ES2241491B1 - COSMETIC AND / OR PHARMACEUTICAL COMPOSITION, REGULATOR OF FAT LEVELS IN ADIPOCYTES AND / OR REGULATOR OF ADIPOCITARY DIFFERENTIATION. - Google Patents

Abstract

Cosmetic and / or pharmaceutical composition, regulator of fat levels in adipocytes and / or regulator of adipocyte differentiation, comprising at least one dihydroflavonol compound of formula I, in free form and / or one of its salts and / or derivatives , ** FIGURE ** where R1 can be H, C1-nalkyl, COC1-nalkyl, (CH2) nCH (NH2) COOH; R2 can be H, C1-nalkyl, COC1-nalkyl, (CH2) nCH (NH2) COOH; R3 can be H, C1-nalkyl, COC1-nalkyl, (CH2) nCH (NH2) COOH; R4 can be H, C1-nalkyl, COC1-nalkyl, (CH2) nCH (NH2) COOH, a sugar such as glucose, rhamnose, rutinose, galactose, fructose, or other acceptable sugars; R5 can be H, C1-nalkyl; COC1-nalkyl, (CH2) nCH (NH2) COOH; and R6 can be H, C1-nalkyl, COC1-nalkyl, (CH2) nCH (NH2) COOH, a sugar such as glucose, rhamnose, rutinose, galactose, fructose, or other acceptable sugars. Use of a dihydroflavonol of formula I, its salts and / or derivatives, for the preparation of a cosmetic and / or pharmaceutical composition for the treatment of cellulite, the reduction of fat levels in adipocytes and adipocyte differentiation.

Description

Cosmetic and / or pharmaceutical composition, regulator of fat levels in adipocytes and / or regulator of adipocyte differentiation.

Technical sector of the invention

The present invention relates to a cosmetic and / or pharmaceutical composition, regulating the levels of fat in adipocytes and / or regulator of differentiation adipocytic. The invention also relates to the use of composition.

Background of the invention

Adipose tissue is a special type of tissue conjunctiva where there is a great predominance of adipose cells that they store fat. Fat cells are isolated or in small groups in the connective tissue, although most of them are grouped in adipose tissue distributed throughout the body.

We understand by fat that set of substances that are insoluble in water and soluble in solvents organic. In the body and as energy substrates, we find as fats, free fatty acids ("Free fatty acids" FFA) and triglycerides (TG), which are the storage form of FFA.

Regulation of fatty acid levels free and triglycerides is made by balancing the two metabolic pathways called lipogenesis, or lipid generation, and lipolysis, or lipid breakdown.

Lipogenesis is carried out mainly in the liver and adipose tissue and consists of the synthesis of triglycerides from lipid sources, such as fatty acids coming from dietary lipids that are taken to the tissues by chylomicrons and very low lipoproteins density (VLDL); or from non-lipid sources, such as dietary carbohydrates and amino acids.

Triglycerides from the diet are hydrolyzed to FFA by lipoprotein lipase (LPL) which is found in the wall of the capillaries of adipose tissue. These FFA are taken up by adipocytes through transport processes active mediated by specific acid transporter proteins fatty. Once inside the cell, fatty acids are reesterified to form triglycerides. In this process, they play an important role insulin levels because they activate the lipoprotein lipase.

Lipolysis consists of the gradual degradation of TG triglycerides into their components, that is, glycerol and fatty acids to CO 2 and H 2 O. Once lipolysis has been carried out, the resulting products will follow different routes for later use. The main agent that favors the degradation of triglycerides is the demand for energy. The triglycerides of adipose tissue are degraded, but also those of muscle tissue. If the diet is low in caloric content, that is, in carbohydrates and lipids, the degradation of these triglycerides is favored. The mechanisms involved, in the short term, are substrate availability, allosteric modification, and covalent modification of the enzymes involved. Glucagon and adrenaline levels, the latter via the β-adrenergic receptors, have a great influence by exerting a favoring effect on the activity of hormone-sensitive lipase
(HSL).

HSL enzyme in conjunction with entry control from fatty acids to the mitochondria, are the main responsible for the control of lipolysis. Lipase sensitive to hormone presents a covalent modification mechanism, being the phosphorylated form the active form. The enzyme is phosphorylated when increases the intracellular amount of cyclic AMP (cAMP).

Fat is stored mainly in the form of Triglyceride (TG) in adipose tissue (subcutaneous and visceral deep) and in small amounts of other tissues. The body has a large capacity to store fat, therefore the percentage of fat that our body contains is relatively high, being able to range between mean values 5 to 50%.

Adipose tissue is very dynamic and has many variations. The variations are mainly due to the balance or imbalance between caloric intake and expenditure, thus, states nutritional deficiencies and excessive physical exercise will produce a decrease in the size of adipocytes and the amount of fat will decrease. Excess caloric intake will induce a enlargement of adipocytes, because they absorb FFA and stored as TG.

Cellulite is a tissue disorder subcutaneous cell that is accompanied by changes in microcirculation of the connective tissue, giving rise to morphological modifications, histochemical and biochemical tissue. Cellulite and obesity are two different problems that may or may not be associated, but that generally converge. In women, cellulite usually be located on the thighs and buttocks, on top of the arms and lower abdomen. In men it is much less frequent, but if it appears it is located in the nape, the lower area of the abdomen and upper arms. There is no origin recognized of cellulite but a set of factors that predispose.

