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    • 专利摘要:
    The present invention provides compositions comprising an extract with at least 5% by weight of anthocyanins and an extract with at least 15% by weight of phenylpropanoids which are especially indicated for use in periods of weight reduction because they reduce Appetite and increase satiety by a modification of the levels of hormones secreted by the digestive tract. In a parallel or complementary manner, these compositions act on lipid metabolism in adipose tissue cells by activating AMPK and the processes of lipid oxidation and elimination. Said composition is obtained by the hydroalcoholic extraction of rosella flower and herbuisa leaf, using in the case of lemongrass alcohol concentrations (ethanol, methanol, propanol, isopropanol or butanol) greater than 70 and up to 100% by volume. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2661579A1
    申请号:ES201731147
    申请日:2017-09-25
    公开日:2018-04-02
    发明作者:María HERRANZ LÓPEZ；Enrique ROCHE COLLADO；Vicente Micol Molina；Jonathan Richard JONES BARBERÁ；Nuria CATURLA CERNUDA
    申请人:Monteloeder S L；MONTELOEDER SL；
    IPC主号:
    专利说明:

    Composition for weight control through modulation of the levels of peptides involved in satiety and / or appetite.
    TECHNICAL FIELD OF THE INVENTION
    The present invention is framed within the following technical fields: nutritional supplements, functional feeding, pharmaceutical preparation, and animal use; to control body weight and appetite, in particular, useful for people who are overweight or obese. In particular, it refers to a composition that includes two polyphenolic plant extracts.
    BACKGROUND OF THE INVENTION
    Obesity has been described by the World Health Organization (WHO) as a global epidemic associated with several metabolic imbalances, which cause an increase in adipose tissue mass, endothelial dysfunction, dyslipidemia, hypertension, atherosclerosis and insulin resistance . Together, these imbalances constitute a complex pathology known worldwide with the term metabolic syndrome.
    Obesity is defined as a condition of excessive accumulation of fat in adipose tissue, which carries a health risk and predisposes to resistance to insulin, hypertension and dyslipidemia. It is generally accepted that the only option to prevent the development of obesity-derived disorders is to restrict calorie intake and increase physical activity. However, these changes in the way of life are not feasible because aging is associated with a reduction in muscle mass and resting metabolic expenditure.
    Obesity is generally treated by a combination of methods, which include adopting a healthy diet, exercise and psychological support to achieve tangible goals of weight reduction and efficient metabolic control of this pathology. However, these methods are difficult to carry out among the overweight population in the long term, because the individuals under treatment tend to abandon it and regain lost weight. In addition, a period of weight loss is accompanied by a body adaptation to the new metabolic requirements. There is an energy deficit between consumption and caloric expenditure that stimulates the production of ghrelin to induce appetite and cause greater food consumption, while reducing metabolic energy consumption in lethargy and increasing energy efficiency (reduction of energy consumption when active). In the long term, this implies that the body tends to consume fewer calories and increases appetite considerably, which produces the well-known "rebounds", in which the same or more food is consumed again than before starting the diet and The body fails to burn calories at the same rate as before the diet, with the consequence that the lost weight is regained or even overcome. It is therefore necessary to modulate the metabolism to avoid increasing appetite during periods of


    diet and weight reduction and avoid or reduce relapses in the treatment of obesity and overweight.
    Obesity is a very complex pathology in which numerous factors are involved such as the presence of a chronic and low intensity inflammatory component, an altered energy metabolism and an oxidative imbalance. This complexity is part of the reason why weight readjustments and metabolism are very slow processes. On numerous occasions this entails the use of complementary methods to achieve the desired weight reduction, such as bariatric surgery, pharmacological treatments and nutritional intervention with dietary or nutraceutical supplements.
    The drawbacks of bariatric surgery are those of any surgical intervention, leading to even death during the procedure. The disadvantages of pharmaceutical treatments are also well known for the so-called "side effects", which can always occur in greater
    or lesser extent In addition, there are numerous nutraceutical products and dietary supplements on the market that advocate their effects of weight reduction, of "burning" fat and even reducing appetite, whose effects are not scientifically proven, are doubtful or at best of the cases is due to a placebo or autosuggestion effect.
    SUMMARY OF THE INVENTION
    A first aspect of the invention provides a composition for use in modulating an individual's food intake by reducing appetite and / or increasing satiety through a modification of appetite-related peptide levels segregated by the digestive tract and adipose tissue of the individual, characterized in that said composition comprises an extract with at least 5% by weight of anthocyanins and an extract with at least 15% by weight of phenylpropanoids.
    A composition that contains both extracts mentioned above is enormously effective in modulating food intake because it produces satiating and / or appetite-reducing effects, through its effect on the levels of peptides secreted by the digestive tract, which cause feelings of satiety and / or appetite With this composition it is possible to reduce the negative effects of weight control and maintenance strategies; for example, it is possible to reduce the appetite during a period of weight reduction and thus avoid or reduce the chances that the dietary individual begins to eat food in greater amounts than recommended and regain weight. Likewise, this composition is very useful in weight maintenance strategies, once the desired weight has been reached, this composition helps to maintain the feeling of satiety for a longer time, and therefore, it is possible to modulate food intake at the desired level, for example, to maintain weight (or reduce it further, if necessary).


