the non-esterified fatty acids by a technique which is available to the person skilled in the art, and    e) a comparison is made of the capacity of inhibition of the release of a fatty acid of the triacylglycerol, or non-esterified fatty acid, resulting from the activity of the lipoprotein lipase in the presence of the potentially active substance tested, either compared with the result obtained in the absence of the potentially active substance tested or compared with the result obtained in the presence of a known inhibitor acting as reference.    
     
     
         13 . The method of  claim 12 , wherein the lipoprotein lipase cofactor comprises or is essentially constituted by apolipoprotein C-II.  
     
     
         14 . The method according to  claim 12 , wherein the determination of the non-esterified fatty acids is made upon the reaction medium by an enzymatic technique.  
     
     
         15 . The method of  claim 14 , wherein the enzymatic technique is observed by colorimetry at a wavelength determined by the enzymatic technique selected, and the lowering of the optical density obtained at this wavelength is determined with respect to the control or to the reference inhibitor.  
     
     
         16 . The method of  claim 12 , wherein the determination of the non-esterified fatty acids is made upon the reaction medium by an enzymatic technique which is observed by colorimetry at 550 nm and an inhibition is determined of the optical density at 550 nm which expresses a decrease in the fatty acids synthesized in the reaction medium, which is compared with the control or with a reference inhibitor, and the positive or negative activity is determined of said substance tested by the observation of a significant or non-significant inhibition effected by said substance tested with respect to the control or to the reference inhibitor.  
     
     
         17 . The method of  claim 1 , wherein the potentially active substance is selected from the group consisting of an extract of fucus; an extract of dulse palmaria palmata; an extract of wheat proteins; an extract of spiruline; an extract of honeysuckle; an extract of St. John's wort; an extract of rice proteins; an extract of liana; an extract of potato; an extract of shiitake; an extract of fresh salmon; an extract of pumpkin; and an extract of lemon.  
     
     
         18 . The method of  claim 17 , wherein said extract is selected from the group consisting of an aqueous or water extract, a hydro alcoholic extract, a hydro glycolic extract, a hydro ethanolic extract, a hydro propylene glycol extract, a hydro butylene glycol extract, and mixtures thereof.  
     
     
         19 . The method of  claim 1 , wherein the potentially active substance is an extract of liana.  
     
     
         20 . The method of  claim 19 , wherein the liana is liana  Uncaria tomentosa.    
     
     
         21 . The method of  claim 1 , wherein the potentially active substance is an extract of St. John's wort.  
     
     
         22 . The method of  claim 1 , wherein said inhibition test is used for selecting a substance potentially having an activity selected from a lipolytic activity and a slimming activity.  
     
     
         23 . The method of  claim 1 , for evaluating the activity of a substance which can be used in a cosmetic composition for a cosmetic care selected from the care of fatty deposits and a slimming care.  
     
     
         24 . The method of  claim 1 , for evaluating the effectiveness of a slimming treatment or care performed on a person in need thereof.  
     
     
         25 . A substance or plant extract active in the field of lipolysis, wherein the activity thereof in the field of lipolysis has been evaluated by a test method as defined in  claim 1  or  12   
     
     
         26 . A method of skin care selected from decreasing, slowing down or reabsorbing fatty deposits, from a slimming activity, from increasing the blood microcirculation, from improving the appearance of the skin, from improving the tone of the skin and from diminishing <<orange peel>> appearance, comprising applying to skin zones in need thereof, a cosmetic composition comprising a lipolysis active substance or lipolytic plant extract, said lipolysis activity having been evaluated by the method of  claim 1  or  12 .  
     
     
         27 . A method of treating a pathology resulting from an excess of fatty deposit, comprising performing a treatment of said fatty deposit with a pharmaceutical composition comprising a lipolytic substance, or lipolytic plant extract wherein the lipolytic activity has been evaluated by the test method of  claim 1  or  12 .  
     
     
         28 . A method of skin care selected from decreasing, slowing down or reabsorbing fatty deposits, from a slimming activity, from increasing the blood microcirculation, from improving the appearance of the skin, from improving the tone of the skin and from diminishing <<orange peel>> appearance, comprising applying to skin zones in need thereof, a cosmetic composition comprising a lipolytic active agent comprising an extract of liana  Uncaria tomentosa.    
     
     
         29 . A method of skin care selected from decreasing, slowing down or reabsorbing fatty deposits, from a slimming activity, from increasing the blood microcirculation, from improving the appearance of the skin, from improving the tone of the skin and from diminishing <<orange peel>> appearance, comprising applying to skin zones in need thereof, a cosmetic composition comprising a lipolytic active agent comprising an extract of St. John's wort.  
     
     
         30 . A cosmetic composition, comprising as one of its lipolytic active agents, a lipolytic substance or a lipolytic plant extract having a lipolytic activity determined by the test method of  claim 1  or  21 .  
     
     
         31 . The composition of  claim 30 , wherein said lipolytic substance, or said plant extract containing it is present at a concentration ranging between 0.01 and 70% by weight of the composition.  
     
     
         32 . A cosmetic composition, comprising a cosmetically effective amount of an extract of liana  Uncaria tomentosa , in a cosmetically acceptable excipient.  
     
     
         33 . The cosmetic composition of  claim 32 , wherein said extract is present at a concentration ranging between 0.01 and 70% by weight of the composition.  
     
     
         34 . The cosmetic composition of  claim 32 , wherein said extract is present in a concentration ranging between 0.01 and 30% by weight of the composition.  
     
     
         35 . A cosmetic composition, comprising a cosmetically effective amount of an extract of St. John's wort, in a cosmetically acceptable excipient.  
     
     
         36 . The composition of  claim 35 , wherein said extract is present at a concentration ranging between 0.01 and 70% by weight of the composition.  
     
     
         37 . The cosmetic composition of  claim 32 , wherein said extract is present in a concentration ranging between 0.01 and 30% by weight of the composition.  
     
     
         38 . A pharmaceutical composition, comprising a pharmaceutically effective amount of an extract of liana  Uncaria tomentosa , in a pharmaceutically acceptable excipient.  
     
     
         39 . The pharmaceutical composition of  claim 38 , wherein said extract is present at a concentration ranging between 0.01 and 70% by weight of the composition.  
     
     
         40 . The pharmaceutical composition of  claim 38 , wherein said extract is present in a concentration ranging between 0.01 and 30% by weight of the composition.  
     
     
         41 . A pharmaceutical composition, comprising a pharmaceutically effective amount of an extract of St. John's wort, in a pharmaceutically acceptable excipient.  
     
     
         42 . The composition of  claim 41 , wherein said extract is present at a concentration ranging between 0.01 and 70% by weight of the composition.  
     
     
         43 . The pharmaceutical composition of  claim 41 , wherein said extract is present in a concentration ranging between 0.01 and 30% by weight of the composition.