The term cellulite was used for the first time in 1920 to describe an aesthetic alteration of the skin surface. Since then, other names such as nodular liposclerosis, edemato-fibrosclerous panniculopathy, panniculosis, lipodystrophy, among others, have been suggested. Etymologically cellulite is defined as a local disorder of the subcutaneous tissue metabolism that causes an alteration of the woman's shape. In this process, the hyperpolymerization of the connective tissue, the primary alteration of the fatty tissues and the microcirculation converge.

Predisposing (genetic) factors, hormonal factors and existing conditions (physical activity, food intake, ...) act on four functional units of the fatty tissue, described below.

The matrix-interstitial unit is made up of cells, especially fibroblasts, which are responsible for the synthesis of matrix macromolecules extracellular. These include fibrous tissue (collagen, fibers elastic and reticular) and the ground substance (proteoglycans, glycoproteins and hyaluronic acid). While the fibrous tissue is responsible for resistance and support, the fundamental substance allows the diffusion of nutrients, metabolites and hormones from the circulatory system through tissue interstitial. Glycosaminoglycans have properties hydrophilic and help maintain osmotic pore pressure. Proteoglycans play a role in collagen production by fibroblasts, as well as in their three-dimensional distribution.

The microcirculatory unit includes five components: arterioles, venules, capillaries, vessels lymphatics and interstitial tissue. There is usually a balance between the arterial capillary filter and the capillary venous absorption. A alteration of this balance may be due to an increase in the capillary pressure, a decrease in plasma osmotic pressure, an increase in interstitial fluid pressure or a decrease of lymphatic flow that is capable of leading to intercellular edema. Factors capable of influencing microcirculation can be endogenous (central nervous system, adrenergic nervous system sympathetic, humoral factors) and exogenous (drugs, clothing adjusted, etc.).

The neurovegetative unit is formed by the Sympathetic innervation of the dermis and subcutaneous tissue. This acts on α and β receptors to induce response through the adenyl cyclase system. He is able to modify the cAMP / cGMP ratio that regulates fibroblasts, microcirculation and the differentiation of adipocytes. The common cosmetic and dermopharmaceutical treatments for reshape and smooth the figure, are based on the receptor pathway adrenergic drugs and activate fat metabolism, on the one hand through an increase in cAMP, and consequently an increase in lipase levels that will promote hydrolysis of the triglycerides; and on the other, by inhibiting the phosphodiesterase, which will lead to an increase in the processes lipolytics.

The fat energy unit is made up of collections of adipocytes, nourished by arterioles and surrounded by septa of connective tissue. Each adipocyte associates with a layer of glycoprotein, reticular fibrils, and other cells (fibroblasts, mast cells, and macrophages), as well as adjacent capillaries. Fat tissue is divided into two layers separated by a superficial connective tissue layer. The outermost layer (in contact with the dermis) is called the areolar stratum and is made up of large, globular adipocytes located vertically: the blood vessels are very numerous and fragile. The deeper layer, called the lamellar layer , the cells are spindle-shaped, small and horizontally distributed; the vessels are more extensive. This second layer, which increases in thickness when people gain weight, is mainly due to the increase in volume of adipocytes capable of invading the superficial connective tissue layer. The relative size of the two layers varies according to the thickness of the skin, according to the body region, sex and age.

Hormonal factors may play a role in predisposition or aggravation in a cellulite process, being especially important during adolescence. Estrogens are the most important hormones that can initiate, aggravate and perpetuate a cellulite process.

Estrogens act via the cyclic nucleotides, stimulating the proliferation of fibroblasts to produce collagenase and influencing the turnover of macromolecules. Estrogens increase response of adipocytes against antilipolytic α receptors and thus stimulate lipoprotein lipase, the main enzyme responsible for lipogenesis, which causes adipocyte hypertrophy leading to the formation of nodules. The joint effect of the action of the collagenase and activation of lipogenesis in tissues fatty, causes the structures to weaken and the appearance of typical raised areas of fat deposit, or otherwise called cellulite.

Taking advantage of the knowledge that phytoestrogens (flavones and isoflavones) can interact with estrogen receptors, in patent application US 2002/0106388 A1, a composition with flavones and isoflavones for the cellulite treatment. Preferably hydroxyflavone used is the compound quercetin and fisetin, and isoflavone genistein is preferred. With a composition like the one described, claims that phytoestrogens block the occupation of estrogen receptors, which, in turn, blocks the action thereof.

Flavones and isoflavones belong to the group of chemical compounds called flavonoids, These compounds comprise a large group of secondary plant metabolites, responsible in several cases for their coloring and also used as free radical scavengers, UV light, or as antioxidants. A subgroup within the flavonoids, are the dihydroflavonols of general structure X:

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One of these dihydroflavonols is dihydromyricetin and corresponds to the general formula Y:

3

Dihydromyricetin is used in compositions for the treatment of obesity due to its effects on the digestive system. An example of this is patent CN 1393443, where a dihydromyricetin extraction procedure is described and where it is detailed that it is useful for the treatment of obesity among other pathologies. In US 4321262, a composition with dihydromyricetin with antiarthritic character is described. Said compound also appears in the literature as an antioxidant for cosmetic products. Thus, patent JP 63208506 describes a composition with dihydromyricetin antioxidant and absorber of UV radiation. In JP 2002065201 dihydromyricetin is used as an antioxidant in food products. In all these compositions and uses, dihydromyricetin can have a synthetic origin or come from extracts of plant species, the most commonly used species Myrica cerifera and Ampelopsis grossedentata .

In the present invention, a new composition and use of dihydromyricetin and its derivatives, not deductible from the state of the art and with surprising effects.

