FR2908309A1 - Obtaining biologically active compositions by extracting plants, comprises separately treating medicinal plants by extracting processes to obtain total crude extract/fractions and selecting extracts/fractions having metabolic activity - Google Patents

Abstract

Obtaining biologically active compositions (I) by extracting plants, comprises separately treating medicinal plants, selected with respect to a given activity, comprising extracting processes using organic solvents, particularly alcoholic solvent, cyclohexane, apolar solvents, dichloromethane or chloroform, to isolate specific fractions to obtain total crude extract or fractions enriched with active ingredients, i.e. (a)polar fractions and selecting extracts or fractions having a metabolic activity in Methyl Thiazol Tetrazoline (MTT) test and a biological activity in a specific test. Obtaining biologically active compositions (I) by extracting plants, comprises separately treating medicinal plants or their parts, selected with respect to a given activity, comprising extracting processes using organic solvents, particularly alcoholic solvent, cyclohexane, apolar solvents, dichloromethane or chloroform and many of these solvents or all these solvents can be used repeatedly for isolating specific fractions to obtain total crude extract or fractions enriched with active ingredients, preferably (a)polar fractions and selecting extracts or fractions having a metabolic activity in Methyl Thiazol Tetrazoline (MTT) test and a biological activity in a specific test of that activity. An independent claim is included for a dietary supplements comprising (I) for a nutraceutical application with an excipient. ACTIVITY : Antidiabetic. MECHANISM OF ACTION : None given. Tests details are described but no results given.

Description

1 Biologically active compositions, process for obtaining them, and

  The invention relates to new biologically active compositions that 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 further relates to the biological applications of these compositions for the treatment 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.

  For thirty years, sedentariness and over-nutrition have caused an explosion in the incidence of obesity in the industrialized countries. Obese people have an increased risk of developing other metabolic diseases, particularly type 2 diabetes. Despite the existence of a large number of therapeutic tools, there is a great medical need for this metabolic disease. There are nearly 200 million people with diabetes worldwide, about 90% of whom have type 2 diabetes. It is estimated that this number will double in 25 years, affecting just over 5% of the world's population. People with diabetes are 2-4 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 - particularly with a suitable 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. Recent studies (Petersen et al, 2004, Petersen et al, 2003) have shown that the decrease in metabolic activity itself, and particularly in the mitochondria, was one of the major and early disturbances observed in the development of metabolic diseases.

2908309 2 Patients suffering from 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. This is the main reason why plant products appear particularly satisfactory for the management of these diseases. 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 of the newly developed or recently marketed new drugs also have side effects and it is therefore urgent to develop more effective effective products. 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 15 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 a manner technically and economically optimized their active ingredients has been described. As a result, 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 relating to food supplements which is the transposition into French law of European Directive 2002/46 / EC.

For the sake of obtaining accurate scientific data and development of original assets, the inventors have developed means for evaluating the effect of a composition on metabolic activity at the mitochondrial level by diverting a biochemical test from its use. usual. Indeed, 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 origin Synthetic or natural (Mosmann, 2908309 1983), could be a tool of great interest to select active compositions from plants. This test is based on the ability of the dehydrogenase enzymes contained in the mitochondria to convert the yellow solution molecule MTT 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 for screening novel molecules for their potential to correct a metabolic defect, using the MTT assay, not only to measure the cytoxicity of extracts and compounds of selected plants but, in an original way, to measure the ability of these extracts and compounds to increase metabolic activity. The object of the invention is therefore to provide a method of high reliability and easy to implement to obtain new compositions that are useful for preventing, accompanying a diet and treating metabolic diseases. It is also intended to provide such compositions as novel products and to use their properties for the manufacture of drugs or food supplements for the treatment and prevention of metabolic diseases. The process for obtaining, according to the invention, biologically active compositions, by extraction from one or more plants, is characterized in that it comprises the steps a) of treatment, separately, of the selected medicinal plants with respect to a given activity, or parts of these plants. with one or more solvents to obtain total extracts or fractions enriched in active principles, more especially apolar fractions and polar fractions b) selection of extracts or fractions having a metabolic activity in the MTT test and a biological activity in at least one specific test for this metabolic activity.