    Preferably the extract with at least 5% by weight of anthocyanins contains about 10% in anthocyanins.
    Preferably the extract with at least 15% by weight of phenylpropanoids contains about 30% of phenylpropanoids.
    These concentrations in anthocyanins and phenylpropanoids ensure that the composition used is not cytotoxic.
    Preferably the extract with anthocyanins comprises a rosella extract (Hibiscus sabdariffa).
    Preferably, the phenylpropanoid extract comprises a herb extract (Lippia citriodora).
    The effects on appetite and satiety of herbal extracts and rosella extracts separately are not clearly defined in the state of the art. There are sources that consider that they have effects of increasing appetite and reduce satiety and sources that consider that they decrease appetite and increase satiety. Here, it can be verified that a composition with both extracts has a potent effect of modifying the expression of peptides that are involved in the regulation of food intake (appetite and satiety), such as ghrelin and leptin, and that in Compared with equivalent doses of these extracts administered separately, they produce a very significantly greater effect. Given the ambiguous effects of both extracts separately, the satiating and appetite reduction effect achieved by the compositions containing both extracts is highly surprising and unexpected.
    Rosella is a plant rich in anthocyanins and its extract combined with the extract of Hierbaluisa, which is a plant rich in phenylpropanoids produces a marked satiating effect and / or appetite reduction.
    The composition may comprise a minimum of 50% by weight of the herb extract (Lippia citriodora) and a minimum of 25% by weight of the rosella extract (Hibiscus sabdariffa).
    Preferably the composition comprises about 65% by weight of the herb extract (Lippia citriodora) and about 35% by weight of the rosella extract (Hibiscus sabdariffa).
    This percentage of both extracts causes the composition to have a greater effect on the activation of fat metabolism mediated by a greater activation of AMPK compared to a composition where the extracts are mixed in equal parts.
    The composition can be administered in a dose of 250 to 1000 milligrams per day.


    Preferably the composition is administered in a dose of 500 mg per day.
    These doses ensure an effect on the peptides related to appetite and segregated by the digestive tract and adipose tissue of the individual, which translates into a reduction in appetite and / or increased satiety that is reflected in a decrease in the need for eating of food.
    A second aspect of the invention provides a composition for use in reducing the weight of an individual by means of a modification in lipid metabolism mediated by an activation of the AMP-activated protein kinase (AMPK) energy sensors of the translated individual. in a reduction of the percentage of body fat, characterized in that said composition comprises an extract with at least 5% by weight of anthocyanins and an extract with at least 15% by weight of phenylpropanoids.
    A composition containing both extracts mentioned above is enormously effective in reducing the weight of an individual through a change
    or improvement in lipid metabolism through the activation of AMP-activated protein kinase (AMPK) energy sensors. This composition is very useful in the treatment of obesity, being able to reduce the amount of adipose tissue of an individual and improve other parameters typically related to obesity or overweight.
    AMPK is a serine / threonine kinase that plays a very important role in the maintenance of cellular homeostasis. Its activation involves the activation of catabolic processes such as lipolysis and fatty acid oxidation and the inhibition of anabolic processes such as lipogenesis and glycogenesis.
    Preferably the extract with at least 5% by weight of anthocyanins contains about 10% in anthocyanins.
    Preferably the extract with at least 15% by weight of phenylpropanoids contains about 30% of phenylpropanoids.
    These concentrations in anthocyanins and phenylpropanoids ensure that the composition used is not cytotoxic.
    Preferably the anthocyanin extract comprises a rosella extract (Hibiscus sabdariffa).
    Preferably the phenylpropanoid extract comprises an extract of Hierbaluisa (Lippia citriodora).
    Rosella anthocyanins have been shown to be particularly effective in reducing inflammation linked to metabolic stress by inhibiting secretion of leptin and monocyte chemoattractant protein 1 (MCP-1), which are


    important adipokines that regulate the migration of non-resident macrophages to adipose tissue and general systemic inflammation. Rosella polyphenolic extracts also prevent liver steatosis in hyperlipidemic mice through modulation of the expression of genes involved in glucose and lipid homeostasis. In mice fed a high-fat diet, Rosella anthocyanins attenuate the increase in blood glucose and the apparent increase in insulin resistance, as well as increase the respiratory rate, which is closely related to the basal metabolic rate (BMR ). In this same animal model, a reduction in the expression of lipogenic genes, such as fatty acid synthase (FASN) and the sterol regulatory element binding protein (Srebp-1c), was also observed, simultaneously with an activation of hepatic AMPK.
    It is known that the activation of hepatic AMPK induces the expression of the PPARGC1A gene and directly improves its activity by phosphorylation, which increases biogenesis and mitochondrial function. This implies that Rosella's anthocyanins act on the modulation of fat use, most likely by the inhibition of lipogenesis and the activation of lipolysis at the level of mitochondrial biogenesis. All these processes resemble a metabolic situation that reflects a high cellular energy demand, an improved energy expenditure and a higher basal metabolic rate.
    On the other hand, the phenylpropanoids of the herb extract and its major component verbascoside improves the metabolic alterations induced by a high level of glucose. These effects are mediated by a positive transcriptional regulation dependent on PPAR and adiponectin and a potent activation of AMPK, a positive regulation of PPAR-α expressed by mRNA and a negative regulation of FASN. Experiments in mice indicate an improvement in fat metabolism (cholesterol and triglycerides), especially in the elimination of triglycerides.
    This indicates a complementary effect hitherto unknown of both extracts on fat metabolism and activation of AMPK. This implies that a composition like the one described above has an unexpected and very significant effect on reducing weight of people with obesity or overweight, as explained in the examples.
    The composition may comprise a minimum of 50% by weight of the herb extract (Lippia citriodora) and a minimum of 25% by weight of the rosella extract (Hibiscus sabdariffa).
    Preferably the composition comprises about 65% by weight of the herb extract (Lippia citriodora) and about 35% by weight of the rosella extract (Hibiscus sabdariffa).


    This percentage of both extracts causes the composition to have a greater effect on the activation of fat metabolism mediated by a greater activation of AMPK compared to a composition where the extracts are mixed in equal parts.
    The composition can be administered in a dose of 250 to 1000 milligrams per day.
    Preferably the composition is administered in a dose of 500 mg per day.
    This dose ensures a significant effect on the activation of AMPK that results in an activation of lipid metabolism and consequently a reduction in body fat percentage.
    In a more particular embodiment, the administration of the composition of the present invention is selected from parenteral, transdermal, oral, topical, intracolonic or vaginal.
    In a more particular embodiment, the 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 composition of the present invention is administered intramuscularly, intraperitoneally or intravenously.
    In a more particular embodiment, the 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, normally ranges from 250


