WO2013004856A1 - Extracts of hibiscus sabdariffa, lippia citriodora and/or stevia rebaudiana for the treatment and/or prevention of hepatic steatosis and/or inflammatory and metabolic disorders - Google Patents

Abstract

Extract of Hibiscus sabdariffa, Lippia citriodora and/or Stevia rebaudiana and combinations thereof for the treatment and/or prevention of hepatic steatosis and/or inflammatory and metabolic disorders, and also pharmaceutical and nutraceutical compositions that contain said extracts.

Description

 EXTRACTS OF HIBISCUS SABDARIFFA, LIPPIA CITRIODORA AND / OR STEVIA REBAUDIANA FOR THE TREATMENT AND / OR PREVENTION OF HEPATIC ESTEATOSIS AND / OR DISORDERS OF INFLAMMATION AND METABOLISM

FIELD OF THE INVENTION

The present invention falls in general in the field of medicine and in particular refers to extracts of Hibiscus sabdariffa, Lippia atriodora and Stevia rebaudiana for the treatment and / or prevention of hepatic steatosis and / or disorders of inflammation and metabolism.

 BACKGROUND OF THE INVENTION

 Phenolic compounds are secondary metabolites of plants that perform various physiological functions. Among them, they intervene in the growth, reproduction and adaptation of plants and in defensive processes against external aggressions. They are present in the plant kingdom and in foods they are usually conjugated with sugars. Currently, this group of compounds is of great interest for its contribution to the maintenance of human health, and they are attributed beneficial biological activities such as antioxidants, vasodilators, anticarcinogenic, anti-inflammatory, bactericidal, immune response stimulants, antiviral, estrogenic effects or inhibitors of prooxidant enzymes, such as cyclooxygenase, lipoxygenase or xanthine oxidase.

Hibiscus sabdariffa (HS) flowers are rich in anthocyanins. Anthocyanins are responsible for the attractive red color of some fruits and vegetables consumed in Western diets, where they represent the most abundant intake of flavonoids. The aqueous extract of HS is also rich in hydroxycitric acid, phenolic acids and various flavonoids such as quercetin and other flavonols (Rodríguez-Medina, I. C, Beltrán-Debón, R., Micol, V., Alonso-Villaverde, C , Joven, J., Menéndez, JA, Segura-Carretero, A., and Fernández-Gutiérrez, A. (2009) Journal of Separation Science 32, 3441-3448). It also contains a high proportion of low molecular weight Arabinoan and Arabinogalactan polysaccharides and pectins. This extract has been commonly used in traditional remedies against hypertension, fever, inflammation, liver disorders and obesity. However, the relationship between the chemical structure of these phenolic compounds, the corresponding biological activity and their consequent health benefit is not known in depth (Jing, P., Bomser, JA, Schwartz, SJ, He, J., Magnuson , BA, and Giusti, MM n. (2008) Journal of Agricultural and Food Chemistry 56, 9391-9398). It is possible that its effects are not exclusively due to its majority compounds, and that other accompanying phenolic compounds are involved. Thus, the combined action of the compounds present in the extract could be more beneficial than that observed by the isolated polyphenols. In this sense, the search for those biologically more active fractions that can be incorporated into a normal diet, could become an alternative strategy for certain pathologies. Particularly, our invention eliminates those compounds that with current knowledge do not seem to have activity and can even give complications, especially polysaccharides.

 Hierbaluisa (Aloysia triphylla, Lippia citriodora) is a plant widely used as a medicinal herb and also for food purposes. The most abundant compounds in the leaves of this plant are iridoids, flavones and phenylpropanoids, with verbascoside being the majority compound. The extract of the leaves of this plant as well as pure verbascoside have strong antioxidant properties. This compound and the extract of Lippia citriodora have been shown to be effective anti-inflammatory through various in vitro tests and in animal models. Recently, this extract has shown the ability to enhance the activity of antioxidant enzymes in human blood cells, as well as reduce plasma oxidative damage due to intense physical exercise.

 Stevia rebaudiana leaves have been used as a source of natural sweeteners for decades in several Asian countries. Stevioside is the main compound of this plant with a sweetening power 300 times greater than sucrose, although it has a bitter aftertaste. This problem has been solved through the enzymatic glycosylation of the Stevia extract or by purifying one of the diterpenoids of this plant, rebaudioside A. Various pharmacological studies have shown that all glycosylated diterpenoids become the intestinal tract. its steviol aglycone, which appears to be the pharmacologically active form. Recent studies propose that stevioside and rebaudioside A may have interesting therapeutic properties. Specifically, these compounds have been shown to reduce postprandial glucose and glucacon levels, as well as having insulinotropic effects in various cellular and animal models. Diterpenes derived from Stevia have demonstrated a wide spectrum of therapeutic effects on human health such as antihypertensives, antihyperglycemic agents, antioxidants, antivirals, anti-inflammatory and anti-tumor properties. In addition, stevioside has been shown to have positive effects on the regulation of glucose metabolism and renal function (Jutabha, P., Toskulkao, C, and Chatsudthipong, V. (2000) Canadian Journal of Physiology and Pharmacology 78, 737- 744).