Explanation of the invention

The cosmetic and / or pharmaceutical composition, regulator of fat levels in adipocytes and / or regulates of adipocyte differentiation according to the invention, it is characterized because it comprises at least one dihydroflavonol compound of formula I and / or one of its salts and / or derivatives,

4

where R1 can be H, C 1-n alkyl, COC 1-n alkyl, (CH2) n CH (NH2) COOH;

R2 can be H, C 1-n alkyl, COC 1-n alkyl, (CH2) n CH (NH2) COOH;

R3 can be H, C 1 -n alkyl, COC 1-n alkyl, (CH2) n CH (NH2) COOH;

R4 can be H, C 1-n alkyl, COC 1-n alkyl, (CH2) n CH (NH2) COOH, a sugar such as glucose, rhamnose, rutinose, galactose, fructose, u other acceptable sugars;

R5 can be H, C 1-n alkyl, COC 1-n alkyl, (CH2) n CH (NH2) COOH; and

R6 can be H, C 1-n alkyl, COC 1-n alkyl, (CH2) n CH (NH2) COOH, a sugar such as glucose, rhamnose, rutinose, galactose, fructose, u other acceptable sugars.

According to another characteristic of the invention, proportion by weight of dihydroflavonol of formula I, its salts and / or derivatives is between 0.01% and 10%.

According to another characteristic of the invention, composition is characterized in that the dihydroflavonol of formula I is dihydromyricetin in free form.

Another object of the invention is a cosmetic and / or pharmaceutical composition in which dihydroflavonol of formula I is dihydromyricetin in salt form.

The composition is also characterized because the Dihydroflavonol of formula I is dihydromyricetin in the form of glycoside.

The cosmetic and / or pharmaceutical composition is also characterized in that it also comprises one or more compounds selected from caffeine, xanthine, theophylline and / or theobromine.

According to another characteristic of the invention, composition is in the form of gel, cream, ointment, soap, lotion, microcapsules or in solid form applicable to tissues.

It is also characteristic of the composition according to the invention that is adapted for oral administration.

The invention also relates to the use of al less one dihydroflavonol compound of formula I, its salts and / or derivatives, for the preparation of a cosmetic composition and / or pharmaceutical, for the regulation of fat levels in adipocytes.

Also characteristic of the invention is the use dihydromyricetin in free form and / or in the form of salts and / or derivatives for the preparation of a cosmetic composition and / or pharmaceutical, for the regulation of fat levels in adipocytes.

According to another characteristic of the invention, reduction of fat levels in adipocytes consists of inhibition of insulin-induced lipogenesis.

According to another characteristic of the invention, reduction of fat levels in adipocytes consists of inhibition of adipogenesis.

According to another characteristic of the invention, reduction of fat levels in adipocytes consists of activation of lipolysis in these cells.

Also characteristic of the invention, the use of at least one dihydroflavonol compound of formula I, its salts and / or derivatives, for the preparation of a cosmetic composition and / or Pharmaceutical for the regulation of differentiation adipocytic.

The use of at least one dihydroflavonol compound of formula I, for the preparation of a cosmetic composition and / or pharmaceutical for the regulation of adipocyte differentiation, is also characterized in that the dihydroflavonol of formula I is dihydromyricetin in free form and / or in the form of its salts and / or derivatives.

The use of at least one dihydroflavonol compound of formula I, is also characterized in that said compound is used for the preparation of a cosmetic and / or pharmaceutical composition for the treatment of cellulite.

Brief description of the drawings

With the aim of exemplifying the effects of dihydroflavonol compound of formula I and its use for the manufacture of anti-cellulite compositions, regulators of differentiation of adipocytes and / or fat levels in adipocytes, Figures 1 to 6 are attached, which present the results of different laboratory tests.

Fig. 1 is a graph that represents the 3T3-L1 cell viability at short exposure with different concentrations of free dihydromyricetin;

Fig. 2 is a diagram of the viability of the 3T3-L1 cells at long exposure with different free dihydromyricetin concentrations;

Fig. 3 is a graph that relates the nanomoles of glycerol released from cells stimulated said effect by dihydromyricetin;

Fig. 4 is a graph detailing the effect of dihydromyricetin on the lipogenic pathway in cells 3T3-L1;

Fig. 5 corresponds to the results of the analysis to determine the effect of dihydromyricetin on the pathway insulin signaling;

Fig. 6 corresponds to photographs of phase contrast microscope of cultures of 3T3-L1, treated with dihydromyricetin (B) or without treat (A); and

Fig. 7 corresponds to the results of "western blots" carried out to determine the presence of proteins characteristic of cell differentiation adipocytes.

Detailed description of the invention

In said invention, the applicant has discovered that surprisingly dihydroflavonol compounds of formula I exert important effects on fatty tissues, especially in adipocytes, inhibiting the accumulation of fat in the same, thus reducing the known unsightly effect like cellulite. The inventors have discovered that mainly the effects of these dihydroflavonols are derived from their action on the insulin signaling pathway in adipocytes.

In the present invention, fat is understood any accumulation of triglycerides and / or lipid derivatives in the structures of the cells destined to this function, mainly in the structures of adipocytes known as drops of fat.

The subject cosmetic and / or pharmaceutical composition of the invention, comprises as dihydroflavonol of formula I preferred, dihydromyricetin. In this composition, the Dihydromyricetin is used in the free form, or one of its derivatives such as salts or glycosides. Dihydromyricetin in the form free is represented by formula I when the substituents R1, R2, R3, R4, R5, and R6 correspond to a hydrogen atom (H). Dihydromyricetin glycosides are understood as those compounds of formula I, in which R6 is a sugar and R1 to R5 correspond likewise to a hydrogen atom (H).