    milligram up to 1000 milligrams, preferably at 500 mg of substance to be administered during one or several ingestions.
    A third aspect of the invention provides a method of producing a composition comprising an extract with at least 5% by weight of anthocyanins and an extract with at least 15% by weight of phenylpropanoids characterized in that it comprises a hydroalcoholic herb extract ( Lippia citriodora) and a hydroalcoholic extract of rosella (Hibiscus sabdariffa).
    This method uses two natural sources rich in the active compounds of the composition. The hydroalcoholic extraction of these plants allows obtaining the required asset content with a remarkable and economically viable yield.
    Hydroalcoholic extraction of Hierbaluisa can be carried out using an alcohol-in-water solution with an alcohol content greater than 70% and up to 100% by volume of ethanol for two hours at a temperature between 40 and 80 ° C.
    Preferably the solution of ethanol in water contains about 70% by volume of alcohol.
    This composition of the extraction agent and the extraction conditions maximize the obtaining of the phenylpropanoic compounds, in particular the verbascoside of the leaves of Hierbaluisa in economically viable conditions without having to distill an excess of alcohol or water.
    BRIEF DESCRIPTION OF THE FIGURES
    Figure 1. Graph representing the percentage of triglyceride accumulation in hypertrophic insulin resistant adipocytes.
    Figure 2. Graph representing the percentage of activation of AMPK in hypertrophic insulin resistant adipocytes treated with different concentrations of rosella and lemongrass extracts.
    Figure 3. Graph depicting the percentage of AMPK activation in hypertrophic insulin resistant adipocytes treated with different concentrations of rosella and herb extract extracts, in two different proportions.
    Figure 4. Graphs showing the levels of total cholesterol, LDL cholesterol (bad) and HDL cholesterol (good) in the blood of mice fed a high-fat diet and treated with extracts of lemongrass and rosella, separately, and with two different doses of a mixture of herbs and rosella extracts in proportion
    65:35 by weight respectively, and from mice fed a high-fat diet untreated and from mice fed a normal diet.
    Figure 5. Graph and table representing the ratio of food efficiency in mice fed a high-fat diet and treated with the extracts of


    Hierbaluisa and Rosella, separately, and with two different doses of a mixture of extracts of Hierbaluisa and Rosella and mice fed a high-fat untreated diet and mice fed a normal diet.
    Figure 6. Graph representing the blood glucose level of mice fed a high-fat diet and treated with the extracts of Hierbaluisa and Rosella, separately, and with two different doses of a mixture of extracts of Hierbaluisa and Rosella and of mice fed an untreated high-fat diet and mice fed a normal diet.
    Figure 7. Graph representing the level of leptin in the blood of mice fed a high-fat diet and treated with the extracts of Hierbaluisa and Rosella, separately, and with two different doses of a mixture of extracts of Hierbaluisa and Rosella and mice fed an untreated high-fat diet and mice fed a normal diet.
    Figure 8. Graph representing the level of leptin in the adipose tissue of mice fed a high-fat diet and treated with the extracts of Hierbaluisa and Rosella, separately, and with two different doses of a mixture of extracts of Hierbaluisa and Rosella and of mice fed a high-fat untreated diet and mice fed a normal diet.
    Figure 9. Graph depicting the body fat weight of mice fed a high-fat diet and treated with the extracts of Hierbaluisa and Rosella, separately, and with two different doses of a mixture of Hierbaluisa and Rosella extracts and mice fed on an untreated high-fat diet and mice fed on a normal diet.
    Figures 10a, 10b, 10c and 10d. Graphs that represent metabolic markers of abdominal adipose tissue and liver of mice fed a high-fat diet and treated with extracts of Hierbaluisa and Rosella, separately, and with two different doses of a mixture of extracts of Hierbaluisa and Rosella and of mice fed an untreated high-fat diet and mice fed a normal diet.
    Figure 11. Graph depicting the Western Blot analysis to determine the amount of protein in the adipose tissue of mice fed a high-fat diet and treated with extracts of lemongrass and rosella, separately, and with two different doses of a mixture of extracts of Hierbaluisa and Rosella and of mice fed with a high-fat untreated diet and of mice fed with a normal diet.
    Figure 12. Graphs representing the genes corresponding to the metabolic markers of immortalized, undifferentiated (ND) and differentiated (MDI) human adipocytes, cultured in vitro treated with different doses of Hierbaluisa (LV) and Rosella (HS) extracts. separately, and with different doses of a mixture of herbal extracts and rosella.


    Figure 13. Graphs showing the decrease in the lipid content of hypertrophic adipocytes treated with the composition with rosella and lemongrass extracts and the parallel increase of AMPK activation in said adipocytes.
    Figure 14. Scheme of the production method of an extract of Hierbaluisa.
    Figure 15. Scheme of the method of production of the composition according to an aspect of this invention.
    DETAILED DESCRIPTION OF THE INVENTION AND EXAMPLES. EXAMPLE 1. Determination of the proportion of extracts of Hierbaluisa and Rosella.
    3T3-L1 preadipocytes were grown in 96-well plates and in complete culture medium (DMEM with low glucose at 1g / L) and sodium pyruvate, enriched with glutamine and supplemented with 10% bovine serum and antibiotic (100 µg / mL of streptomycin and 100 U / mL of penicillin).
    Adipocyte differentiation was induced by adding adipogenic agents (0.5 mM IBMX, 1 µM DEX and 1 µM insulin) to the culture medium for two days. The medium was changed to fresh medium every 48 hours.
    The phenotypic change of adipocytes was observed with a microscope. In all experiments, more than 90% of the cells were mature adipocytes after 8-10 days of incubation. To induce cell hypertrophy, the adipocytes were exposed to a medium high in glucose (25 mM).
    Cell cultures were carried out under sterile conditions using a laminar air flow hood for cell cultures and a 37 ° C incubator with a humidified atmosphere with 5% CO2.
    The effect of the extracts and that of the composition with both extracts on triglyceride accumulation and cytoplasmic activation of AMPK in 3T3-L1 adipocytes were studied. Extracts were added after the adipocytes had differentiated until the plates were analyzed. The extracts were previously prepared with the culture medium and filtered with a 0.2 um filter to sterilize them.
    Hypertrophic adipocytes with 29 days of differentiation were exposed to rosella extracts (~ 10% by weight anthocyanins) and herbal extracts (~ 30% by weight phenylpropanoids) separately and to mixtures of 50:50 by weight and 35:65 in weight. The extracts were added on day 24 after adipocyte differentiation and were incubated at 50, 200 and 500 µg / mL for 5 days.
    After these five days, the lipid content of the adipocytes was evaluated with the commercial AdipoRed ™ reagent and using the multi-modal microplate reader with BioTek Cytation 3 cell image capture with an excitation wavelength of 485 nm and emission of 572 nm. The absence of cytotoxicity was evaluated by the violet crystal method. The results are shown in the


