EP2100139A2

EP2100139A2 - Biologically active compositions, methods for producing the same and biological applications

Info

Publication number: EP2100139A2
Application number: EP07870278A
Authority: EP
Inventors: Charles Mioskowski; Annelise Lobstein; Alain Wagner; Régis SALADIN
Original assignee: Phytodia; Centre National de la Recherche Scientifique CNRS; Universite de Strasbourg
Current assignee: Phytodia; Centre National de la Recherche Scientifique CNRS; Universite de Strasbourg
Priority date: 2006-11-14
Filing date: 2007-11-13
Publication date: 2009-09-16
Also published as: FR2908310A1; JP2010509390A; WO2008074935A3; WO2008074935A2; FR2908310B1

Abstract

The invention relates to compositions and products that can be obtained from plants, such as extracts, fractions and/or molecules. They can be used in the production of drugs for human beings or animals, essentially for preventing or treating metabolic disorders, obesity and/or diseases associated therewith such as the X syndrome (metabolic syndrome), type-2 diabetes, or for producing food additives for human beings or animals.

Description

Biologically active compositions, process for obtaining them, and biological applications

The subject of the invention is novel biologically active compositions which can be obtained from plants. It also relates to a process for obtaining these compositions by extraction from selected plants with regard to a given activity. The invention also relates to the biological applications of these compositions for the treatment in humans or animals of diseases which result from a disturbance of the metabolism and / or the energy balance, called metabolic diseases, in particular the treatment of type 2 diabetes. In the last 30 years, sedentary lifestyle and over-nutrition have caused an explosion in the incidence of obesity in industrialized countries. Obese people have an increased risk of developing other syndromes or metabolic diseases, such as syndrome X, insulin resistance, and type 2 diabetes. There are about 200 million people worldwide with diabetes, including about 90% have type 2 diabetes. It is estimated that this figure will double in 25 years, affecting just over 5% of the world's population.

People with diabetes are 2 to 4 times more likely to develop cerebrovascular and cardiovascular complications. In addition to these macro-vascular problems, patients also develop a number of microvascular complications: retinopathies, neuropathies and nephropathies. After 10 years of disease progression, one-third of diabetic patients suffer from these complications, which could be considerably reduced by both behavioral management - especially with an adapted diet and accompanied by food supplements - and medication. There are different classes of drugs available for the treatment of metabolic diseases, which have in common to act on one of the components of metabolism. Nevertheless, the multi-symptomatic nature of metabolic diseases implies a multi-medication, which, in addition to its high cost for health organizations, increases the risk of side effects by drug interaction. This is why, despite the existence of a large number of therapeutic tools, there is a very great medical need for these metabolic diseases. Patients with metabolic diseases see their disease evolve over periods of 30 or even 40 years. During these years, there is no real feeling of a sick state and most people suffering from metabolic diseases find it very difficult to adhere to multi-drug treatments, which are often a source of side effects.

In addition, the products used to date to treat diabetes do not have a truly satisfactory profile, their side effects sustained and many restricting their use. Most newly developed or newly marketed new drugs also have side effects and it is therefore urgent to develop more effective and effective products.

Research is therefore continuing in this field, with a particular interest for alternative solutions, based in particular on products of plant origin, considered as natural, as opposed to synthetic compounds. This research leads to the discovery of herbal medicines and / or plant origin, but also to the identification of herbal dietary supplements. It is clearly established that the combination of a suitable diet incorporating the use of food supplements based on certain plants and adequate physical activity, prevents the development of metabolic diseases, and in particular that of obesity and diabetes.

Globally, nearly 1,200 plants have been attributed effects on metabolic diseases, particularly type 2 diabetes. However, in most cases, the non-toxicity of the plants has not been demonstrated. they contain have not been identified, their pharmacology to determine which therapeutic class the plant belongs to and possibly identify a new therapeutic class has not been performed or yet no extraction method to obtain in an optimized manner technically and economically their active ingredients has been described.

It follows that this lack of precise scientific knowledge of their effects prevents their therapeutic use. Without this scientific information, the use of nutraceuticals is also difficult, since the regulatory framework governing this field has recently been strengthened since the implementation of the new decree 2006-352 on to food supplements which is the transposition into French law of the European Directive 2002/46 / EC.

For the sake of obtaining precise scientific data and development of original assets, the inventors have developed means for evaluating the effect of compositions of plant origin on metabolic activity at the mitochondrial level. Indeed, recent studies have shown that the decrease of the metabolic activity itself, and in particular at the level of the mitochondria, was one of the major and early disturbances observed in the development of metabolic diseases, notably in the pathogenesis type 2 diabetes [Petersen et al].

According to a first approach, the inventors have found that the MTT test

(bromide 2 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium), commonly used to evaluate the cytotoxicity of a given chemical, of synthetic or natural origin (Mosmann), could constitute a a tool of great interest for selecting active compositions from plants and thus diverting it from its usual uses.

This test is based on the ability of the dehydrogenase enzymes contained in mitochondria to convert MTT, a solution-colored yellow molecule, into blue formazan crystals in solution. It makes it possible to obtain a measurement of the continuous dehydrogenase activity in the mitochondria after treatment with a given product and therefore to measure the amount of ATP produced by the mitochondria, which is the signature of the metabolic activity at the level of the mitochondria. of a cell.

The inventors have thus developed a method of screening novel molecules for their potential to act on the metabolic activity, using the MTT test, not only to measure the cytoxicity of extracts, fractions and compounds of selected plants, but also original, to measure the ability of these extracts, fractions and compounds to increase metabolic activity.