The results of the examples of the present invention demonstrated that the polyphenolic fraction of the HS extract (FPHS), as well as the polyphenolic fraction of Lippia citriodora (iridoids, phenylpropanoids, flavones) inhibited programmed adipogenesis and reduced the accumulation of triglycerides in both adipocytes mature as in a cellular model of adipocytic hypertrophy. In addition, preliminary results obtained in knock-out LDLr mice fed HS extract, Lippia citriodora extract, or extracts from Stevia rebaudiana and high cholesterol diet, demonstrated a decrease in body weight, decrease in triglycerides and plasma cholesterol , as well as the prevention of hepatic steatosis. The polyphenolic fraction from the plants Hibiscus sabdariffa, Lippia citriodora and Stevia rebaudiana, in isolation or in any of their combinations, were effective in preventing liver steatosis and other disorders related to inflammation and metabolism.

 DESCRIPTION OF THE INVENTION

Thus, in a first aspect, the present invention relates to the extract of Hibiscus sabdariffa, Lippia citriodora and / or Stevia rebaudiana for the treatment and / or prevention of hepatic steatosis and / or disorders of inflammation and metabolism.

 More particularly, the present invention relates to the polyphenolic extract of Hibiscus sabdariffa, Lippia citriodora and / or diterpenic extract of Stevia rebaudiana characterized in that the polyphenolic extract does not comprise polysaccharides, for the treatment and / or prevention of hepatic steatosis and / or disorders of inflammation and metabolism.

 In the present invention, a polyphenolic extract refers to the polyphenolic fraction obtained from the extract of Hibiscus sabdariffa, Lippia citriodora, using either a polyphenolic extract or a polyphenolic fraction. And diterpenic extract means the diterpenic fraction obtained from the Stevia rebaudiana extract using either diterpenic extract or diterpenic fraction.

In the present invention by disorders of inflammation and metabolism we refer to hypertension, atherosclerosis, hyperlipemia, hepatic steatosis.

 In a more particular embodiment, the polyphenolic extract of the present invention comprises phenolic acids, anthocyanins, flavonols, iridoids, phenylpropanoids, flavones and / or mixtures thereof.

In a more particular embodiment, the diterpenic extract of the present invention comprises steviolglycosides.

 In a more particular embodiment, the diterpenic extract of the present invention comprises stevioside, rebaudioside A, B, C and D, dulcoside A, stiviolbioside and / or mixtures thereof.

 In a second aspect, the present invention relates to a pharmaceutical and / or nutraceutical composition comprising the extract of Hibiscus sabdariffa, Lippia citriodora and / or Stevia rebaudiana and at least one pharmaceutically acceptable excipient for the treatment of hepatic steatosis and / or disorders of inflammation and metabolism.

In a more particular embodiment, the present invention relates to a pharmaceutical and / or nutraceutical composition comprising the polyphenolic extract of Hibiscus sabdariffa, Lippia citriodora and / or diterpenic extract of Stevia rebaudiana described above and at least one pharmaceutically acceptable excipient for the Treatment of liver steatosis and / or disorders of inflammation and metabolism. In a more particular embodiment, the polyphenolic extract in the pharmaceutical composition of the present invention is in a proportion comprised between 1-60% by weight.

 In a more particular embodiment, the pharmaceutical composition of the present invention is presented in a formulation selected from the group consisting of tablets, dragees, capsules, tablets, oily or aqueous solutions, suspensions, emulsions and powders.

 The pharmaceutical and / or nutraceutical composition of the present invention can be produced by conventional procedures known to those skilled in the art. The pharmaceutical or nutraceutical composition may vary depending on the route of administration.

 In a more particular embodiment, the administration of the pharmaceutical composition of the present invention is selected from parenteral, transdermal, oral, topical, intracolonic and vaginal routes.

 In a more particular embodiment, the pharmaceutical composition of the present invention is administered parenterally in combination with conventional injectable liquid carriers, such as water or suitable alcohols. Conventional pharmaceutical adjuvants for injection, such as stabilizing agents, solubilizing agents and buffers can be included in such injectable compositions. In a more particular embodiment, the pharmaceutical composition of the present invention is administered intramuscularly, intraperitoneally or intravenously.