Below, and as an example no limiting, different types of compositions are detailed according to the invention. In all of them the proportions of the compounds components of the formulations are expressed as a percentage in weight.

Example 1 Bath gel

Water 24.7% Lauryl ether sulfate sodium 28% 55.0 Preservative 0.05 PEG-4 Rapessedamide 0.3 Cocamido propyl betaine 30% 16.0 PEG-7 Glycerylcocoate 1.0 Dihydromyricetin ( Myrica cerifera Extract) 2.0 Fragrance 0.5 Triethanolamine 99% 0.06 Cl 16035 0.04 Cl 19140 0.2 Chloride sodium 0.15

Example 2 Liquid soap

Water 55.87% Chloride Hydroxypropyl Guar Hydroxypropyltrimonium 0.2 Acid citric 0.03 TEA Lauryl sulfate 40% 30.0 Preservative 0.5 Cocamidopropyl Betaine 30% 8.0 Dimethicone Copolyol 0.5 PEG-7 Glyceryl cocoato 0.5 Decile Polyglucose fifty% 2.0 Fragrance 0.4 Dihydromyricetin ( Amelopsis grossedentata Extract) 2.0

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Example 3 Sun cream SPF 20

PEG-100 Stearate 50%; Glyceryl Stearate fifty% 6.0% Petrolatum 5.0 Cetyl Alcohol 2.5 Paraffin liquid 9.0 Octyl Methoxycinnamate 9.0 Benzophenone-3 3.5 Triticum vulgar 1.5 B HT 0.1 Water 55.4 Propylene glycol 2.0 Preservative 0.7 Fragrance 0.3 Dihydromyricetin 2.0 Dioxide titanium 3.0

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Example 4 Medium protection sun milk

Glyceryl Stearate; Ceteth-20 3.0% Stearil alcohol; Steareth 7; Steareth 10 2.5 Cetyl Alcohol 1.0 Caprylic / Capric Triglyceride 4.0 B HT 0.1 Triticum vulgar 3.0 Benzophenone-3 1.0 Paraffin liquid 7.0 Octyl Methoxycinnamate 4.0 Water 72.35 Fragrance 0.35 Preservative 0.7 Dihydromyricetin 1.0

Example 5 Moisturizing-anti-cellulite cream

Steareth-2 2.4% Steareth-21 1.6 PEG-15 Stearyl Ether 4.0 Paraffin liquid 3.0 Preservative 0.6 Cetyl alcohol 3.0 Benzophenone-3 0.5 Triticum vulgar 1.0 B HT 0.1 Wax Sunrise 1.0 Propylene glycol 2.5 Water 75.9 Dimethicone Copolyol 2.0 Fragrance 0.4 Dihydromyricetin (dihydromyricetin glucoside) 2.0

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Example 6 Gel-cream

Glycerin 2.5 Water 77.3 Acrylates C / 10-30 Alkyl-Acrylate polymer crisscrossed 0.4 Carbomer 0.35 Preservative 0.5 B HT 0.1 Octyl cocoate 6.0 Dimethicone copolyol 1.0 Caprylic / Capric Triglyceride 3.0 Triticum vulgar 3.0 Fragrance 0.2 Triethanolamine (99%) 0.65 Dihydromyricetin 5.0

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Example 7 Atomizing water

Water 91.4% Acrylates C / 10-30 Alkyl-Acrylate polymer crisscrossed 0.05 Preservative 0.05 Quarternium-15 0.05 Fragrance 0.2 PEG-40 Beaver oil hydrogenated 1.1 Water 1.0 Dihydromyricetin 5.0 Dimethicone copolyol 0.4 Triethanolamine (99%) 0.75

Example 8 Firming-anti-cellulite gel

Carbomer 0.5 Water c.s. 100.0 Triethanolamine 99% c.s. pH = 5.5 Dimethicone Copolyol 1.5 Glycerin 2.0 Alcohol Denat. 20.0 Centella extract Asian 4.0 Dihydromyricetin 5.0 Fragrance c.s. Preservative c.s.

Example 9 Anti-cellulite-activating gel circulation

Hydroxyethylcellulose 1.8 Alcohol ethyl 3.0 Water c.s. 100.0 Alcohol ethyl 10.0 Glycerin 2.5 Dimethicone copolyol 2.5 Rough Extract H.G. 3.0 Dihydromyricetin 5.0 Fragrance c.s. Preservative c.s.

Example 10 Anti-cellulite gel

Carbomer 0.5 Water c.s. 100.0 Triethanolamine 99% c.s. pH = 5-5.5 Dimethicone copolyol 1.5 Glycerin 2.0 Alcohol denat. 20.0 Dihydromyricetin 5.0 Gel Aloe Vera 2.0 Fragrance c.s. Preservative c.s.

In all examples, the amount of Free dihydromyricetin and / or in salt form and / or in the form of glucoside, is comprised between 0.01% and 10% by weight.

It can be appreciated that preferably uses dihydromyricetin in free form as responsible anti-cellulite or as a reducer of fat levels in adipocytes.

These compositions are expected to add one or more compounds selected from xanthine, caffeine, theophylline, theobromine, or others known as activators of lipolysis and microcirculation in adipose tissue, due to its inhibitory effect of phosphodiesterase on adipocytes. They can also plant extracts containing these compounds as coffee extract, tea extract, cocoa extract, mate or guarana, etc. Along the same lines, compounds derived from saponins, which stimulate the blood circulation in adipose tissue. Examples of saponins They are ruscogenin, aescin, hederin, etc. They are also used flavonoid compounds whose function is to improve the capillary permeability and thus reduce edema.