The continuation of their work then focused on the search for products of plant origin specifically recognizing a biological target interfering with the mitochondrial function, the TGR5. The TGR5 receptor belongs to the family of G-protein coupled receptors (GPCRs for short). It is the first membrane receptor activated by bile acids. TGR5 is described as a mediator of certain endocrine functions of bile acids.

Thus, by using a CHO cell line stably transfected with a TGR5 expression vector and a reporter plasmid (for example the gene coding for luciferase), the inventors found that the TGR5 activity was increased, relative to a control, when the CHO cells were treated with extracts and / or fractions obtainable from plants, or with molecules that could be obtained from these extracts or fractions. These extracts, fractions and molecules are generally designated by the terms compositions or "products" in the description and claims.

The use of TGR5 as a biological target in accordance with the invention makes it possible to have products with a high specificity in terms of their effects on metabolism.

The invention therefore aims to provide a method of high reliability and easy to implement to obtain new compositions useful to prevent, accompany a diet and treat metabolic diseases.

It is also intended to provide such compositions as new products and to use their properties for the manufacture of drugs or herbal supplements or plant active ingredients for the treatment and prevention of metabolic diseases .

The purpose of the invention is in particular to provide agonist products of TGR5, and aims at the applications of these products, in humans and animals, in particular in the therapeutic, nutritional and nutraceutical fields and, in general, to prevent and treat treat conditions, disorders and syndromes related to metabolic disorders.

According to a first aspect, the invention relates to a process for obtaining biologically active compositions, by extraction from one or more plants, characterized in that it comprises the steps a) separately treating the plants selected with respect to a given activity, or parts thereof, with one or more solvents to obtain total extracts or fractions enriched in active principles, especially apolar fractions and fractions; polar

b) selecting extracts or fractions having a metabolic activity in the MTT test and a biological activity in at least one test specific for this metabolic activity.

This method advantageously comprises, besides the steps a) and b) defined above, a step c), implemented after step a), and / or after step b), this step c) comprising the analyzing said extracts or said selected fractions to determine the components they contain and isolate and purify, if desired, the active principle (s) carrying the bulk of the biological activity. This analysis is carried out more particularly by HPLC profiles.

This provision on the identification and isolation of active compounds opens the prospect of obtaining such compounds by synthesis in the context of a pharmaceutical or nutraceutical development.

In step a), the plants or parts of plants are preferably used in the form of a fine powder, obtained after grinding and drying.

It is more particularly the leaves, the barks, the roots, the aerial parts, the fruits, the seeds ...

Stage a) generally comprises one or more extractions, these extractions being carried out using supercritical CO ₂ or with organic solvents, in particular an alcoholic solvent (in particular with ethanol or methanol) or cyclohexane or any other apolar solvent (petroleum ether, heptane, etc.), dichloromethane or chloroform, and several of these solvents or all these solvents, which can be used successively for the isolation of specific fractions

It will be noted that the use of the MTT test according to step b), applied to plants to which biological effects were recognized, but could not be used effectively because of lack of means for their characterization, makes it possible to reveal extracts and fractions which had never been identified until the invention. The compositions and products, extracts, compound fractions and molecules obtainable by the process defined above are new and as such are also within the scope of the invention.

The invention is illustrated hereinafter by its application to obtain compositions having antidiabetic activity and useful in particular for the prevention and treatment of type 2 diabetes.

For this purpose, the biological tests carried out according to step b) of the process defined above are tests for determining an antidiabetic activity, in particular on the insulin secretion.

Preferred compositions and products in this regard are obtainable from plants selected from genera and species: Strychnos myrtoldes, Obetia radula, Trema orientalis, Sclerocarya birrea, Voacanga thouarsii Moringa oleifera (= Moringa moringa, Moringa pterygosperma, Guilandina moringa), Agrimonia eupatoria, Arctium majus = Lappa major, Alchemilla vulgaris, Phaseolus vulgaris. Vaccinium myrtillus, Cichorium intybus, Cynara scolymus, Geranium robertianum, Juglans regia, Kalanchoe crenata, Leptodenia madagascariensis, Morus nigra, Olea europaea, Rubus fructicosus, Spathodea campanulata.

Advantageously, these are compositions and products characterized in that they are capable of being obtained from plants chosen from the genera: Strychnos, Betia, Dema, Sclerocarya, Voacanga, Moringa, Agrimonia, Arctium, Alchemilla, Phaseolus ,

Vaccinium, Cichorium, Cynara, Geranium, Juglans, Kalanchoe, Leptodenia, Morus, Olea,

Rubus, Spathodea.

Particularly preferred compositions and products are obtainable from plants selected from the following families: Loganiaceae, Urticaceae, Ulmaceae, Anacardiaceae, Apocynaceae, Moringaceae, Rosaceae, Asteraceae, Fabaceae, Ericaceae, Geraniacaea, Juglandacaea, Crassulaceae, Oleaceae, Bignoniacea .

Other particularly preferred compositions are fractions of Tréma orientalis

Ulmaceae, Strychnos myrtoides Loganiaceae, Obetia radula Urticaceae, Vaccinium myrtillus Ericaceae, Sclerocarya birrea Anacardiaceae, Agrimonia eupatoria Rosaceae, Alchemilla vulgaris Rosaceae and Olea europaea Oleaceae, showing, respectively, HPLC profiles of Figures 1-8, as well as biologically active compounds derived from these fractions.

In another aspect, the invention is particularly directed to the use of products obtainable from plants, such as extracts, fractions, compounds and / or molecules as agonists specific for TGR5.