This method advantageously comprises, in addition to the steps a) and b) defined above, a step c), carried out 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 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, barks, roots, aerial parts, fruits, seeds, etc. Step a) generally comprises one or more extractions, these extractions being carried out using 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 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 means for their characterization, allows to reveal extracts and fractions that had never been identified until the invention. Compositions obtainable by the process defined above are also within the scope of the invention as novel products. The invention thus relates to the extracts, fractions and compounds that can be obtained by implementing the process of the invention.

The invention also aims at making use of the biological properties of these compositions. 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 as defined above, in association with a pharmaceutically acceptable vehicle. These compositions 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, etc. For topical administration, they are advantageously in the form of creams, milks, lotions , oils or patches. Suitable galenic formulations include aerosols. For injectable administration, they are in the form of intravenous, subcutaneous or intramuscular injection solutions made from sterile or sterilizable solutions, or suspensions or emulsions. According to a second aspect, the invention relates to food supplements, characterized in that they contain an effective amount of at least one composition as defined above for a nutraceutical application, in combination with an inert excipient. . These supplements are advantageously in liquid form, gum, powder, tablets, capsules, decoctions, infusions and others. The invention is more particularly 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 US Pat. step b) are tests for determining an antidiabetic activity, in particular on the secretion of insulin.

Preferred compositions in this regard are capable of being obtained from plants selected from the genera and species: Strychnos myrtoides, Obetia radula, Trema orientalis, Sclerocarya birrea, Voacanga thouarsii Moringa oleifera (= Moringa moringa, Moringa pterygosperma, Guilandina moringa), Agrimonia eupatoria, Arctium majus = 5 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 characterized in that they are capable of being obtained from plants chosen from the genera: Strychnos, Obetia, Trema, Sclerocarya, Voacanga, Moringa, Agrimonia, Arctium, Alchemilla, Phaseolus, Vaccinium, Cichorium, Cynara, Geranium, Juglans, Kalanchoe, Leptodenia, Morus, Olea, Rubus, Spathodea. Particularly preferred compositions are obtainable from plants selected from the families Loganiaceae, Urticaceae, Ulmaceae, Anacardiaceae, Apocynaceae, Moringaceae, Rosaceae, Asteraceae, Fabaceae, Ericaceae, Geraniacaea, Juglandacaea, Crassulaceae, Oleaceae, Bignoniacea. Other particularly preferred compositions are fractions of 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, showing, respectively, the HPLC profiles of Figures 1-8, as well as the biologically active compounds from these fractions. As the examples show, these preferred compositions are of great interest for the treatment of diabetes. The above preferred compositions are also of great interest in nutraceuticals and are advantageously used for the preparation of food supplements. These supplements allow in particular to accompany a diet of a patient under treatment or not.

In yet another aspect, the invention is directed to a method for preventing and / or treating metabolic diseases, characterized in that it comprises the administration of a therapeutically or nutraceutically effective amount of at least one composition as defined above.

In general, the invention therefore proposes an alternative for the prevention and treatment of these metabolic diseases because these products of natural origin are at least psychologically associated with a better quality of life and, secondly, their use often. age-old in the traditional pharmacopoeia provides a comprehensive knowledge of 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. In these examples, reference is made to FIGS. 1 to 9, which represent the HPLC profiles of plant extracts according to the invention (FIGS. 1 to 8) and give an MTT test result (FIG. 9).