The examples described may also contain any of the active ingredients commonly used in cosmetics for reaffirm the tissues, such as free radicals, moisturizing compounds, etc.

Obviously, for the formulation of the compositions described in the examples, the excipients and compounds known to those skilled in the art.

The composition that is the subject of the invention can be applied on a textile surface, such as such as stockings, T-shirts or socks, allowing the absorption of the dihydromyricetin compound while the user is wearing the aforementioned textile garment. In this case, the active substance is generally It is encapsulated in microcapsules adapted to be adsorbed on the fibers of the textile material, and using the compounds, excipients and preservatives commonly used in this type of formulations.

Similarly, a composition like the one described in the invention it may be in the form of a tablet or capsule for its oral administration as a dietary supplement. In this case, as excipients and preservatives are also used the usual ones for said type of formulations and that are widely known by the skilled.

Dihydroflavonol of formula I is used to the preparation of cosmetic and / or pharmaceutical compositions for the cellulite treatment, derived from this effect, from the ability of the same to regulate the levels of fat in adipocytes and to regulate the differentiation of adipocyte cells into adipocytes mature, which in turn, contribute to the formation of characteristic roughness of cellulite.

Specifically, it has been shown that dihydroflavonols of formula I, and in particular dihydromyricetin, act on the cells that make up the fat storage tissues at least three different levels. In the first place, it has demonstrated that the anti-cellulite effect and the reduction of Fat levels in adipocytes are caused by the action of dihydromyricetin and / or one of its derivatives in the route of lipolysis, acting as a powerful activator of it. I also know decreases cellulite and fat levels in adipocytes by the inhibitory effect of adipogenesis or differentiation of potential cells in adipocytes. Finally, dihydromyricetin it is also used in cosmetic and / or pharmaceutical compositions anti-cellulite due to its inhibitory effect on induced lipogenesis by insulin insulin.

The tests carried out are detailed below carried out to study the action of dihydromyricetin and that, surprisingly, they demonstrated the new uses of dihydroflavonols of formula I.

Test No. 1

Determination of the range of concentrations in which the Dihydromyricetin is safe in acute studies in adipocytes rat isolated

This test evaluates the range of concentrations in which the dihydromyricetin of formula I has no effects toxic in fat cells. For this, adipocytes are used rat isolates (200,000 adipocytes in 1 ml of incubation) that are incubated acutely (120 minutes) in presence of free dihydromyricetin 1 mM, 500 µM, 250 µ, 100 µM, 50 µM, 25 µM and 10 µM. For the last 60 min, cells are incubated in the presence of the MTT marker (3, [4,5-dimethylthiazol-2-yl] 2,5, -diphenyltetrazolium-bromide) (100 µl of a 5 mg / ml solution are added). MTT is a salt soluble which gives rise to a yellow coloration; however in the cell interior, MTT becomes an insoluble derivative of Purple color. The intensity of coloring of the purple derivative soluble in DMSO (dimethylsulfoxide) gives us an estimate of the number of viable cells present in the assay. At the end of the incubation, the medium is aspirated and the adipocytes are lysed in DMSO. After centrifuging the lysate at 16,000 g for 15 min, the absorbance at 570 nm and at 630 nm, the difference between the two being readings proportional to the amount of MTT transformed. How I know shown in Table 1 and Figure 1, incubation with Free dihydromyricetin does not reduce the amount of transformed MTT. These results therefore show that in the range of concentrations studied (from 10 µM to 1 mM) and in an incubation 2 hours (acute study) free dihydroxymericitin does not reduce cell viability.

TABLE 1 Acute effect of dihydromyricetin on cell viability in rat adipocytes

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Test No. 2

Determination of the range of concentrations in which the Dihydromyricetin is safe in longer-term studies in isolated rat adipocytes

In these assays, adipocytes are incubated 3T3-L1 (7 days after completing the differentiation) with different concentrations of dihydromyricetin free (2 mM, 1 mM, 500 µM, 250 µM, 100 µM, 50 µM, 25 µM and 10 µM) for 24 hours. After that time, the cells are incubated in the presence of MTT (0.5 mg / ml in DMEM colorless-10% FBS) for 60 min. At the end of the incubation with MTT, cells are solubilized in isopropanol acidic (0.1N CIH in absolute isopropanol) and the absorbance at 570 nm and at 630 nm. After centrifuging the lysate at 16,000 g 15 min the absorbance is measured at 570 nm and 630 nm, the difference being between the two readings proportional to the amount of MTT turned. As shown in Figure 2, incubation with 2 mM dihydromyricetin significantly reduces (p ≤ 0.05) the amount of MTT transformed, the reduction of the MTT transformed from 500 µM.

Test No. 3

Study of the lipolytic effect of dihydromyricetin in isolated rat adipocytes

A compound with anti-cellulite activity can be if it increases the rate of degradation of triglycerides in fat cells. In this sense, this essay assesses whether the active principle in question exerts a rapid action (60 minutes of incubation) on the degradation of triglycerides in adipocytes rat. This test is based on the fact that stimulation of lipolysis leads to an increase in cellular release of glycerol, which is determined spectrophotometrically. In this Assay isolated rat adipocytes are incubated at 37 ° C (200,000 adipocytes in 1 ml of incubation buffer) in the absence or in presence of four concentrations of free dihydromyricetin (500 µM, 250 µM, 100 µM and 50 µM) and in parallel in presence of 100 nM epinephrine (lipolysis stimulator and used as a positive assay control). After 60 minutes of incubation, the incubation media are obtained and after deproteinize them, the glycerol concentration is determined. Of according to Table 2 and Figure 3 the free dihydromyricetin induces lipolysis, this effect being remarkable (more than 6 times stimulation) at 100 µM.