We will measure the originality of this aspect of the invention based on the demonstration of the mechanism of action of said products that allows the development of specific applications.

The products used in the use according to the invention are biologically active products, characterized in that they have a chemical structure different from that of the bile acids, known agonists of TGR5; have an EC ₅₀ greater than or equal to that of a natural ligand of TGR5, such as lithocholic acid (LCA); and furthermore do not activate the FXR nuclear receiver. In addition, these products lead to a reduction in fat-induced weight gain in a mouse model of metabolic syndrome and also lead to a reduction in the mass of epididymal fat pads in the same model. They also lead to improved glucose tolerance.

Thanks to these specific effects, they are particularly suitable for preventing or treating patients suffering from pathologies such as obesity, the metabolic syndrome (or syndrome X) or type 2 diabetes.

The products used as TGR5 agonists according to the invention are more particularly capable of being obtained from plants chosen from the group comprising Olea europaea, Alchemilla vulgaris, Juglans regia, Agrimonia eupatoria, Vaccinium myrtillus and Obetia radula.

Particularly preferred molecules in view of their activating effect of TGR5 include pentacyclic triterpenes. They are more particularly pentacyclic triterpenes of the oleanane type, such as oleanolic acid, of the ursane type, such as corosolic acid, or of the lupane type.

The demonstration of the antihyperglycaemic, antihyperinsulinemic and obesity effects of oleanolic acid is particularly surprising since, to date, the antihypertensive and hypoglycaemic properties of olive leaves have only been attributed to oleuropein.

Advantageously, said TGR5 agonists according to the invention are highly innocuous and do not give rise to harmful side effects.

The invention therefore aims generally at making use of the compositions and products defined above, namely extracts, fractions and compounds of the invention.

According to a first aspect, the invention thus relates to novel pharmaceutical compositions, characterized in that they contain an effective amount of at least one composition or a product as defined above, in association with a pharmaceutically acceptable vehicle.

The invention also relates to the use of said biologically active compositions and products for the manufacture of medicaments intended for the prevention and / or treatment of metabolic disorders / syndromes, more particularly of obesity and / or associated diseases such as Syndrome X ( metabolic syndrome), type 2 diabetes.

In one embodiment, the invention is directed to the use of pentacyclic triterpenes of the ursane type, pentacyclic triterpenes of the oleanane type, or pentacyclic triterpenes of the lupane type.

In another embodiment, the invention provides in particular the use of said extracts or fractions, or corosolic acid for the prevention and / or treatment of type 2 diabetes ₅ of obesity and / or metabolic syndrome .

In yet another embodiment, the invention relates to the use of said extracts or fractions, or oleanolic acid for the prevention and / or treatment of obesity or diabetes, particularly type 2 diabetes, and / or the metabolic syndrome. The medicaments of the invention are advantageously in galenic forms for oral, topical or injection administration.

For oral administration, the pharmaceutical compositions are more particularly in the form of tablets, tablets, capsules, granules, drops, syrup, alcohol syrup.

For topical administration, they are advantageously in the form of creams, milks, lotions, oils or patches.

Suitable galenic formulations include aerosols.

For administration by injection, they are in the form of solutions in injectable ampoules intravenous, subcutaneous or intramuscular, developed from sterile or sterilizable solutions, or suspensions or emulsions.

The dosage in the various forms and for the daily application, will be established, in a conventional manner, depending on the type of effects to be treated.

By way of example, said drugs based on said agonists will be administered at a rate of 0.1 mg to 5 g / day.

The antihyperglycemic, antihyperinsulineuric and weight gain effects of said products are also advantageously used in dietetics.

According to a second aspect, the invention thus relates to food supplements, characterized in that they contain an effective amount of at least one composition or a product as defined above for a nutraceutical application, in association with an inert excipient.

The invention thus particularly targets the use of TGR5 agonists defined above as food supplements intended for humans (adults or children) or animals. Complements of the invention are incorporated into the diet during the preparation of food or during their packaging, or by using preparations already formulated with said products, to perform the desired supplementation.

They may also be in an ingestible form such as capsules, capsules, in liquid infusions, gum, powder, tablets, capsules, decoctions, this enumeration is not limiting.

In general, the invention therefore proposes an alternative for the prevention of these metabolic diseases because these products of natural origin are associated with a better quality of life and secondly their often centuries-old use in the traditional pharmacopoeia ensures an exhaustive knowledge. side effects.

Other features and advantages of the invention are given with the following examples of detailed embodiments of the invention. These examples serve to illustrate the invention without limiting its scope. Reference is made in these examples:

- A: to Figures 1 to 9 which represent the HPLC profiles of plant extracts according to the invention (Figures 1 to 8) and give an MTT test result (Figure 9), and

FIGS. 10 to 24 which illustrate the results obtained with fractions of 6 plant extracts (Olea europaea, Alchemilla vulgae, Juglans regia, Agrimonia eupatoria, Vaccinium myrtillus, Obetia radula), namely, respectively:

FIG. 10: the effect of fractions of Olea europaea on the activity of the TGR5 receptor; FIG. 11: the HPLC profile of the active Olea europaea fraction on the TGR5,

FIG. 12: the NMR spectrum of oleanolic acid, active principle on TGR5, extracted from Olea europaea,

- Figure 13: the effect of fractions to? Alchemilla vulgaris on the activity of the TGR5 receptor,

- Figure 14: HPLC profile of the ^fraction: Alchemilla vulgaris active on the TGR5, - Figure 15: the NMR spectrum of corosolic acid, the active ingredient on the TGR5, extract of Alchemilla vulgaris,