    Figure 1, using a graph representing the percentage of triglyceride accumulation in hypertrophic insulin resistant adipocytes. HS represents adipocytes treated with rosella extract with 10% anthocyanins, LV represents adipocytes treated with herb extract with 30% phenylpropanoids, HS-LV (1: 1) represents adipocytes treated with a mixture of extracts of Hierbaluisa and Rosella in proportion one by one in weight and HS-LV
    (35:65) represents the adipocytes treated with a mixture of the herb and rosella extracts in proportion 65 to 35 by weight, respectively. The values of p are: * p <0.05, ** p <0.01 and *** p <0.001.
    AMPK and phosphorylated (activated) AMPK levels were determined by a quantitative immunofluorescence experiment using the AMPK and phosphorylated AMPK antibodies. In addition, the nuclei were labeled with the Hoechst 33342 reagent. Once the cells were labeled, the fluorescence of the AMPK was quantified with an excitation wavelength of 490 nm and emission of 520 nm, that of the phosphorylated AMPK with a length of excitation wave of 593 nm and emission of 614 nm and that of the nuclei with an excitation wavelength of 350 nm and emission of 461 nm, using the multi-modal microplate reader.
    The results are shown in Figures 2 and 3 that show graphs representing percentages of AMPK activation in hypertrophic insulin resistant adipocytes treated with different doses of rosella and herb extract separately and in combination in two different proportions. It can be seen that the greatest activation of AMPK occurs with the highest dose of the composition containing the two extracts in a proportion of 35 to 65 by weight of rosella extract to herb extract, respectively. P values are: * p <0.05, ** p <0.01 and *** p <0.001, which indicate statistically significant differences with control adipocytes (untreated adipocytes). The legends have the same meaning as in Figure 1. EXAMPLE 2. Evaluation of fat reduction, hyperlipidemia, hyperglycemia and adipogenesis in obese mice treated with a composition containing extracts of lemongrass and rosella.
    In vivo experiments were performed on normal mice that were given a high fat diet (HFD) for 8 weeks. Total blood cholesterol levels were analyzed, as well as HDL (good cholesterol or high density lipoprotein) and LDL (bad cholesterol or low density lipoprotein).
    The results are shown in Figure 4 where C57bl / 6-Nr corresponds to mice with normal diet, HFD-CTL to mice with high fat diet (HFD), HFD-LV to mice with high fat diet and taking extract of Hierbaluisa with ~ 30% by weight of phenylpropanoids, HFD-HS to mice with a high-fat diet and taking rosella extract with ~ 10% by weight of anthocyanins, and HFD-MetA to mice taking the composition with both extracts in proportion 65: 35, respectively.


    The results show that the mice taking the composition with both extracts had lower levels of total cholesterol, coinciding with a decrease in LDL and a rise in HDL. These values were lower than with extracts ingested separately (* p <0.05, ** p <0.01 with respect to the HFD-CTL control).
    The amount of food the mice ate per day was analyzed, with respect to the weight of the animal. This value is known as the Food Efficiency Ratio. A lower amount implies a lower intake of food with respect to its weight. The results can be seen in Figure 5. (* p <0.01 with respect to the HFD-CTL control)
    The blood glucose level was also analyzed, since mice with a high-fat diet are hyperglycemic. As can be seen in Figure 6 (* p <0.05 with respect to the HFD-CTL control), the mice that took the highest concentration of the composition with both extracts were the only ones with a significant reduction in blood glucose, close to the levels of mice with normal diet.
    Leptin hormone levels in mice were analyzed by eating a high-fat diet. Specifically, blood hormone levels were analyzed, as can be seen in Figure 7 (* p <0.05, ** p <0.01 with respect to HFD-CTL).
    Comparing the control with normal diet (C57bl / 6J Nr) with respect to the one who is taking a high fat diet (HFD-CTL), we can verify that leptin has a significantly higher expression in the case of the high fat diet. Leptin induces satiety, however, it is known that membrane receptors of said hormone have a high resistance in obese individuals, and require high levels of leptin to be stimulated and therefore activate the cascade of signals that induce satiety . As a consequence, detecting high levels of leptin in mice with a high-fat diet, and consequently obese, confirms such resistance to the hormone.
    Taking any of the extracts or mixtures tested (Hierbaluisa, Rosella, combination or mixture) decreases the expression of leptin in blood, although only in the case of the composition with both extracts can we see values similar to those detected in mice with diet normal, that is, not obese. Therefore, the taking of this composition is capable of reversing this overexpression of leptin, since it cancels the resistance to said hormone, thus allowing mice to have satiety levels similar to normal.
    This data coincides with the results observed with the FER (Food Efficiency Ratio), where the mice that took the composition with both extracts would consume less food with respect to their weight.
    A similar result can be seen in Figure 8 regarding the expression of leptin in adipose tissue, where it can be seen that only in mice that took the composition with both extracts decreased their leptin expression in adipose tissue (* p <0.05 vs HFD-CTL), reaching the levels observed in mice with a normal diet. This is due to a possible sensitization of leptin receptors in mice that took the composition with both