TABLE 2 Lipolysis in rat adipocytes

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Test No. 4

Effect of dihydromyricetin on lipogenesis

A compound with anti-cellulite activity can be so if the rate of fatty acid synthesis decreases and, definitive, triglycerides in adipose cells. In this sense, this test assesses whether the active ingredient in question exerts a rapid action on the synthesis of fatty acids from glucose in rat adipocytes. These tests are carried out in absence or presence of four different concentrations of free dihydromyricetin and in the presence of 1 nM insulin.

Insulin is a polypeptide secreted by the cells of the pancreas, whose mission is to reduce glucose levels in blood, causing a set of biochemical reactions, which result in the introduction of glucose molecules into the interior of cells. The action of insulin starts as consequence of the binding between insulin with its receptor located on the cell membrane. This receptor is a protein with tyrosine kinase activity. Activation of the receptor insulin in turn stimulates other proteins. One of them is the IRS-1 protein, or receptor substrate insulin-1, which has a high number of phosphorylation centers in tyrosine residues. Phosphorylation o IRS-1 activation consistently allows that has an affinity to bind to other proteins involved in insulin signal transduction. One of them is the phosphatidylinositol 3-kinase (or PI 3-kinase). In turn, this protein allows activating other important proteins, among which the protein kinase B or PK-B, which, among other routes, intervenes in the regulation of the cyclic AMP synthesis pathway (cAMP), in concrete causing the decrease of cAMP which blocks the lipolysis or degradation of triglycerides. Alternatively, the activation of phosphatidylinositol 3-kinase promotes GLUT-4 glucose receptors to be transported to the membrane in order to introduce glucose into the inside the cell, which leads to a stimulation of the pathway lipogenic, or synthesis of lipids and fatty acids.

This assay is based on the fact that glucose is the main substrate for the synthesis of fatty acids, so that it is possible to radioactively label the intracellular "pool" of fatty acids present in triglycerides when cells are incubated in the presence of glucose. radioactive (D- [U- <14> C] -glucose). For this, isolated rat adipocytes (200,000 cells / ml) are incubated in plastic tubes at 37 ° C in Krebs-Ringer buffer, pH 7.4, containing 3% BSA, 10 mM Hepes, 0.5 µCi of D- [U- ^ {14} C] glucose (specific activity 11.8 GBq / mmol, Amersham) and 5.56 mM unlabeled glucose in the absence or presence of 1 nM insulin and four different concentrations of free dihydromyricetin (500 µM, 250 µM, 100 µM and 50 µM). Incubations are carried out for 90 min at 37 ° C and after this time the reaction is stopped by adding 5 ml of Dole extraction solution (isopropanol: heptane: 1 N H2SO4 40:10: 1, v / v). The tubes are shaken and then 2 ml of H2O and 3 ml of heptane are added. After shaking, the samples from the upper phase are transferred to scintillation vials and the radioactivity is determined.
associated.

As shown in Figure 4, the Free dihydromyricetin does not modify, at the concentrations tested, lipogenesis in the absence of insulin. However, when analyzes lipogenesis in the presence of 1 nM insulin, this is seen highly stimulated and dihydromyricetin causes a reduction concentration dependent.

Test No. 5

Effect of dihydromyricetin on lipogenesis (treatment of 24 hours)

The same test described as Test No. 4, is performed but with an incubation time of the adipocytes with 24-hour dihydromyricetin. According to Table 3 attached.

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TABLE 3 Lipogenic effect of adipocyte treatment 3T3-L1 with dihydromyricetin for 24 hours

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The data in Table 3 indicate that different dihydromyricetin concentrations moderately induce lipogenesis (about 2.5 times) in the absence of insulin. The Insulin produces a 6-fold increase in activity of lipogenesis and under these conditions, dihydromyricetin inhibits concentration-dependent manner the stimulatory effect elicited by insulin, such that at 250 µM dihydromyricetin the effect of insulin is completely inhibited.

To determine at what levels the dihydromyricetin insulin-induced lipogenesis is studied the effect of free dihydromyricetin on the insulin receptor in adipocytes; activation of the insulin receptor substrate IRS-1; the degree of association of the phosphatidylinositol 3-kinase to IRS-1 and the activation of protein kinase B.

study 1

Determination of the effect of dihydromyricetin on the activation of the insulin receptor in adipocytes 3T3-L1

The study consists of cultivating adipocytes 3T3-L1 for 24 hours in the presence of 250 µM dihydromyricetin. This concentration, according to Table 3 blocks the action of insulin on lipogenesis. Elapsed this time, the cells are incubated for 30 minutes in the presence or absence of 100 nM insulin. The cells are then lysed and the extracts are subjected to immunoprecipitation with an antibody anti-phosphotyrosine. Immunoprecipitates are Western blot and developed with antibody anti-insulin receptor.

The results are depicted in Figure 5A. The lane corresponding to cells previously treated with insulin and subsequent incubation with 250 µM dihydromyricetin, shows less precipitation of the insulin receptor compared to the lane of cells treated with insulin and without incubation of dihydromyricetin. This indicates that previous exposure in the presence of dihydromyricetin causes a marked decrease in phosphorylation of the receptor.
insulin.