FIG. 16: the effect of fractions of Juglans regia on the activity of the TGR5 receptor,

FIG. 17: the HPLC profile of the Juglans regia fraction pool active on the TGR5, ^ ^

FIG. 18: the effect of Agrimonia eupatoria fractions on the activity of the TGR5 receptor,

FIG. 19: the HPLC profile of the active fraction of Agrimonia eupatoria on the TGR5, FIG. 20: the effect of Vaccinium myrtillus fractions on the activity of the TGR5 receptor,

FIG. 21: the HPLC profile of the active Vaccinium myrtillus fraction on TGR5,

FIG. 22: the effect of Obetia r adula fractions on the activity of the TGR5 receptor, FIG. 23: the HPLC profile of the active Obetia r adula fraction on TGR5, and

- Figure 24: the effect of oleanolic acid (OA), corosolic acid (AVA) and active fraction TGR5 of Juglans regia (JRL) on the glucose tolerance test

(IPGTT) in the mouse on a fat diet.

Examples A

Material and method

plants

The parts of dried plants (leaves, roots, etc.) are crushed in order to obtain a fine powder intended for extraction.

Extraction-purification

The powder is exhausted by percolation or maceration, or by sonication with polar or apolar solvents. These solvents may be cyclohexane, dichloromethane, chloroform, acetone, ethyl acetate, butanol, ethanol, methanol, water or a mixture of these various solvents. The extracts are obtained with yields of between 2 and 10%. They are evaporated to dryness under reduced pressure. Then fractionated either by liquid-liquid extraction, or by successive extractions by solvents of increasing polarity. The pure compounds can be obtained in particular by fractionation by adsorption chromato graphy, exclusion chromato graphy, affinity chromatography, ion exchange chromatography, or by fractional crystallization. Chromatographic profiles

Extracts, enriched fractions, more or less purified fractions or pure compounds are analyzed by high pressure chromatography using a reverse phase column (RP-HPLC). The stationary phase used corresponds in particular to a grafted silica C8 or Cl8, 5 to 10 granulometry. The eluting mixtures correspond in particular to acidified water (pH 3) added with methanol or acetonitrile. The gradient used varies from 98% to 0% acidified water over at least 60 min.

MTT test

The INSl pancreatic transformed line used for this test is a donation from the European Diabetes Center (Hautepierre, France). The cells are seeded at a density of 3 × 10 ³ / well in 96-well plates and cultured in RPMI 1640 medium enriched with fetal calf serum (10%). The cells are treated with the various plant extracts (200 μg / ml) for 24 hours and the MTT test then carried out: MTT (bromide 2 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium ) is a tetrazolium salt giving a yellow solution when diluted in a medium. It is converted into insoluble violet formazan in the culture medium after cleavage of the tetrazolium ring by the active mitochondrial dehydrogenases of living cells alone. Dead cells can not be the seat of this change. The MTT solution (final concentration of 0.5 mg / ml) is added to the cells for 4 hours of incubation. The medium is then sucked carefully so as not to suck the formed formazan crystals, which will be solubilized in 100 .mu.l of DMSO. The dissolved material is measured by spectrophotometry giving an absorbance proportional to the concentration of converted dye. The absorbance of each well is measured at 540-630 after subtraction of the background noise on the spectrophotometer. The results are expressed as a percentage of the control (DMSO alone).

Insulin secretion test

Langerhans islets of pork pancreas are obtained from the European Diabetes Center (Hautepierre, France). 100 cells / well are seeded in 24-well plates and cultured in RPMI 1640 medium enriched with fetal calf serum (10%). The cells are treated with the various plant extracts (200 μg / ml) for 60 minutes and the insulin secretion measured using an Elisa kit (Mercodia) according to the supplier's recommendations. The results are expressed as a percentage of the control (DMSO alone).

Example 1 A: Study of the effect of selected plants on the metabolic activity of cells

INS1 cells were treated for 24 hours with 200 μg / ml of extract of plant obtained by extraction with ethanol, methanol or dichoromethane. The MTT conversion rate was calculated and the effect of the extracts expressed as a percentage of the control.

The results obtained are shown in Table 1: ethanolic extracts (total), methanol (enriched in polar compounds) and dichloromethane (enriched in apolar compounds). In this table, EtOH = ethanol fraction; MeOH = methanol fraction; DCM = dichloromethane fraction.

Table 1

Examination of these results shows that all the selected plants have an effect on the metabolic activity of the cells. Example 2 A: HPLC Profiles

The active fractions are subjected to analytical RP-HPLC in order to have the chromatographic fingerprint of the extracts with respect to their biological activity. FIGS. 1-8 give, respectively, the RP-HPLC profiles of the ethanolic fractions of Tréma orientalis, Strychnos myrtoides, Obetia radula, Vaccinium myrtillus, Sclerocarya birrea, Agrimonia eupatoria, Alchemilla vulgaris and Olea europaea.

The fractions are then purified by semi-preparative RP-HPLC and then subjected to the MTT test to confirm and identify the active ingredient. For this test, INS 1 cells were treated with 100, 200, 400 or 800 μg / ml of plant extract for 24 hours. An example of a result obtained with Tréma orientalis is presented in Figure 9. The abbreviations given in this figure have the following meanings: TC: cell-only control; DMSO: cell-only control + solvent (DMSO); A-J: fractions obtained after RP-HPLC semi-preparative (details of the fractions presented in FIG. 1). The conversion rate is presented after measuring the optical density at 560 nm.