    extracts, so that a lower concentration of leptin in the blood is enough to produce a feeling of fullness.
    The effect of herbal extracts and rosella extracts separately and combined in the composition object of the invention on the metabolism of adipose tissue has been studied. First, the amount of adipose tissue that the mice had was checked, isolating and weighing fat from the abdomen, epididymis, kidney and intestine.
    As can be seen in Figure 9, only mice that took the composition with both extracts had significantly less fat, in the case of abdominal, epididymal, and intestine (* p <0.05, ** p <0, 01). These values are consistent with the results discussed above, that is, that mice eat less food, by regulating satiety, which results in weight loss through loss of adipose tissue.
    In addition to weight, metabolic markers in abdominal adipose tissue and liver were studied (because in the case of overweight / obesity there is an abnormal accumulation of adipose tissue in the liver, called "fatty liver") related to the regulation of accumulation or production of triglycerides and fatty acids. The markers analyzed, by RT-PCR, were PPARγ, SREBP1c (ADD1), C / EBPα and AMPK.
    PPARγ is a nuclear membrane receptor found in adipocytes, as well as colon and macrophage cells. In the case of adipose tissue, its function is to activate lipogenesis and lipid uptake. SREBP1c regulates lipid homeostasis, and its activation induces cholesterol synthesis when it is low. C / EBPα is a transcription factor that regulates both adipogenesis and normal adipocyte function, and regulates the expression of PPARγ. Finally, AMPK (AMP kinase) inhibits lipid uptake, cholesterol synthesis and lipogenesis, among other functions, although to perform its function the protein must be phosphorylated (p-AMPK).
    As can be seen in the histograms of Figures 10a to 10d, the expression of PPARγ and SREBP1c (ADD1) decreases significantly only in the mice that took the composition with both extracts, studied in both the liver and abdominal adipose tissue (* p <0.05). As for the C / EBPα, there is also a diminishing effect on its expression with Hierbaluisa and Rosella, but only in the liver, while the composition with both extracts decreased the expression of this transcription factor in both tissues analyzed. In these three genes, the resulting expression in mice taking the composition with both extracts was similar to that obtained in control mice taking a normal diet.
    On the other hand, AMPK transcriptional expression was increased in mice taking the composition with both extracts (in the liver, mice taking rosella also appreciated an increase in their expression, but not in adipose tissue).
    This result was also confirmed by a Western Blot analysis, which allows analyzing the amount of protein present in adipose tissue and whose results are


    shown in Figure 11. The legend is interpreted as: N mice with normal diet, CT mice with high fat diet untreated, LV mice with high diet oils treated with lemongrass extract, HS mice with high diet oils treated with extract Rosella, MA1 mice with high diet oils treated with 35:65 mixture of extracts of rosella and lemongrass at a dose of 50 mg / kg and MA2 mice with high diet oils treated with 35:65 mixture of extracts of rosella and lemongrass at a dose 100 mg / kg
    The active form of AMPK is when it is phosphorylated (p-AMPK). As can be seen in the figure, the adipose tissue of the mice taking the composition with both extracts were those with the highest concentration of p-AMPK in the form of active protein.
    Therefore, mice taking a high-fat diet for 8 weeks (HFD-CTL) have an activation of genes involved in fat accumulation, lipogenesis, triglyceride and cholesterol synthesis, and adipogenesis, which results in a higher Body fat content, compared to control mice with a normal diet (C57bl / 6J Nr). However, when these mice take the composition with both extracts, the expression of these genes is reversed, towards concentrations similar to those observed in non-obese mice with normal diet. In addition, the activation of p-AMPK indicates that it increases the metabolism of fatty acids, decreasing their storage while increasing their degradation.
    These in vivo results in mice were corroborated with in vitro studies using a human adipocyte cell line, where different concentrations of the various ingredients were tested and cultured for 10 days. The same genes as those studied in mice for fatty acid metabolism were analyzed. The results are shown in Figure 12. (ND: undifferentiated cell cultures, MDI: differentiated cell cultures, LV: lemongrass, HB: rosella, MA: composition with both extracts, 125-1000: ingredient concentrations, expressed in µg / ml).
    In the cases of PPARγ, SREBP1c (ADD1), C / EBPα, a decrease in its expression is observed in the three cases studied with respect to differentiated adipocytes (MDI), specifically in the concentration of 1 mg / ml of lemongrass, of rosella, and both 0.5 and 1 mg / ml of the composition with both extracts. Therefore, the composition with both extracts can be used at lower concentrations to induce a repressive effect of said transcripts, compared with the extracts individually.
    As for AMPK, a significant increase in expression can be observed in the case of the composition with both extracts at 0.5 and 1 mg / ml, while with the extracts separately, no significant differences are observed with respect to the differentiated cell culture (MDI).
    EXAMPLE 3. Reduction of appetite and / or increase in satiety by changes in peptides secreted by the digestive tract and adipose tissue induced by consumption of composition containing an extract of Hierbaluisa with a content of 30% by weight of phenylpropanoids and a

    Rosella extract with a content of 10% by weight of anthocyanins, in a proportion of 65 to 35 by weight.
    54 women aged between 30 and 75 years were selected with a body mass index between 25 to 35 kg / m2. The study is a randomized, double-blind, 8-week, placebo-controlled trial conducted at the Miguel Hernández de Elche University in Spain. After enlistment, the volunteers were randomly assigned to the placebo control group L2 (n = 26) or the experimental group L1 (n = 28).
    Members of the L1 group (mean age 51 years) received a daily dose of 500 mg of a composition containing herb extract and rosella extract.
    Members of the L2 group (mean age 51 years) received a daily dose of 500 mg of microcrystalline cellulose.
    The placebo capsules and the satiating effect composition had the same size, smell, color and weight. The volunteers took one capsule 20 or 30 minutes before breakfast every day for two months.
    Volunteers were required to follow a balanced isocaloric diet and walk for at least 30 minutes a day.
    Compliance with these requirements was monitored during clinical visits or by telephone interviews every week. A total of 3 and 4 participants were excluded from groups L2 and L1 respectively.
    Several parameters were monitored during the study. Anthropometric measurements were taken at the start of the study, after one month and after two months from the start of the study, which include body weight, height, measurement of the triceps, biceps and abdominal skin folds and measurement of the arm and arm perimeter abdomen. The perimeter of the abdomen (AC) was measured at two different sites: ahead midway between the xiphoid bulge of the sternum and the navel and laterally midway between the end of the ribcage and the iliac crests (AC1) and at the height of the navel (AC2). The percentage of body fat was obtained from abdominal measurements using the Weltman equation. In addition, systolic and diastolic blood pressure and resting heart rate were also measured at baseline, after one month and after two months of study.
    The validated visual analogue scale (VAS test) was used to record hunger, satiety, the feeling of filling, the consumption of prospective food, the desire to eat something fatty, salty, sweet or not sweet and the appreciation of the taste of meals . The VAS test was completed at the beginning of the study at rest and after 15, 20, 45 and 60 days of study. In addition to the subjective assessment of the state of health, the volunteers filled out an SF-36 questionnaire at the beginning and at the end of the study. Finally, blood samples were obtained from the vein of the forearm after an overnight fast at the beginning, after 30 and after 60 days of study. The plasma was processed immediately and the samples were stored at -80 degrees Celsius.