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study 2

Determination of the effect of dihydromyricetin on IRS-1 activation in rat adipocytes

3T3-L1 adipocytes are cultured for 24 hours in the presence of 250 µM dihydromyricetin. After this time, the cells are incubated for 30 minutes. in the presence or absence of 100 nM insulin. Then the cells are lysed and the extracts are immunoprecipitated with a anti-IRS-1 antibody and Immunoprecipitates are subjected to Western blot which is developed with anti-phosphotyrosine.

The results are depicted in Figure 5C. The lane corresponding to cells previously treated with insulin and subsequent incubation with 250 µM dihydromyricetin, presents less precipitation of IRS-1 compared to lane of cells treated with insulin and without incubation of dihydromyricetin. This indicates that previous exposure in the presence dihydromyricetin causes a marked decrease in the insulin receptor substrate phosphorylation IRS-1.

study 3

Determination of the effect of dihydromyricetin on the interaction of the proteins phosphatidylinositol 3-kinase i IRS-1 in rat adipocytes

The study consists of cultivating adipocytes 3T3-L1 for 24 hours in the presence of 250 µM dihydromyricetin. This concentration, according to Table 3 blocks the action of insulin on lipogenesis. Elapsed this time, the cells are incubated for 30 minutes in the presence or absence of 100 nM insulin. The cells are then lysed and the extracts are subjected to immunoprecipitation with an antibody anti-p85 subunit of phosphatidylinositol protein 3-kinase. Immunoprecipitates are subjected to Western blot and develop with antibody anti-phosphotyrosine.

The results are depicted in Figure 5B. The lane corresponding to cells previously treated with insulin and subsequent incubation with 250 µM dihydromyricetin, shows less precipitation of the p85 subunit of the protein phosphatidylinositol 3-kinase, with respect to the cells treated with insulin and without dihydromyricetin incubation. This indicates that previous exposure in the presence of dihydromyricetin causes a marked decrease in activation of the p85 subunit by IRS-1, while Insulin is shown to exert a potent stimulation of the association of the two proteins.

study 4

Determination of the effect of dihydromyricetin on activation of protein kinase B in rat adipocytes

The study consists of cultivating adipocytes 3T3-L1 for 24 hours in the presence of 250 µM dihydromyricetin. This concentration, according to Table 3 blocks the action of insulin on lipogenesis. Elapsed this time, the cells are incubated for 30 minutes in the presence or absence of 100 nM insulin. The cells are then lysed and the extracts are subjected to Western blot developed with an antibody anti-phosphoSer473 protein kinase B.

The results are depicted in Figure 5D. It can be seen that insulin exerts a powerful stimulation of phosphorylation of protein kinase B in control adipocytes C. However, it can be seen that previous exposure with dihydromyricetin causes a marked decrease in the phosphorylation, and therefore non-activation, of the protein kinase B.

Test No. 6

Inhibitory effect of dihydromyricetin of formula I on the adipogenesis in rat adipocytes

A compound with anti-cellulite activity can be if it represses the differentiation of preadipocytic cells into mature adipocytes. These types of effects can be rehearsed using mouse 3T3-L1 preadipocytes which, when incubated under certain conditions, they differentiate into adipocytes with ability to accumulate triglycerides.

3T3-L1 cells are incubated and differ in the presence of different concentrations of Free dihydromyricetin (1 mM, 500 µM, 250 µM, 100 µM, 50 µM, 25 µM and 10 µM). Differentiation starts in fibroblasts two days after having a culture post-confluence. For this, the mixture of isomethylbutylxanthine / dexamethasone / insulin (d1 medium) for 3 days and then with insulin (medium d2) for 2 more days in presence of dihydromyricetin. Cells are cultured for 4 additional days in DMEM FBS 10% (d3 medium) in the absence of stimulators of differentiation. The different concentrations dihydromyricetin are tested for 9 days. After that time, I know determines the morphology of cells by phase contrast and after staining with Oil Red O (Figure 6) as well as the concentration of triglycerides inside the cells (Table 4). The Cell morphology is studied using the method Colorimetric Triglyceride Enzymatic Trinder test (Biotrol Diagnostic).

TABLE 4 Cellular concentration of triglycerides in cells 3T3-L1

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9

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The data obtained in this test No. 6 show that dihydromyricetin at concentrations of 100 µM and 250 µM significantly reduces the differentiation of 3T3-L1 fibroblasts, both when the cellular concentration of triglycerides is determined or when the differentiated cells are visualized. by morphological analysis of the
Figure 6.

To determine at what levels the dihydromyricetin adipocyte differentiation, are performed studies that aim to determine the concentration of proteins involved in critical biochemical processes for mature adipocyte.

study 5

Determination of glucose transporter concentration GLUT-4

According to Test No. 6, the dihydromyricetin blocks adipogenic differentiation to concentrations of 100 to 250 µM.

A culture of 3T3-L1 cells in 2-day post-confluence, it is treated with a isomethylbutylxanthine / dexamethasone / insulin mixture for 2 days. It is then incubated with more insulin for 2 days and finally the cells are cultured for another four days without molecules that stimulate cell differentiation. During the 10 days that the experiment lasts, the cells are exposed to a dihydromyricetin concentration of 100 µM. After this time, the morphology of the cells is determined, they are lysed and determines the expression of the glucose transporter GLUT-4 by Western blot with antibodies specific.

The results in Figure 7B show that in presence of 100 µM dihydromyricetin, the expression of GLUT-4 (45 kDa) in cells 3T3-L1 is less than without dihydromyricetin.