Material and Method Plants Parts of dried plants (leaves, roots, etc.) are milled to obtain a fine powder for extraction. Extractionpurification 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 with solvents of increasing polarity. The pure compounds can be obtained in particular by fractionation by adsorption chromatography, exclusion chromatography, affinity chromatography, ion exchange chromatography, or by fractional crystallization. 2908309 Chromatographic profiles Extracts, enriched fractions, fractions more or less purified or the 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 C18, with a particle size of 5 to 10. 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 minutes. MTT Test The INS1 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 3 103 / 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 g / 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 aspirated with care so as not to suck the formed formazan crystals, which will be solubilized in 100 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 pig pancreas are obtained from the European Center for Diabetes (Hautepierre, France). 100 cells / then 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: Study of the effect of selected plants on the metabolic activity of cells INS1 cells were treated for 24 hours with 200 g / ml of plant extract obtained by extraction with ethanol, methanol or dichoromethane. The conversion rate of the MTT 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; McOH = methanol fraction; DCM = dichloromethane fraction.

2908309 Table 1 Genus / Species / Part Percent Control (%) EtOH DCM MeOH Strychnos myrtoides / leaves 128 107 121 Obetia radula / bark, stem 112 127 116 Trema orientalis / roots 319 103 355 Voacanga thouarsii / bark 120 91 111 Arctium majus / leaves 63 80 ND Arctium majus / roots 156 77 162 Cynara scolymus / leaves 72 75 123 Geranium robertianum / aerial 172 174 164 Kalanchoe crenata / aerial 107 ND Rubus fructicosus / leaves 130 151 220 Spathodea campanulata / bark 114 ND ND Sclerocarya birrea / leaves 284 119 263 Alchemilla vulgaris / aerial part 189 117 201 Cichorium intybus / leaves 122 139 129 Cichorium intybus / roots 105 83 98 Leptodenia madagascariensis / leaves 109 132 125 Moringa ofeifera / leaves 104 118 102 Agrimonia eupatoria / aerial part 205 121 221 Phaseolus vulgaris / pods 103 104 86 Vaccinium myrtillus / leaves 163 172 147 Juglans regia / leaves 152 110 223 Morus nigra / leaves 104 11 7 110 Olea europaea / feuilles [130 135 133 Examination of these results shows that all the selected plants have an effect on the metabolic activity of the cells.

Example 2: 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 Trema 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, 1NS 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 Trema orientalis is shown 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 semipréparative (details of the fractions presented in FIG. 1). The conversion rate is presented after measuring the optical density at 560 nm.

EXAMPLE 3 Effect of Selected Plant Extracts on Insulin Secretion There are four distinct classes of antidiabetic therapies for treating type 2 diabetes. Among them, sulfonylureas, the leading drug in the United States for the treatment of diabetes of type 2. Sulfonylureas promote the secretion of insulin by the pancreas and thereby prevent hyperglycemia. Since the plants are active on cellular metabolism, in order 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 islands. of porcine Langherans. 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.

11 12 2908309 Table 2 Genus / Species / Part Percent Control (%) EtOH MeOH DCM Strychnos myrtoides / leaves 641 604 364 Obetia radula / bark, stem 493 392 134 Trema orientalis / roots 235 408 201 Voacanga thouarsii / bark 352 256 384 Arctium majus / leaves 118 ND 136 Arctium majus / roots 157 140 119 Cynara scolymus / leaves 101 105 252 Geranium robertianum / aerial part 83 138 226 Kalanchoe crenata / aerial part ND ND Rubus fructicosus / leaves 86 98 168 Spathodea campanulata / bark ND ND ND Sclerocarya birrea / leaves 81 108 118 Alchemilla vulgaris / aerial part 110 162 139 Cichorium intybus / leaves 100 105 171 Cichorium intybus / roots 100 123 114 Leptodenia madagascariensis / leaves 197 161 271 Moringa ofeifera / leaves 216 152 336 Agrimonia eupatoria / aerial part 129 137 178 Phaseolus vulgaris / pods 117 134 115 Vaccinium myrtillus / leaves 171 134 122 Juglans regia / leaves 100 180 76 Morus nigra / leaf s 114 189 380 Olea europaea / leaves 109 139 230 2908309 References Impaired Mitochondrial Activity in the Insulin-Resistant Offspring of Patients with Type 2 Diabetes. KF Petersen, S Dufour, D Befroy, R Garcia, and GI Shulman. N Engl J Med, 350: 664-671 (2004) Mitochondrial Dysfunction in the Elderly: Possible Role in Insulin Resistance. KF Petersen, D Befroy, S Dufour, J Dziura, C A.riyan, DL Rothman, L DiPietro, GW Cline and GI Shulman. Science, 300: 1140-1142 (2003). Rapid colorimetric assay for cell growth and survival: application to proliferation and cytotoxicity assays. Mosmann, J. Immunol Methods, 65 (1-2): 55-63 (1983). 13 10 14