 In a more particular embodiment, the pharmaceutical and / or nutraceutical composition of the present invention is administered orally containing one or more physiologically compatible carriers or excipients, in solid or liquid form. These compositions may contain conventional components such as binding agents, fillers, lubricants and physiologically acceptable wetting agents. The compositions may take any appropriate form, such as tablets, dragees, capsules, lozenges, oily or aqueous solutions, suspensions, emulsions or in dry powder form suitable for reconstitution with water or other suitable liquid medium before use, for immediate or controlled release.

 In a more particular embodiment of the present invention, the oral liquid forms for administration may also contain certain additives such as sweeteners, flavorings, preservatives and emulsifying agents. Non-aqueous liquid compositions for oral administration may also be formulated, containing, for example, edible oils. Such liquid compositions can be conveniently encapsulated in, for example, gelatin capsules in a unit dosage amount.

In a more particular embodiment, the dosage of the pharmaceutical composition of the present invention is daily for humans and animals and may vary depending on age, weight or degree of disease etc. The daily dosage for mammals, including humans, ranges normally from 1 milligram to 2000 milligrams, preferably from 1 to 1500 mg, more preferably from 1 to 1000 mg of substance to be administered during one or more ingestions.

In a third aspect, the present invention relates to the use of the extract of Hibiscus sabdariffa, Lippia citriodora and / or Stevia rebaudiana for the preparation of a food ingredient.

In a more particular embodiment, the present invention relates to the use of the polyphenolic extract and / or diterpenic extract of the present invention for the preparation of a food ingredient.

 BRIEF DESCRIPTION OF THE FIGURES

 Figure 1. Base peak chromatogram in negative ionization mode obtained by high performance liquid chromatography with mass spectrometry detection of the HS extract (A) and its isolated polyphenolic fraction (FPHS) (B). The data presented correspond to the compounds indicated in Table 1.

 Figure 2. (A) UV chromatogram obtained at 280 nm and base peak chromatogram (50-1000 m / z) in negative ionization mode of the aqueous extract of Lippia citriodora obtained by high performance liquid chromatography with mass spectrometry detection .

Figure 3. Antiadipogenic effect of the polyphenolic extract of Hibiscus sabdariffa. Representative images of untreated 3T3-L1 cells (A), in the presence of 1000 μg / ml of HS extract (B), or in the presence of 40 μg / mL of FPHS (C). The effect proved to be dose dependent for both extracts (D and E) and without toxic effects. *** (p <0.001).

Figure 4. Antiadipogenic effect of the polyphenolic extract of Lippia citriodora. Study of adipogenesis in 3T3-L1 cells analyzed by AdipoRed staining in adipocytes treated with increasing concentrations of an extract of Lippia citriodora valued at 10% phenylpropanoids (w / w). A dose-dependent anti-adipogenic activity of the extract of Lippia citriodora is shown. The differences between the groups and the differentiated control are marked with *** (p <0.001).

 Figure 5. Reduction of triglyceride levels in mature adipocytes using Hibiscus sabdariffa extract. Mature adipocytes were treated with HS or FPHS, and the accumulation of triglycerides quantified using Adipored, a statistically significant decrease for FPHS being observed at 40 μg / ml, with respect to the differentiated control and not treated with the extract. No significant effect was observed with HS. The statistical differences with respect to the positive control are marked with ** (P <0.01).

Figure 6. Reduction in triglyceride levels by means of the Hibiscus sabdariffa polyphenolic extract in an adipocyte hypertrophy model. Lipid accumulation was determined by staining with Adipored. Morphological changes of preadipocytes 3T3-L1 were observed under a microscope and photographed after 22 days from the start of differentiation. (B) Hypertrophic adipocytes, (C) and (D) adipocytes treated with 1000 μg / mΙ of HS extract and 40 μg / mΙ of FPHS, respectively. Adipocytes treated with 500-1000 μg / mΙ of HS and 20-40 μg / mΙ of FPHS after differentiation for 22 days showed a lower relative content of post-treatment lipids (D). No toxic effects were observed at the concentrations tested (data not shown). Differences from the control are marked with ** or *** (p <0.01 or p <0.001, respectively).

 Figure 7. Demonstration that the effect on non-weight gain with continued administration of HS is dose-dependent in the LDL receptor deficient experimentation model (A). The effect became evident regardless of the diet used although it was quantitatively more important in animals subjected to diets rich in fat and cholesterol (B, C) Figure 8. The effect observed in weight maintenance, described in Figure 7, was lost when using the white variant of H. sabdariffa collected in the same place as the red variant, but with a different polyphenol composition. The data indicates that the active substances have to be looked for in the red variant.