Example 3 A: Effect of selected plant extracts on insulin secretion

There are four distinct classes of anti-diabetic therapies for treating type 2 diabetes. These include sulfonylureas, the flagship drug in the United States for the treatment of type 2 diabetes. Sulfonylureas promote the secretion of insulin by the pancreas. thereby prevent hyperglycemia. Since the plants are active on cellular metabolism, to determine if the active ingredients belonged to one of the 4 known therapeutic classes, in particular the sulfonylureas, the activity of these extracts was verified on an insulin secretion test on Langherans pigs.

Pork Langherans islets were treated for 60 minutes with 200 μg / ml of plant extract. Insulin secretion is measured by ELISA and the effect of extracts expressed as a percentage of control.

The results are given in Table 2 below. In this table, EtOH, MeOH and DCM correspond, respectively, to the ethanol (total), methanol (enriched in polar compounds) and dichloromethane (enriched in nonpolar compounds) extracts. Table 2

Examples B

Products and processes Products

Oleanolic acid is a product marketed by Extrasynthèse (Genay, France) and oleuropein and lithocholic acid are products marketed by Sigma-Aldrich (Saint-Quentin Fallavier, France).

Extraction from plant

The extraction with ethanol is carried out, exhaustively, on 10 g of air-dried and pulverized olive leaves (Olea europaea L. Oleaceae, PhytoEst, France) with an aqueous solution of ethanol at 80%, by maceration at room temperature for 3 hours.

The mixture is then filtered using a suction pump and the filtrate is concentrated by evaporation under reduced pressure.

For extraction with dichloromethane and methanol, the powdered plant material (10 to 20 g) is successively extracted with cyclohexane (100 ml), dichloromethane (100 ml) and methanol (100 ml), using an extractor. Automatic Soxhlet (2055 Aventi Soxtex, Foss Tecator). This protocol makes it possible to obtain three types of extract in which the highly lipophilic, non-polar and polar constituents of the plant are respectively concentrated.

The solvent is removed by evaporation under reduced pressure and the corresponding residues are weighed and stored in the freezer until use.

HPLC analysis

For the analytical profiles of each extract, a methanolic solution of the residue (5 mg / mL) was subjected to analytical HPLC (Varian Pro Star) coupled to a DAD detector (Varian) using a reverse phase column (Nucleodur 100-10). -Cl 8,250 / 4,6, Chew ey-Nagel). For elution, a mixture of acetonitrile and acidified water (0.1% trifluoroacetic acid) is used.

Under analytical conditions, the flow rate is 1 mL / min with the following gradient: H ₂ O / CH ₃ CN (20:80) for 30 min, CH3CN (100%) for 5 min, and H ₂ O / CH ₃ CN

(20:80) for 5 min. For LC / MS analysis (Agilent 1200SL), the PDA is registered in the gamma from 200 to 500 nm, using 254 nm as the detection wavelength. The analyzes are performed with an Application Layer Protocol Control Information (APCI) coupled to an electrospray ionization interface (ESI) in negative mode.

NMR analysis

A DPX300 NMR spectrometer operating at 300 MHz for ¹ H is used. The chemical shifts are expressed in δ (parts per million) with reference to the solvent peak of 7.58 for the pyridine-d5 C5D5N.

Determination of TGR5 activity by a luciferase test

Chinese hamster ovary (CHO) cells were obtained from ATCC (Manassas, Virginia) and maintained in Minimum Essential Alpha Medium (α-MEM) supplemented with 10% (v / v) serum fetal calf (FCS), 100 μM non-essential amino acids (NAA), 100 units / mL of penicillin and 100 μg / mL of streptomycin sulphate.

For the determination of the TGR5 activity, a stable cell line obtained by transfecting CHO cells with 3.8 μg of a plasmid, TGR5 expression vector (pCMVSPORT6 / TGR5), 3.8 μg of a luciferase reporter plasmid whose expression is controlled by the cAMP Response Element (CRE, pCRE-Luc) and 0.4 μg of a plasmid for expression of the neomycin resistance gene (pcDNA3.1 (+)) using of the

Lipofectamine 2000. The transfected cells are selected with 400 μg / mL of sulfate

G418 and the individual clones are grown independently on a 96-well plate. The CHO cells expressing TGR5 are treated with 10 μM of lithocholic acid (LCA) or plant extracts, and then subjected to luciferase assays [Picard et al.]. Luminescence is determined on a Centro XS3 LB960 (Berthold Technologies,

Bad Wildbad, Germany).

FXR assay by luciferase

To evaluate the FXR activity of the plant extracts, COSF (ATCC) cells are transfected with Lipofectamine 2000 with 25 ng of hFXR expression vector plasmid. (pCMX-hFXR), 25 ng of mouse rexinoid receptor expression vector (RXRα) plasmid (m) (pCMX-mRXRα), luciferase reporter (pEcREx7 -Luc) and 50 ng of pCMVβ used as internal standard in each well.

About 18 hours after the transfections, the cells are incubated for 5 hours with different concentrations of each extract in fresh MEM (or DMEM). After this treatment, the cells are lysed and the standardized luciferase activity is determined by operating according to Picard et al.

Animals and diet

Six-week old male C57BL / 6 mice obtained from Charles River are used.

Normal and balanced high-carbohydrate food (SAFE, D04) and high-fat food (60% Kcal Fat, D12492) are obtained from Research Diets (New Brunswick, New Jersey).