    Table 1 shows the anthropometric parameters at the beginning and during the study. No significant differences are observed at the beginning of the study between both groups. The results in Table 1 indicate a statistically significant improvement of the anthropometric parameters after two months of study in the group (L1) that took the
    5 composition with extracts of lemongrass and rosella, especially thebody fat percentage, triceps skin fold, body weight andhip perimeter (AC2). These differences can be observed with moreclarity in table 1a.
    L2 Group Group L1
    Anthropometric parameters StartMonth 1Month 2StartMonth 1Month 2
    Body weight (kg) 75.64 ± 12.9274.20 ± 12.62 ****73.56 ± 12.57 ****75.26 ± 9.0672.80 ± 9.45 ****71.78 ± 9.06 ****
    Body mass index (kg / m2) 29.78 ± 4.1929.15 ± 4.08 ****28.95 ± 4.01 ****29.60 ± 3.4028.60 ± 3.52 ****28.26 ± 3.46 ****
    Arm perimeter (cm) 31.20 ± 3.8031.23 ± 3.8030.98 ± 4.1030.58 ± 1.6730.47 ± 1.8330.26 ± 1.76 ****
    AC1 (cm) 94.02 ± 13.0392.84 ± 12.86 ***92.05 ± 13.16 ****90.96 ± 9.0388.96 ± 9.18 ****88.01 ± 8.90 ****
    AC2 (cm) 100.7 ± 14.01100.5 ± 14.2199.90 ± 14.54 **96.42 ± 7.9394.84 ± 7.87 ****93.85 ± 7.95 ****
    Hip perimeter (cm) 108.8 ± 8.73108.15 ± 8.55 **107.5 ± 8.48 ***110.4 ± 7.23108.2 ± 7.56 ****106.9 ± 7.48 ****
    Cutaneous triceps fold (mm) 43.25 ± 9.2843.15 ± 9.3443.10 ± 9.4241.62 ± 8.1840.96 ± 8-08 **39.98 ± 7.97 ****
    Cutaneous bicep fold (mm) 41.53 ± 14.4641.25 ± 14.35 *41.09 ± 14.35 *38.33 ± 10.6337.61 ± 10.87 *36.87 ± 10.52 ****
    Abdominal skin fold (mm) 35.72 ± 11.1535.39 ± 10.6834.39 ± 10.6841.45 ± 12.8939.72 ± 12.95 ***38.60 ± 13.08 ***
    % body fat 44.98 ± 2.7144.69 ± 2.65 ***44.53 ± 2.64 ****44.66 ± 2.0444.10 ± 2.10 ****43.83 ± 2.06 ****
    Vital signs
    Heart rate (bpm) 71.41 ± 8.8971.95 ± 8.8572.09 ± 9.2473.32 ± 9.7070.84 ± 7.54 *68.64 ± 7.07 ****
    Systolic Tension (mmHg) 114.5 ± 23.26115.1 ± 23.55 *115.2 ± 23.13 *117.4 ± 12.13115.1 ± 12.82 **113.9 ± 12.5 ****
    Diastolic Tension (mmHg) 73.73 ± 10.5774.05 ± 11.5073.73 ± 10.7073.40 ± 5.0071.28 ± 5.89 ****69.48 ± 6.62 ****
    Table 1. Changes in anthropometric parameters during the study expressed in mean values plus / minus standard deviations. The intragroup statistical analysis was determined in each period compared to the data at the beginning of the
    20 study. The significance was established at: * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001.


    Differences after 1 month Differences after 2 months
    Anthropometric parameters Placebo (L2, n = 22)MetabolAid® (L1, n = 25)Placebo (L2, n = 22)MetabolAid® (L1, n = 25)
    Body weight (kg) -1.44 ± 0.27-2.46 ± 0.28 *-2.08 ± 0.30-3.48 ± 0.40 *
    Body mass index (kg / m2) -0.63 ± 0.14-1.00 ± 0.15 *-0.83 ± 0.12-1.37 ± 0.16 *
    Arm perimeter (cm) 0.03 ± 0.03-0.11 ± 0.06-0.22 ± 0.23-0.32 ± 0.08 **
    AC1 (cm) 1.18 ± 0.29-2.00 ± 0.27 *-1.97 ± 0.37-2.95 ± 0.36
    AC2 (cm) 0.20 ± 0.54-1.58 ± 0.25 **-0.80 ± 0.55-2.57 ± 0.34 **
    Hip perimeter (cm) 0.65 ± 0.23-2.20 ± 0.32 ***-1.30 ± 0.28-3.50 ± 0.37 ****
    Cutaneous triceps fold (mm) -0.10 ± 0.16-0.66 ± 0.18 *-0.15 ± 0.15-1.64 ± 0.29 ****
    Cutaneous bicep fold (mm) -0.28 ± 0.14-0.62 ± 0.22-0.44 ± 0.19-1.46 ± 0.29 **
    Abdominal skin fold (mm) -0.33 ± 0.36-1.73 ± 0.37 *-1.33 ± 0.29-2.84 ± 0.53 *
    % Body fat 0.29 ± 0.06-0.56 ± 0.06 **-0.45 ± 0.07-0.83 ± 0.08 ***
    Cardiac pulse (bpm) 0.54 ± 0.28-2.48 ± 0.89 ****0.68 ± 0.36-4.68 ± 0.36 ****
    Systolic pressure (mmHg) 0.60 ± 0.28-2.30 ± 0.64 ****0.70 ± 0.30-3.50 ± 0.61 ****
    Diastolic Pressure (mmHg) 0.32 ± 0.43-2.12 ± 0.39 ****0 ± 0.50-3.92 ± 0.63 ****
    Table 1a. Differences between the anthropometric parameters during the study with the initial values in mean values plus / minus standard deviations. The analysis
    5 intra-group statistic was determined in each period compared to the data at the start of the study. The significance was established at: * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001.
    10 According to the scores shown in Table 2, the consumption of the composition with the extracts of Hierbaluisa and Rosella decreases the sensation of hunger consistently throughout the two months of study. The average hunger score decreased from 5.92 on day 15 to 2.58 on day 60 in group L1, while in group L2 it increased from 6.18 on day 15 to 6.41 on day 60 in
    15 the placebo group. A similar trend is observed in the scores of question 4.
    Satiety increased from a value of 5.04 on day 15 to a value of 7.58 on day 60 in group L1, while it decreased from a value of 4.82 on day
    20 15 to a value of 4.22 on day 60 in the L2 placebo group. A similar trend was observed in the score of question 3.