Figure 9. The effect on the prevention of hepatic steatosis in mice deficient in LDL receptor is only observed in the presence of metabolic stress, exemplified here with the continued intake of diets rich in fat and cholesterol.

 Figure 10. The consumption of H. sabdariffa in mice deficient in the LDL receptor, after an initial period of induction of hepatic steatosis by eating a diet rich in fat and water as the only liquid intake (A), reduces the accumulation of fat in the liver (B).

Figure 11. The consumption of an extract of Lippia citriodora (10% phenylpropanoids) also had a preventive effect on weight gain, independent of the amount of food ingested (A) in addition to a significant lipid lowering effect (B). This effect is only observed in the presence of metabolic stress, exemplified here with the continued intake of diets rich in fat and cholesterol.

 Figure 12. C: Effect of the extract of Lippia citriodora in the prevention of hepatic steatosis in the same experimental model of Figure 9; A: Control, B: Lippia.

Figure 13. In a model of hepatic steatosis caused by obesity derived from leptin deficiency (ob / ob), glycosides derived from Stevia rebaudiana also showed a significant effect in the prevention of hepatic steatosis. Both steviol (B) and stevioside (C) and rebaudioside A (D) had beneficial effects, although quantitatively different. A: control. DETAILED DESCRIPTION OF THE INVENTION EXAMPLES OF EMBODIMENT

 The following specific examples provided herein serve to illustrate the nature of the present invention. These examples are included for illustrative purposes only and should not be construed as limitations on the invention claimed herein.

High performance liquid chromatography (HPLC) analyzes were performed with a 1200 series RRLC device (Agilent Technologies, Palo Alto, CA, USA) equipped with a binary pump supply system, a degasser, an autosampler, a Ultraviolet detector visible by diode network (DAD, UV-VIS array diode detector) and a mass spectrometry detector (MSD). The semi-preparative HPLC column used was a Phenomenex Luna column (C ₁₈ ), 10 μm di, 25 cm x 10 mm and the flow rate was 3 ml / min. The analytical HPLC column used was a Luna C ₁₈ column, 5 μm di, 25 cm x 3.0 mm (Phenomenex, Torrance, CA, USA), with a C ₁₈ pre-column filter (Phenomenex). The flow rate of the mobile phase was 0.5 ml / min.

 HPLC quality solvents were from Merck (Darmstadt, Germany). Distilled water with a conductivity of 18.2 Ω was deionized using a Milli-Q system (Millipore, Bedford, MA, USA).

Dry calyces of Hibiscus sabdariffa (Senegal or Sudan origin), extract of Lippia citriodora (10% verbascoside) and commercial extracts of Stevia rebaudiana were used for cell and animal tests.

 EXAMPLE 1: Global process of obtaining the bioactive polyphenolic fraction of the Hibiscus sabdariffa calyx.

 Extraction:

 The compositions according to the present invention were obtained through the aqueous or hydroalcoholic extraction of the Hibiscus sabdariffa calyx by agitated tank extraction or percolation in a subsequent enrichment phase.

The complex distribution of active compounds contained in the Hibiscus sabdariffa calyx required the use of a specific hydroalcoholic medium, specifically water-ethanol, for a complete and global extraction in the following range: water [90-75%] : ethanol [10-25%].

 The extraction was carried out at a temperature between 25 and 45 ° C, using, to achieve greater process efficiency, a stirred tank, and a solid-liquid ratio of 1:20. The extraction proceeded in a nitrogen-rich environment to inhibit the potential oxidation processes that some of the active ingredients might undergo during extraction.

Polysaccharide removal: The polysaccharide fraction was removed from the extract either by filtration or by alcohol precipitation. The solid-liquid separation was carried out using a centrifuge or filter press using polypropylene or polyamide filter cloth. The solution thus obtained was concentrated in a vacuum evaporator (reduced pressure) until a syrup with a solids concentration in the range 40-70% was obtained which was loaded in a vacuum dryer (reduced pressure) type Guedu or oven trays and dried to obtain a crystalline solid with a water content of less than 5%.

 The solid powder obtained was ground and dissolved in 96% ethanol in a 1:50 weight-volume ratio. The medium was kept under stirring for 10-15 hours until complete precipitation of the non-active fractions (mainly polysaccharides) occurred.

The suspension was filtered and the ethanol solution containing the active compounds was concentrated in a vacuum evaporator until a concentrated syrup with 60-70% solids was obtained and dissolved in water in a solid-liquid 1:20 ratio. The aqueous solution was clarified using a plate filter and a silica and cellulose filter medium.

 Enrichment or purification:

 The clarified solution obtained was purified by an affinity chromatography column using hydrophobic resins Amberlite type. After the adsorption process, the column was washed with water until the color in the column eluted disappeared.