The mice are in cages of 4 or 5 animals under stable temperature, with a day / night cycle of 12 hours, and with 1 free access to food and water. The animals are kept for 10 weeks either in a normal and balanced diet (n = 5) or in a diet rich in lipids (n = 32). After 10 weeks under a diet rich in lipids, the animals are divided into four groups of 8 animals: one group is maintained under a diet rich in lipids only, another group receives the same diet supplemented with oleanolic acid at a dose of 100 mg / kg / day (food intake is measured throughout the study and the amount of compound adjusted to maintain the dosage stable), another group receives the same diet supplemented with corosolic acid at one dose of 100 mg / kg / day, finally a last group receives the same diet supplemented with active fraction of Jugions regia at a dose of 100 mg / kg / day. Body weight and food intake are determined every other day. This experience is carried out respecting ethical principles.

Glucose tolerance test

The test is performed on animals that have been allowed to fast for 10 hours. The blood glucose is monitored using a portable blood glucose meter (Maxi Kit Glucometer 4, Bayer Diagnostics). For this test, an intraperitoneal injection of a sterile solution containing 2 g glucose / kg body weight. Blood glucose is assessed by drawing blood from the tail vein at 0, 15, 30, 45, 60, 90, 120, 150 and 180 minutes.

Example IB: Identification of TGR5 agonists from Olea europaea

• Extraction from leaves of Olea europaea

Total ethanolic extracts, extracted with DCM (dichloromethane) and extracted with MeOH (methanol). The total extracts exhibiting TGR5 activation similar to that observed with

LCA (used as a positive control) on the luciferase assay in CHO-TGR5 cells were first screened.

The isolation of the active principle is continued by testing enriched extracts either in hydrophobic molecules (DCM extraction) or in hydrophilic molecules (MeOH). For Olea europaea, most of the activity is present in the DCM extract.

Splitting of the hydrophobic extract

The fractionation of the hydrophobic fraction of the Olea europaea extract is continued by

Preparative reverse phase HPLC or thin layer chromatography. Ten fractions numbered from 1 to 10 are obtained and the luciferase activity in the CHO-TGR5 is measured with a control, that of LCA and that of oleuropein at doses of 1.5,

5, 15 and 50 μg / mL.

As shown in FIG. 10, the activity is present only in fractions 6 to 9. With oleuropein at doses ranging from 1.5 to 50 μg / ml, no agonist effect on TGR5 is observed. These data demonstrate that the active molecule contained in the DCM extract of Olea europaea is different from oleuropein.

• Active molecule of Fraction 7

Surprisingly, the active fraction 7 has a high purity. Detection

UV reveals only one main peak with a retention time of 21.9 minutes (Figure 11). The LC / MS analysis of fraction 7 makes it possible to identify the molecular weight of the molecule at 455.3 g / mol. This analysis is carried out in negative mode, which means that a hydrogen proton is missing on the molecule. It is therefore estimated the actual molecular mass of the peak detected at around 456 to 457 g / mol. Identification is complete by analyzing fraction 7 by NMR (Figure 12). The spectrum obtained is characteristic of a triterpenoid-type molecule. Analytical data of HPLC, LC / MS and NMR combined with those available for the olive leaf triterpenes already described, lead to the conclusion that the molecule of fraction 7 is oleanolic acid. This identification is confirmed by performing a co-injection into HPLC with a commercial standard (Extrasynthesis, Genay, France) and by comparison of the NMR spectrum obtained with that already reported of oleanolic acid [Seebacher et al.]. The EC50 value obtained for oleanolic acid in the human TGR5 luciferase activity test, which is 1.42 μM, is comparable to the EC50 of 0.89 μM obtained for LCA, a natural ligand of

TGR5.

• Study of the selectivity of oleanolic acid with respect to TGR5

To rule out any possible involvement of the bile acid nuclear receptor in the explanation of the biological activity of oleanolic acid, it is then determined whether oleanolic acid activates FXR in an FXR-dependent reporter gene (luciferase) assay. The results confirm that oleanolic acid does not activate FXR, demonstrating that this triterpene is a selective agonist of TGR5, clearly differentiating it from bile acids, which activate both TGR5 and FXR.

Example 2 B: Identification of agonists of TGR5 from Alchemilla vulgar ± s

Extracts and fractions are prepared by operating as above with Oleα europαeα.

The results obtained with the fractions of the DCM extracts are given in FIG.

The very high reactivity of fraction 9 is found in the TGR5 test.

The HPLC profile of this fraction is given in FIG.

Figure 15 reports the NMR spectrum of corosolic acid, which is the active molecule contained in fraction 9. Example 3 B: Identification of agonists of TGR5 from Juglans regia

The procedure is as indicated in Example 1 in connection with Olea europaea. The results obtained with the fractions of the DCM extracts are given in FIG. 16 which shows the strong activity of the fractions 7, 8 and 9. The HPLC profile of the pool of these fractions is given in FIG.

Example 4 B: Identification of agonists of TGR5 from tfAgrimonia eupatoria

The procedure is as indicated in Example 1 in connection with Olea europaea. The results obtained with 8 fractions of the DCM extracts are given in FIG. 18 which shows the strong activity of fraction 4. The HPLC profile of these fractions is given in FIG.

Example 5 B: Identification of TGR5 agonists from Vaccinium myrtillus

The procedure is as indicated in Example 1.

The results obtained on 9 DCM fractions are given in FIG. 20. The examination of this figure shows that the fractions 3, 5, 6, 7, and 8 exhibit a higher activity than that of LCA, especially with regard to the fraction 6. The HPLC profile of this fraction is given in FIG.

Example 6 B: Identification of TGR5 agonists from Obetia radula

The procedure is as indicated in Example 1.