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study 6

Perilipin protein analysis

3T3-L1 cells were cultured in the same way as in study 5. After 10 days of culture with 100 µM dihydromyricetin, the cells are lysed and performs a Western blot with antibodies anti-perilipin.

As can be seen in Figure 7A, in presence of 100 µM dihydromyricetin, the expression of perilipin (57 kDa) in 3T3-L1 cells is lower than without dihydromyricetin.

study 7

Analysis of protein expression caveolin-1 in the presence of dihydromyricetin

3T3-L1 cells were cultured in the same way as in study 5. After 10 days of culture with 100 µM dihydromyricetin, the cells are lysed and determines the expression of caveolin-1 by Western blot with specific antibodies.

As can be seen in Figure 7C, in presence of 100 µM dihydromyricetin, the expression of caveolin-1 (22 kDa) in cells 3T3-L1 is less than without dihydromyricetin.

Trials No. 1 to No. 7, as well as the respective studies 1 to 7, indicate that in lipid or adipocyte reserve cells, dihydromyricetin acts in the early stages of reactions promoted by insulin, which supposedly indicates that Due to the blockage of the initial intracellular pathways, the rest of the effects promoted by insulin are also blocked. In relation to the effect of dihydromyricetin on adipocyte cell differentiation, tests show that it inhibits differentiation at the level of the GLUT-4 transporter, or of perilipin and caveolin, which prevents the formation of caveolae on the cell surface in order to locate the conveyors
GLUT-4.

In all the compositions and uses described in the invention, dihydromyricetin and / or its salts or derivatives, comes from chemical synthesis or from a plant extract, the most widely used extracts being those of the Myrica ceriferaa and Ampelopsis grossedentata species. In the case that a vegetable extract is used, the dihydromyricetin content must be previously analyzed, to ensure that in the final cosmetic and / or pharmaceutical composition, it is in the proportion by weight established to exert the desired effects and previously described.

A composition as described is applicable for cosmetics intended to smooth the figure or to improve the unsightly effect caused by accumulations of fat on the areas below the epidermis.

Claims (16)

1. Cosmetic and / or pharmaceutical composition, regulating fat levels in adipocytes and / or regulating adipocyte differentiation, characterized in that it comprises at least one dihydroflavonol compound of formula I and / or one of its salts and / or derivatives, 10 where R1 can be H, C 1-n alkyl, COC 1-n alkyl, (CH2) n CH (NH2) COOH; R2 can be H, C 1-n alkyl, COC 1-n alkyl, (CH2) n CH (NH2) COOH; R3 can be H, 1-n alkyl, COC 1-n alkyl, (CH2) n CH (NH2) COOH; R4 can be H, C 1-n alkyl, COC 1-n alkyl, (CH2) n CH (NH2) COOH, a sugar such as glucose, rhamnose, rutinose, galactose, fructose, u other acceptable sugars; R5 can be H, C 1-n alkyl, COC 1-n alkyl, (CH2) n CH (NH2) COOH; and R6 can be H, C 1-n alkyl, COC 1-n alkyl, (CH2) n CH (NH2) COOH, a sugar such as glucose, rhamnose, rutinose, galactose, fructose, u other acceptable sugars. Composition according to claim 1, characterized in that the proportion by weight of dihydroflavonol of formula I, of one of its salts and / or derivatives is between 0.01% and 10%. Composition according to any one of Claims 1 to 2, characterized in that the dihydroflavonol of formula I is dihydromyricetin in free form. Composition according to claims 1 or 2, characterized in that the dihydroflavonol of formula I is dihydromyricetin in salt form. Composition according to any one of Claims 1 or 2, characterized in that the dihydroflavonol of formula I is dihydromyricetin in glucoside form. 6. Cosmetic and / or pharmaceutical composition according to any one of the preceding claims, characterized in that it comprises one or more compounds selected from caffeine, xanthine, theophylline, and / or theobromine. 7. Cosmetic and / or pharmaceutical composition according to any one of the preceding claims 1 to 6, characterized in that it is in the form of a gel, cream, ointment, soap, lotion, microcapsules or in a solid form applicable to fabrics. 8. Cosmetic and / or pharmaceutical composition according to any one of claims 1 to 6, characterized in that it is adapted for oral administration. 9. Use of at least one dihydroflavonol compound of formula I, its salts and / or derivatives, for the preparation of a cosmetic and / or pharmaceutical composition, for the regulation of fat levels in adipocytes. 10. Use according to claim 9, characterized in that the dihydroflavonol of formula I is dihydromyricetin in free form and / or in the form of salts and / or derivatives. Use according to claims 9 or 10, characterized in that the reduction of fat levels in adipocytes consists in the inhibition of insulin-induced lipogenesis. 12. Use according to claims 9 or 10, characterized in that the reduction of fat levels in adipocytes consists in the inhibition of adipogenesis. 13. Use according to claims 9 or 10, characterized in that the reduction of fat levels in adipocytes consists of the activation of lipolysis. 14. Use of at least one dihydroflavonol compound of formula I, its salts and / or derivatives, for the preparation of a cosmetic and / or pharmaceutical composition for the regulation of adipocyte differentiation. 15. Use according to claim 14, characterized in that the dihydroflavonol of formula I is dihydromyricetin in free form and / or in the form of salts and / or derivatives. 16. Use according to any one of claims 9 to 15, characterized in that the dihydroflavonol of formula I, its salts and / or derivatives is for the preparation of a cosmetic and / or pharmaceutical composition for the treatment of cellulite.