 The desorption of the bioactive compounds was carried out by an organic solution, preferably ethanol, acetone or ethyl acetate.

 Recrystallization:

 The final polyphenolic fraction was obtained by recrystallization from highly nonpolar solvents.

The solution obtained after desorption, containing the bioactive compounds, was concentrated in a vacuum evaporator (reduced pressure) until a syrup with a solids concentration in the range 40-70% was obtained. The syrup obtained was loaded into a vacuum dryer (reduced pressure) type Guedu or tray oven and dried to obtain a crystalline solid with a water content of less than 2%.

 The solid powder obtained was ground and dissolved in an ethanol-ethyl acetate mixture (1: 1) in a 1: 5 weight-volume ratio. The medium was kept under stirring for one hour, after which it was filtered and the precipitate obtained was eliminated.

The clarified medium was concentrated to the consistency of syrup and water was added in a 1:10 ratio. The medium was stirred until completely dissolved. The water was removed in a vacuum evaporator until a syrup with a solids concentration in the range 70-80% was obtained. The syrup obtained was loaded into a Vacuum dryer (reduced pressure) Guedu type or tray stove and dried to obtain a crystalline solid with a water content of less than 5%.

This last bioactive fraction was called a polyphenolic fraction (FPHS). The example composition of this fraction is described in example 2.

EXAMPLE 2: Composition of the extracts obtained by solid-liquid extraction of Hibiscus sabdariffa and subsequent purification of the phenolic compounds by semi-preparative reverse phase chromatography.

EJEMPLO 3: Composición del extracto acuoso de Lippia citriodora (Figura 2). For the H PLC analysis of the HS and FPHS extracts, solutions were prepared at 50 and 5 g / l, respectively. The aqueous extract was homogenized in a vortex until completely dissolved and filtered with a single use filter. The composition of the extracts was analyzed by liquid chromatography RRLC 1200 series (Agilent Technologies, CA) coupled to mass spectrometry by time of flight (TOF) using electrospray as an interface (Bruker Daltonics, GmbH, Germany). The compounds were separated by a Ci ₈ column of 4.6x150 mm and 1.8 μηη pore size, at a flow rate of 0.5 ml / min and with an injection volume of 10 μΙ. The use of prefilters (RRLC in-line filters, 4.6 mm, 0.2 microns, supplied by Agilent Technologies) as a safety column provided some protection against decomposition and blocking of the working column. The array diode detector coupled to the chromatography system was set in a spectrum range between 190 nm and 950 nm. The mobile phase consisted of a gradient with A: formic acid and B: acetonitrile (0 min, 5% B; 20 min 20% B; 25 min 40% B; 30 min 5% B; 35 min, 5% B). The mass spectrometer conditions were an ionization of the compounds in negative mode, a capillary voltage of +4500 V, drying gas temperature at 200 ° C, drying gas flow at 7 L / min and nebulizer gas pressure 1.5 bar Mass spectrometer calibration was done by internal calibration using sodium formate (5 mM sodium hydroxide in 1/1 (v / v) isopropanol / water with 0.2% formic acid) using a high precision quadratic regression (HPC) . The characterization of the compounds was carried out through the Genérate Molecular Formula ™ application developed by Bruker Daltonics in its Data Analysis 3.5 software, which takes into account the distribution and isotopic abundance, the exact mass of the compounds, the electronic configuration and the possible doubles. or triple links to generate the molecular formula (s) / is as accurate as possible. The identification of the compounds was carried out from the molecular formula generated by different databases, especially Scifinder Scholar and ChemID Plus Advance. Figure 1 and Table 1 show the mass spectrometry (MS) data in negative inonization mode and UV absorption bands of the phenolic compounds identified in the Hibiscus sabdariffa (HS) extract and in the polyphenolic fraction (FPHS) of the same extract.

EXAMPLE 3: Composition of the aqueous extract of Lippia citriodora (Figure 2).

Chromatographic separation of the Lippia citriodora compounds was performed at room temperature using a gradient elution at a flow of 0.5 ml min ^-1 . The mobile phase consisted of a mixture of water: acetonitrile (90:10, v / v) with 1% formic acid (A) and acetonitrile (B). The following linear gradient was used for separation: 0 min, 5% B; 25 min, 20% B; 30 min, 40% B; 35 min, 5% B. The initial conditions were maintained for 10 min. The injection volume was 20 L and UV-Vis detection was performed in the range 190-450nm. In order to obtain constant flow conditions at the electrospray interface, a "T" type split diver (split ratio 1: 3) was used, so that the flow supplied to the ESI-TOF-MS detector was 125 I min ^-1 . The HPLC system was coupled to a flight time mass spectrometer (TOF) equipped with an electrospray interface (ESI) operating in negative ion mode. A capillary voltage of +4000 V, drying gas temperature at 190 ° C, drying gas flow at 7 L / min and nebulizer gas pressure at 1.5 bar were used. The detection was carried out considering a mass range of 50-1000 m / z. The characterization of the compounds was carried out through the Genérate Molecular Formula ™ application developed by Bruker Daltonics in its Data Analysis 3.5 software, which takes into account the distribution and isotopic abundance, the exact mass of the compounds, the electronic configuration and the possible doubles. or triple links to generate the molecular formula (s) / is as accurate as possible. The identification of the compounds was carried out from the molecular formula generated by different databases, especially Scifinder Scholar and ChemID Plus Advance (Figure 2).