The results obtained on 8 DCM fractions are given in FIG. 22. The examination of this figure shows that fractions 3, 4 and 6, especially fraction 6, have an activity greater than that of LCA. The HPLC profile of this fraction is given in FIG. Example 7 B: Study of the effect of active fractions of plant extracts on glucose tolerance in mice on a fat diet

The effect of active fractions of plant extracts according to the invention was evaluated in a mouse model of metabolic syndrome, namely the mouse subjected to a fatty diet. C57BL / 6 mice are fed either with normal and balanced food or with high-fat food for 10 weeks before starting treatment with plant extracts. During this period, the fat mice show a 50% increase in their body weight, accompanied by a similar increase in caloric intake.

At the end of the treatment period (14 days) with the fractions of: Olea (OA), Alchemilla (AVA) and Juglans (JRL), the clinical parameters given in Table 1 below are obtained.

Table 1

Normal diet fat diet

OA vehicle AVA JRL 100 mg / kg 100 mg / kg 100 mg / kg

OJ 24.3 ± 0.5 36.1 ± 1 34.5 ± 0.7 34.1 ± 1, 5 35 ± 1.3

Body weight (g) J14 26.3 ± 0.4 38.3 ± 1, 1 30.4 ± 1, 4 35 ± 1, 4 35.9 ± 1, 3

Average food intake 7.20 ± 0.29 14.79 ± 0.88 11, 43 ± 0.98 X X (Kcal / animal / day)

WAT Epididymal (% Body weight) 1, 73 ± 0.09 5.77 ± 0.27 3.7 ± 0.7 * 4.8 ± 0.4 ^* 5.1 ± 0.3

Data represent mean values ± SEM, normal feeds n = 5, Vehicle, OA, AVA, JRL n = 8. (* p <0.001) - WAT = white adipose tissue

A tendency to produce an inhibitory effect on body weight gain is observed, as well as a marked reduction in the mass of fat pads in the epididymis, indicating that active fractions affect the energy metabolism.

It will be appreciated that in these experiments, a high fat diet is maintained for 10 weeks before starting treatment. The study therefore focuses on the ability to correct, rather than prevent, the onset of overweight and insulin resistance. The results of Figure 24 indicate that these fractions are capable of improving metabolic homeostasis in mice on a fat diet. In particular, the insulin resistance induced in this model of high-fat diet and causing a pre-diabetic effect is corrected: indeed after 14 days of treatment with the active fractions of the invention, a hyperglycemia test induced intraperitoneally (IPGTT) is performed. The mean blood glucose levels before IPGTT are 103 ± 7, 154 ± 5, 144 ± 18, 141 ± 2 and 159 ± 3 mg / dL, respectively, in the normal-fed, high-lipid, high-lipid diet groups + oleanolic acid, diet rich in lipids + corosolic acid, and high fat diet + JRL. The IPGTT curves clearly show that the administration of a high-fat diet induces glucose intolerance and insulin resistance (Figure 24). This glucose tolerance is partially corrected when the mice are treated with the active fractions of plants.

The invention thus provides highly specific and powerful TGR5 agonists and the results obtained show the high activity of these molecules, corresponding to pure and / or enriched fractions.

References

KF Petersen, S Dufour, D Befroy, R Garcia, and GI Shulman. N Engl J Med, 350: 664-671 (2004)

KF Petersen, D Befroy, S Dufour, J Dziura, Ariyan C, DL Rothman, L DiPietro, GW Cline and GI Shulman. Science, 300: 1140-1142 (2003).

Mosmann, J. Immunol Methods, 65 (1-2): 55-63 (1983).

F. Picard, M. Gehin, M. C. Annicotte, S. Rocchi, M. F. Champy, B. W. O'Malley, P. Chambon, and J. Auwerx. SRC-I and TIF2 control energy balance between white and brown tissue adipose. CeIl 111 (2002) 931-941.

W. Seebacher, N. Simic, R. Weiss, R. Sat, and O. Kunert. Magn. Reson. Chem. 41 (2003) 636-638.

Claims

1 - Process for obtaining biologically active compositions, by extraction from one or more plants, characterized in that it comprises the steps

a) separately treating the plants selected with respect to a given activity, or parts thereof, with one or more solvents to obtain crude total extracts or fractions enriched in active principles, more especially apolar fractions and polar fractions

b) selecting extracts or fractions having a metabolic activity in the MTT test and a biological activity in at least one test specific for this activity.

2 - Process according to claim 1, characterized in that it comprises, besides the steps a) and b) defined in claim 1, a step c), implemented after step a), and / or after step b), this step c) comprising the analysis of said extracts or said selected fractions to determine the components they contain and isolate, if desired, the compound (s) carrying the bulk of the biological activity.

3 - Process according to claim 1 or 2, characterized in that in step a), the plants or parts of plants are used in fine powder form, obtained after grinding and drying.

4 - Process according to claim 3, characterized in that the plant parts are leaves, bark, roots, aerial parts, fruits and seeds.

5 - Process according to any one of claims 1 to 4, characterized in that step a) comprises one or more extractions, these extractions being carried out using CO ₂ or organic solvents, in particular an alcoholic solvent or cyclohexane or any other apolar solvent, dichloromethane or chloroform, and several of these solvents or all these solvents, which can be used successively for the isolation of specific fractions

6 - Process according to any one of claims 2 to 5, characterized in that the analysis of a fraction according to step c) is carried out by HPLC profiles. 7 - Process according to any one of claims 1 to 6, characterized in that the biological tests carried out according to step b) are tests for determining an antidiabetic activity, in particular on the secretion of insulin.

8 - Biologically active compositions, characterized in that they are extracts, fractions obtainable by the method according to any one of claims 1 to 7.