    15 days 30 days45 days60 days
    Items L1L2L1L2L1L2L1L2
    1. How hungry are you 5.92 ± 2.486.18 ± 2.364.00 ± 2.34 **6.05 ± 1.733.29 ± 2.37 ****6.18 ± 2.112.58 ± 2.35 ****6.41 ± 1.87
    2. How satiated do you feel 5.04 ± 2.074.82 ± 1.895.92 ± 2.02 *4.32 ± 1.956.45 ± 2.32 **4.63 ± 1.507.58 ± 1.72 ****4.22 ± 1.38
    3. How full do you feel 4.65 ± 1.944.45 ± 1.765.76 ± 1.80 *4.30 ± 1.906.42 ± 1.87 ****3.86 ± 1.837.46 ± 1.59 ****3.52 ± 1.91
    4. How much do you think you can eat 5.57 ± 2.837.22 ± 1.524.14 ± 1.98 ****6.95 ± 1.902.83 ± 2.20 ****6.82 ± 1.742.54 ± 1.90 ****7.33 ± 1.90
    5. Would you like to eat something sweet 2.04 ± 2.232.45 ± 2.705.65 ± 2.55 **2.80 ± 2.764.83 ± 2.73 ***1.95 ± 2.136.67 ± 2.18 ****1.82 ± 2.17
    6. Would you like to eat something salty 5.45 ± 3.425.09 ± 3.536.38 ± 2.73 ****2.60 ± 1.797.57 ± 2.27 ****2.86 ± 2.597.75 ± 2.19 ****3.09 ± 2.72
    7. Would you like to eat something not sweet 2.70 ± 2.25 *4.68 ± 2.934.96 ± 2.513.68 ± 2.736.50 ± 2.19 ****2.90 ± 2.757.67 ± 1.40 ****2.71 ± 2.47
    8. Would you like to eat something fatty 4.29 ± 3.58 ** *8.07 ± 2.526.87 ± 3.185.60 ± 3.588.13 ± 2.71 **5.18 ± 3.888.21 ± 2.47 **5.14 ± 3.48
    Table 2. Markers of appetite-related sensations in group L1 (composition) and group L2 (placebo) after 15, 30, 45 and 60 days of study,
    5 expressed as mean values plus / minus standard deviations. The data express the markers of each question compared to placebo. Statistically significant differences were established as: * p, 0.05, ** p <0.01, *** p <0.001, **** p <0.0001. The score varies according to the following scheme:
    10 1. 0 "I'm not hungry" - 10 "I've never been hungry".
    2. 0 “I am completely empty” - 10 “I can't take another bite”
    3. 0 “I am not full of anything” - 10 “I am completely full”
    Four. 0 “Nothing at all” - 10 “A lot”
    5. 0 “Yes, a lot” - 10 “No, not at all”
    15 6. 0 “Yes, a lot” - 10- “No, not at all”
    7. 0 “Yes, a lot” - 10- “No, not at all”
    8. 0 “Yes, a lot” - 10- “No, not at all”


    Placebo (L2, n = 10) Composition with extracts (L1, n = 10)
    START MONTH 2 STARTMONTH 2
    GLP-1 (pg / mL) 5.66 ± 1.274,228 ± 2.04 *5.22 ± 1.436.82 ± 1.78 *
    GHRELINA (pg / mL) 30.64 ± 0.6433.74 ± 3.48 *32.70 ± 4.8232.89 ± 3.23
    LEPTINE (pg / mL) 9669 ± 66175237 ± 48869772 ± 44554311 ± 2304 *
    PYY (pg / mL) 7.84 ± 4.155.82 ± 5.019.80 ± 0.319.81 ± 0.24
    RESISTINE (pg / mL) 15593 ± 671810799 ± 662418101 ± 627412049 ± 4022 *
    Table 3. Blood peptide levels segregated by the digestive tract.
    5 related to appetite and satiety determined at the beginning and end of the study (second month), expressed as mean values plus / minus standard deviations. Significance was established for: * p <0.05.
    It is necessary to mention that some peptides analyzed (such as GLP-1, PYY and
    10 ghrelin) are secreted after food intake. Ghrelin stimulates appetite, while GLP-1 and PYY are secreted together to cause satiety. Because blood samples were taken on an empty stomach in the morning, the determined levels of these peptides in particular correspond to baseline levels. Initially, the basal levels of both groups coincide and
    15 there are no significant differences between them. The other peptides have significant differences in the L1 group. In this context, incretin GLP-1 increased significantly in the L1 group while it decreased in the L2 group and ghrelin increased in the L2 group and remained constant in the L1 group. Therefore, the L1 group volunteers had less appetite and more feeling of
    20 satiety than the volunteers of the L2 group.
    On the other hand, leptin, which is synthesized in adipose tissue and produces a feeling of satiety, decreased significantly in the L1 group while no changes were seen in the L2 group. The resistin decreased significantly in the L1 group while no changes were observed in the L2 group. The high level of leptin in blood in the L2 group is explained by the resistance created in the corresponding receptors to cause satiety in response to the secretion of leptin, it is as if the corresponding receptors had become numb, while in the L1 group the level Normal blood leptin indicates that
    30 corresponding receptors remain with the same or greater sensitivity at the leptin level.
    In general, these changes in hormone levels (peptides) correlate positively with the scores of the appetite and satiety questionnaires and
    35 with the evolution of the anthropometric parameters shown above.