EXAMPLE 4: Cell lines and culture conditions. Models of inhibition of programmed adipogenesis and accumulation of triglycerides in mature and hypertrophic adipocytes.

Maintenance of the 3T3-L1 preadipocyte cell line

The 3T3-L1 preadipocyte line, maintained at low pass number, was grown in DMEM enriched with stable glutamine, supplemented with 10% calf serum, 1% pyruvate, and antibiotic (100 μg / ml streptomycin and 100 units / ml penicillin). The manipulation was conducted under sterile conditions using a laminar flow cabinet for culturing cells and an incubator at 37 ° C with a humidified atmosphere of C0 ₂ and 5%. The preadipocytes were subcultured before reaching a confluence of 80%. The cells were stored in liquid nitrogen, since higher temperature storage results in loss of viability.

Quantification of lipid content by Adipored analysis

To check the effect of the extracts object of the invention on the lipid content of the differentiated adipocytes, the cells were revealed 10 or 20 days after the initiation of differentiation, at which time more than 90% of the cells were identified as adipocytes. mature with drops of fat accumulated in the cytoplasm (according to microscopic evaluation). Lipid content was measured using the AdipoRed commercial reagent for staining intracellular lipids. Briefly, when the cells were ready to be analyzed, the culture supernatant was removed and the cells were carefully washed with 200 ml of phosphate buffered saline (PBS, pH 7.4). Next, the AdipoRed ™ reagent was added with a multichannel pipette diluted in PBS. After 10 minutes, the intracellular lipid content was quantified using a fluorescent plate reader (FLUOstar Omega, BMG LABTECH), with excitation at 485 nm and emission at 572 nm. Lipid droplets in the cells were observed and photographed in a fluorescent microscope with a 10-magnification objective.

 MODEL 1. Induction to differentiation. Inhibition of adipocyte differentiation.

 3T3-L1 cells undergo a conversion of preadipocytes to adipocytes as a progression that goes from a state of rapid division to a state of contact inhibition upon reaching the confluence. A high serum and glucose content in the medium increases the accumulation of fat. The preadipocytes were differentiated using standard protocols. Briefly, the cells were stimulated to differentiate with DMEM completed with 10% fetal bovine serum (FBS) and adipogenic agents (insulin, DEX and IBMX) that were added to the culture medium for 2 days. Next, the cells were maintained in differentiation medium containing insulin in DMEM with 10% FBS. Subsequently, the medium was changed every two days for the next 10 days. To test the effect of different extracts or fractions on adipogenesis, they were added to the medium at different concentrations from the beginning of the induction of differentiation until the cells were revealed. The extracts were prepared with the culture medium and filtered through 0.22 micron filters for sterility. To test the effect of the different HS extracts on the accumulation of cytoplasmic triglycerides of 3T3-L1, the extracts were added after the cells had differentiated, until the plates were revealed. The cytotoxicity of the extracts was measured by the crystal violet method. Briefly, the differentiated cells were stained with violet crystal and after removal of excess and solubilization of the dye, the amount absorbed by the cells was measured spectrophotometrically in a plate reader (SPECTROstar Omega, BMG LABTECH) at 620 nm and correlated with the number of viable cells in the well.

Cells treated with adipogenic agents (Fig. 3A) and also in the presence of 1000 μg / ml of the HS extract (Fig. 3B) or 40 μg / ml of the FPHS (Fig. 3C) accumulated 44.4% and 19.3 %, respectively, of intracellular triglycerides with respect to the control treated with the same agents (Fig. 3A), as evidenced by the quantification of the effect through Adipored staining (Figures 3D and 3E). To exclude possible cytotoxic or antiproliferative effects of the extracts, cell viability was measured under the same conditions. However, both HS extract and FPHS showed no cytotoxic effect. A similar dose-dependent programmed adipogenesis inhibition behavior was also obtained when an extract of Lippia citriodora with a phenylpropanoid content of approximately 10% (essentially verbascoside) was used (Figure 4). The inhibition value of the IC50 adipogenesis obtained for this extract was approximately 200 microg / ml.