9 - Compositions according to claim 8, characterized in that they are compounds capable of being isolated from said extracts or fractions.

10 - Compositions according to claim 8 or 9, characterized in that they are likely to be obtained from plants selected from the genera and species:

Strychnos myrtoides, Obetia radula, Trema orientalis, Sclerocarya birrea, Voacanga thouars i Moringa oleifera (= Moringa moringa, Moringa pterygosperma, Guilandina moringa) Agrimonia eupatoria, Arctium majus (= Lappa major), Aichemiella vulgaris, Phaseolus vulgaris, Vaccinium myrtillus, Cichorium intybus , Cynara scolymus, Geranium robertianum, Jugions regia, Kalanchoe crenata, Leptodenia madagascariensis, Morus nigra, Olea europaea, Rubus fructicosus, Spathodea campanulata.

11 - Compositions according to claim 10, characterized in that they are likely to be obtained from plants selected from the genera: Strychnos, Obetia, Dema, Sclerocarya, Voacanga, Moringa, Agrimonia, Arctium, Alchemilla, Phaseolus, Vaccinium , Cichorium, Cynara, Geranium, Jugions, Kalanchoe, Leptodenia, Morus, Olea, Rubus, Spathodea.

12 - Compositions according to claim 10, characterized in that they are likely to be obtained from plants selected from the families: Loganiaceae, Urticaceae, Ulmaceae, Anacardiaceae, Apocynaceae, Moringaceae, Rosaceae, Asteraceae, Fabaceae, Ericaceae, Geraniacaea , Juglandacaea, Crassulaceae, Oleaceae, Bignoniacea.

13 - Compositions according to claim 11, characterized in that they are fractions purified from Trema orientalis Ulmaceae, Strychnos myrtoides Loganiaceae, Obetia radula Urticaceae, Vaccinium myrtillus Ericaceae, Sclerocarya birrea Anacardiaceae, Agrimonia eupatoria Rosaceae, Alchemilla vulgaris Rosaceae and Olea europaea Oleaceae,

14 - Use of products obtainable from plants, such as extracts, fractions and / or molecules as specific agonists of TGR5.

15 - Use according to claim 14, characterized in that said products are plant extracts, or fractions that can be obtained from these extracts or molecules that can be obtained from these extracts or fractions:

having an EC ₅₀ greater than or equal to that of a natural ligand of TGR5, such as lithocholic acid (LCA),

- not activating the FXR nuclear receiver,

- resulting in a reduction in fat-induced weight gain in a mouse model of metabolic syndrome as well as a reduction in the mass of fat pads of the epididymis in the same model, - resulting in an improvement in glucose tolerance .

16 - Use according to claim 14 or 15, characterized in that the plants are selected from the group consisting of Olea europaea, Alchemilla vulgaris, Jugions Regia, Agrimonia eupatoria, Vaccinium myrtillus and Obetia radula.

17 - Use according to any one of claims 14 to 16, characterized in that the molecules comprise a triterpene pentacyclic.

18. The use as claimed in claim 17, characterized in that the molecules comprise a pentacyclic triterpene of oleanane type, such as oleanolic acid.

19. Use according to claim 17, characterized in that the molecules comprise a ursane-type pentacyclic triterpene, such as corosolic acid.

20. Use according to claim 17, characterized in that the molecules comprise a pentacyclic triterpene lupane type. Pharmaceutical compositions, characterized in that they contain an effective amount of at least one composition according to any one of Claims 8 to 13, or an agonist according to any one of Claims 14 to 20 in association with a pharmaceutically acceptable carrier. .

22 - Compositions according to claim 21, characterized in that they are in galenic forms for oral, topical or injection.

23 - Pharmaceutical compositions according to claim 21, characterized in that they are in the form of tablets, tablets, capsules, granules, drops, syrups, alcoholic syrups for oral administration, or in the form of aerosols.

24 - Pharmaceutical compositions according to claim 21, characterized in that they are in dosage forms for topical administration, such as milks, creams, lotions, oils or patches.

25 - Pharmaceutical compositions according to claim 21, characterized in that they are in the form of intravenous, subcutaneous or intramuscular injection solutions, prepared from sterile or sterilizable solutions, or suspensions or emulsions, for administration by injection.

Pharmaceutical compositions, characterized in that they contain an effective amount of at least one composition according to any one of claims 8 to 13, or an agonist according to any one of claims 14 to 20, in combination with a pharmaceutically acceptable vehicle for the treatment of diabetes.

27 - Use of the agonists according to any one of claims 14 to 20, for manufacturing drugs for humans or animals to prevent and / or treat metabolic disorders, obesity and / or diseases associated therewith like syndrome X (metabolic syndrome), type 2 diabetes.

The use according to claim 27, wherein said extracts, or fractions, or corosolic acid are used to prevent and / or treat type 2 diabetes, obesity and / or metabolic syndrome. The use according to claim 27, wherein said extracts, or fractions, or oleanolic acid are used for the prevention and / or treatment of obesity or diabetes, including type 2 diabetes and / or metabolic syndrome

30 - Food supplements intended for humans or animals, characterized in that they contain an effective amount of at least one composition according to any one of claims 8 to 13, or an agonist according to any of claims 14 to 20 for nutraceutical application with an inert usable excipient.

31 - food supplements according to claim 27, characterized in that they are in unmanageable form, gum, powder, tablets, capsules, capsules, infusions, decoctions.

32 - food complements according to claim 31 or 32 characterized in that they are incorporated into the diet during the preparation of food or when they are packaged, or by using preparations already formulated with said products, allowing to perform the desired supplementation.