Claims (4)

  1 - Process for obtaining biologically active compositions, by extraction from one or more plants, characterized in that it comprises the steps a) of treatment, separately, selected medicinal plants with respect to a given activity, or parts of these plants, comprising one or more extractions, these extractions being carried out using 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 to obtain crude total extracts or fractions enriched in active principles, especially apolar fractions and polar fractions b) selection of extracts or fractions having an activity in the MTT assay and a biological activity in at least one specific test of this quickly.   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. - Process according to any one of claims 2 to 4, characterized in that the analysis of a fraction according to step c) is carried out by HPLC profiles. 6 - Process according to any one of claims 1 to 5, 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. 7 - Biologically active compositions, characterized in that they are extracts, fractions obtainable by the process according to any one of Claims 1 to 6. 8 - Compositions according to Claim 7, characterized in that what are compounds that can be isolated from said extracts or fractions. 9 - Compositions according to claim 7 or 8, characterized in that they are likely to be obtained from plants selected from genera and species: Strychnos myrtoides, Obetia radula, Trema orientalis, Sclerocarya birrea, Voacanga thouars and 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. -Compositions according to claim 9, characterized in that they are capable of being obtained from plants selected from the genera: Strychnos, Obetia, Trema, Sclerocarya, Voacanga, Moringa, Agrimonia, Arctium, Alchemilla, Phaseolus, Vaccinium, Cichorium, Cynara, Geranium, Juglans, Kalanchoe, Leptodenia, Morus, Olea, Rubus, Spathodea. 11 - Compositions according to claim 9, 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. 12 - Compositions according to claim 10, characterized in that they are fractions purified from Trema orientalis Ulmaceae, Strychnos myrtoides Loganiaceae, Obetia radula Urticaceae, Vaccinium myrtillus Ericaceae, Sclerocarya 2908309 16 birrea Anacardiaceae, Agrimonia eupatoria Rosaceae, Ai hemilla vulgaris Rosaceae and Olea europaea Oleaceae, 13 - Pharmaceutical compositions, characterized in that they contain an effective amount of at least one composition according to any one of claims 7 to 12, in association with a pharmaceutically acceptable carrier. 14 - Compositions according to claim 13, characterized in that they are in galenic forms for oral, topical or injection. - Pharmaceutical compositions according to claim 14, characterized in that they are in the form of tablets, tablets, capsules, granules, drops, for oral administration. 16 - Pharmaceutical compositions according to claim 14, characterized in that they are in dosage forms for topical administration, such as milks, creams, lotions or oils. 17 - Pharmaceutical compositions according to claim 14, characterized in that they are in the form of injectable solutions by intravenous, subcutaneous or intramuscular, developed from sterile or sterilizable solutions, or suspensions or emulsions, for administration by injection. 18 - Pharmaceutical compositions, characterized in that they contain an effective amount of at least one composition according to any one of claims 7 to 12, in association with a pharmaceutically acceptable vehicle for the treatment of diabetes. 19 - Food supplements, characterized in that they contain an effective amount of at least one composition according to any one of claims 7 to 12, for a nutraceutical application with an inert usable excipient. - Food supplements according to claim 19, characterized in that they are in liquid form, gum, powder, tablets.