 MODEL 2. Inhibition of triglyceride accumulation in mature adipocytes.

 To observe the effect on the accumulation of triglycerides in mature adipocytes, the culture of adipocytes was maintained after differentiation, providing them with a culture medium with a high concentration of glucose (4.5 g / l) and fetal bovine serum. At 10 days from the induction of differentiation, cells that had accumulated large lipid droplets were used as hypertrophied adipocytes 3T3-Ll. The serum-free medium of the 3T3-L1 hypertrophied adipocytes cultured for 48 hours with the extracts was collected and stored at -20 ° C until use.

 To study the effects of the polyphenolic fraction of hibiscus (FPHS) compared to the total extract in the inhibition of the accumulation of cytoplasmic lipids, several concentrations of HS and FPHS were added to 3T3-L1 cells once differentiated, that is, when microscopic observations confirmed the typical morphology of the mature adipocyte (Figure 5). This happened on the eighth day from the beginning of the induction of differentiation. After 48 hours of treatment the cells were revealed, obtaining a decrease in the accumulation of cytoplasmic triglycerides in adipocytes treated with FPHS of 22%, with respect to the differentiated control (Figure 5).

MODEL 3. Inhibition of triglyceride accumulation in a model of adipocyte hypertrophy.

The 3T3-L1 preadipocytes were differentiated to mature adipocytes and cultured for up to 14 more days after differentiation (a total of 22 days of culture), using them as a 3T3-L1 hypertrophic adipocyte model with larger lipid drops (Fig. 6B ). On the eighth day of induction, small drops of fat in the cytoplasm were already observed under the microscope, a sign that the cells had acquired the typical morphology of the adipocyte. The culture medium supplemented with fetal bovine serum provided the substrate cells in excess to increase the intracellular lipid content. On day 18, after induction of differentiation, several concentrations of original extract (HS) and polyphenolic extract (FPHS) (as shown in Figure 6) were added to the culture for 96 hours, renewing the serum-free medium at the two days.

To investigate the possible inhibitory effects of HS and FPHS extract on the accumulation of cytoplasmic lipids, staining with Adipored was used. Microscopic observations of untreated cells (Figure 6B) and treated cells (Figures 6C and 6D), grown under optimal differentiation conditions, revealed a significant reduction in the accumulation of triglycerides in the cytoplasm at the end of the assay. Cells, 22 days after induction of differentiation treated with 1000 μg / ml of original extract (Fig. 6C) and 40 μg / ml of FPHS (Fig. 6D) accumulated 81.8% and 62.5% respectively, of Intracellular lipid content with respect to the positive controls treated with the same inducers of differentiation, as shown by staining with Adipored.

 EXAMPLE 5. The absence of weight gain in the experimental model deficient in LDL receptor is shown by measuring the percentage of weight gain in the presence of active compounds or in their absence. First, it was determined that the effect depended on the dose of Hibiscus sabdariffa: at doses of 0, 15, 30, 40, 50 and 125 g / day the effects increased linearly. It does not appear that these effects increase significantly once they exceed 125 g / day, but it has to be confirmed. All procedures were approved by the Ethical Committee of animal experimentation, and neither the caregivers nor the experimenters knew the design of the experimental groups. Two diets were used, one considered as maintenance and the other that was the same supplemented with 20% fat and 0.25% cholesterol (Figure 7). There was no mortality in the study or demonstrable toxic effects at necropsy and therefore all the data obtained were included in the analysis. In successive experiments, an amount of Hibiscus sabdariffa was used, which transferred to humans of about 70 kg corresponded to a dose of 10 g / day. The effect was significant with both diets, but it became apparent sooner in the presence of increased caloric intake.

 EXAMPLE 6. To demonstrate that the composition and relative richness in polyphenols was the result of the effect, the experiment of the previous example was repeated with the absence of active compounds and with extracts of H. sabdariffa white variant and H. sabdariffa red variant obtained in the same field . There was no mortality in the study or demonstrable toxic effects on necropsy and therefore all the data obtained were included in the analysis (Figure 8).

 EXAMPLE 7. Using the same model as examples 5 and 6, mice develop hepatic steatosis when fed a diet high in fat and cholesterol. Continued treatment with H. sabdariffa prevents the formation of hepatic steatosis in full according to the data obtained by liver histology following proven methods (Tous M, Ferré N, Camps J, Riu F, Young J. Feeding apolipoprotein E knock-out mice with cholesterol and fat enriched diets may be a model of non-alcoholic steatohepatitis Mol Cell Biochem 2005; 268: 53-8.) (Figure 9).

 EXAMPLE 8. In the same animal model as the previous examples (5 to 7), we study the ability of H. sabdariffa to cure liver steatosis. After being fed 14 weeks with the diet rich in fat (20% palm oil + 0.25% cholesterol) and water as the only liquid intake, a liver biopsy is performed to determine the degree of steatosis (Figure 10A). During the next 10 weeks, the animals continue to receive the same diet and begin to take the extract of H. sabdariffa. After this period of treatment, the decrease in the presence of fat in the liver is evident (Figure 10B).

EXAMPLE 9. Figure 12 shows the effect of consuming an extract of Lippia citriodora (10% phenylpropanoids) on body weight gain in the same model. It also declined despite that the amount of food ingested did not vary in the experimental models. Likewise, despite being a model with experimental hyperlipemia, the plasma concentration of both triglycerides and cholesterol decreased significantly (Figure 11).

 EXAMPLE 10. Using the same model as in Example 7, the continuously administered atria Lippia extract also prevented the formation of fatty liver determined by the intake of diets rich in fat and cholesterol (Figure 12).

 EXAMPLE 11. Finally, the effect of the extracts in an obese mouse model (ob / ob) characterized by the congenital absence of leptin was studied. These mice, even with normal diet develop very evident macrovesicular hepatic steatosis. Figure 14 shows that the continued administration of sweeteners (1, steviol; 2, stevioside; 3, rebaudioside A) at human equivalent doses at lg / day partially prevents the occurrence of fatty liver (Figure 13).

 The data showed that the polyphenolic extract and diterpenoid extract present in Hibiscus sabdariffa, Lippia átriodora and Stevia rebaudiana (ac. Phenolics, anthocyanins, flavonols, iridoids, phenylpropanoids, flavones and steviol glycosides) regulated the mechanisms of adipocyte differentiation and intracellular accumulation triglycerides decreasing the accumulation of triglycerides or exerting a lipolytic effect. This effect resulted in a decrease in cellular secretion of adipokines and / or proinflammatory cytokines, as well as levels of cellular oxidative stress.

 The data showed that the beneficial effects of these extracts were compromised when these polyphenols were accompanied by massive amounts of polysaccharides, such as, for example, the complete extract of Hibiscus sabdariffa.

Claims

 1. Extract of Hibiscus sabdariffa, Lippia citriodora and / or Stevia rebaudiana for the treatment and / or prevention of hepatic steatosis and / or disorders of inflammation and metabolism.  2. Hibiscus sabdariffa, Lippia citriodora and / or Stevia rebaudiana extract according to claim 1, characterized in that the Hibiscus sabdariffa extract, Lippia citriodora is the polyphenolic fraction of the extract and the Stevia rebaudiana extract is the diterpenic fraction of the extract.  3. Hibiscus sabdariffa, Lippia citriodora and / or Stevia rebaudiana extract according to claim 2, characterized in that the polyphenolic fraction of Hibiscus sabdariffa, Lippia citriodora does not comprise polysaccharides. 4. Hibiscus sabdariffa, Lippia citriodora and / or Stevia rebaudiana extract according to claim 3, characterized in that the polyphenolic fraction comprises phenolic acids, anthocyanins, flavonols, iridoids, phenylpropanoids, flavones and / or mixtures thereof.  5. Hibiscus sabdariffa, Lippia citriodora and / or Stevia rebaudiana extract according to claim 3, characterized in that the diterpenic fraction comprises steviolglycosides. 6. Hibiscus sabdariffa, Lippia citriodora and / or Stevia rebaudiana extract according to claim 5, characterized in that the diterpenic fraction comprises stevioside, rebaudioside A, B, C and D, dulcósido A, stiviolbioside and / or mixtures thereof.  7. Pharmaceutical and / or nutraceutical composition comprising the extract of Hibiscus sabdariffa, Lippia citriodora and / or Stevia rebaudiana according to any of claims 1-6 and at least one pharmaceutically acceptable excipient for the treatment of hepatic steatosis and / or disorders of Inflammation and metabolism.  8. Pharmaceutical and / or nutraceutical composition according to claim 7 characterized in that the polyphenolic extract is in a proportion comprised between 1-60% by weight.  9. Pharmaceutical and / or nutraceutical composition according to any of claims 7-8 characterized in that it is presented in a formulation selected from the group consisting of tablets, dragees, capsules, tablets, oily or aqueous solutions, suspensions, emulsions and powders.  10. Pharmaceutical and / or nutraceutical composition according to any of claims 7-8 wherein the administration is selected from parenteral, transdermal, oral, topical, intracolonic and vaginal. 11. Use of the extract Hibiscus sabdariffa, Lippia citriodora and / or Stevia rebaudiana according to any of claims 1-6 for the preparation of a food ingredient.