    EXAMPLE 4. Reduction of body weight through a modification in lipid metabolism mediated by an activation of AMP-activated protein kinase energy sensors (AMPK).
    Triglyceride accumulation and activation of AMPK by polyphenols was evaluated by immunofluorescence with the hypertrophic insulin resistant 3T3-L1 adipocyte model. On the other hand, a clinical study was conducted with volunteers taking a placebo or 500 mg a day of a dietary supplement with extracts of Hierbaluisa and Rosella in a proportion 65:35 respectively, double blind with placebo and randomized control in which 56 volunteers participated Overweight for two months. Anthropometric parameters and biochemical parameters were determined.
    3T3-L1 preadipocytes were cultured in a low-glucose medium (1g / L) DMEM supplemented with 10% bovine serum (CS), 100 µg / mL streptomycin and 100 U / mL penicillin and were incubated at 37 ° C in a humidified atmosphere with 5% CO2 and 95% air by volume. Adipocyte differentiation was induced by culture in a medium high in glucose (4.5g / L) DMEM supplemented with 10% fetal bovine serum (FBS), 1 µM insulin, 1 µM dexamethasone (DEX) and
    0.5 mM of 3 isobutyl-1-methylxanthine (IBMX) for 48 hours. After 48 hours, the cells were maintained in a medium high in glucose with FBS and insulin, and the medium was changed every two days, whereby hypertrophied adipocytes were obtained after 20 days of incubation. Once the hypertrophied adipocytes were obtained, the cells were treated with extracts of Hierbaluisa (~ 30% by weight of phenylrpopanoids) and Rosella (~ 10% by weight of anthocyanins) with a proportion by weight of 65 to 36, at various concentrations for 72 hours The extracts were dissolved in a medium and filtered to be sterilized.
    The lipid content of hypertrophic adipocytes was evaluated using the AdipoRed ™ reagent. The supernatant was removed from the cells and these were carefully washed with phosphate buffered saline (PBS). Next, AdipoRed ™ was added and the cells were allowed to incubate for 15 at room temperature. Triglyceride accumulation was measured using a microplate reader at 485 nm excitation wavelength and at 572 nm emission.
    For the study of AMPK activation in hypertrophied adipocytes, phosphorylated AMPK was quantified in Thr172 (pAMPK) with an immunofluorescence assay. The cells were fixed with a fixation buffer, permeabilized with 0.3% Triton x-100 (Sigma-Aldrich, Spain) and blocked with 4% goat serum. Once this was done, the cells were incubated overnight at 4 ° C with AMPK mouse monoclonal antibodies alpha 1 + alpha 2 (Abcam, Cambridge, UK) and with phospho-AMPK alpha 1 rabbit (Thr172; Cell Signaling) Technology, Danvers, MA, USA). After incubation with the primary antibodies, the cells were washed with PBS and incubated for 6 hours at room temperature with each corresponding secondary polyclonal antibody, goat anti-rabbit IgG CF ™ 594 and anti-mouse FITC (both from Sigma-Aldrich, St. Louis, MO, USA). Cell fluorescence was measured using a multi microplate reader


    mode with cellular image capture (Cytation 3, Biotek, Spain) at 593 nm excitation wavelength and 614 nm emission to measure AMPK levels. AMPK activation is expressed as the ratio of AMPK levels normalized by total AMPK. The results are shown in Figure 13 (* p <0.05, *** p <0.001).
    The human study lasted eight weeks, randomized, double blind and placebo controlled. The exclusion criteria were a total cholesterol level of less than 200 mg / dL, the presence of any pathology related to obesity, the use of medication for cholesterol or hypertension, the consumption of antioxidant supplements or pharmaceutical products, alcohol addiction and pregnant or nursing women. Based on these criteria, 55 healthy women were selected, aged between 36 and 69 years, with a body mass index of 25 to 34 kg / m2, who passed a selection and telephone interview about their health as well as a biochemical and anthropometric evaluation.
    Once enrolled in the study, the volunteers were randomly assigned to the placebo group (n = 26) or the experimental group (n = 29). During the study, 9 volunteers withdrew, 6 in the placebo group and 3 in the experimental group, and a total of 46 volunteers completed the study. The placebo group (mean age 51 years) received two placebo capsules with 400 mg of microcrystalline cellulose each and the experimental group (average age 52) received two capsules each containing 250 mg of the composition with extracts of rosella and lemongrass and 150 mg of excipients (microcrystalline cellulose). Therefore, the experimental group took 500 mg / day of the composition under study. Both types of capsules had the same size, smell, color and weight. The volunteers took two capsules every day 20 or 30 minutes before breakfast for two months. During the selection visit, demographic and lifestyle information (age, diet, physical exercise patterns, alcohol and tobacco consumption) was collected. A doctor asked the volunteer women to follow an isocaloric diet with normal hydration and walk at least 30 minutes a day. Compliance with the study requirements was verified at each visit and by phone, every week, during the two months of study. The measurements of the parameters were taken at the beginning of the study and after 30 and 60 days of study.
    Several parameters were monitored during the study. Anthropometric measurements were taken at the start of the study, after one month and after two months from the start of the study, which include body weight, height, measurement of the triceps, biceps and abdominal skin folds and measurement of the arm and arm perimeter abdomen. The perimeter of the abdomen (AC) was measured at two different sites: ahead midway between the xiphoid bulge of the sternum and the navel and laterally midway between the end of the ribcage and the iliac crests (AC1) and at height of the navel (AC2). The percentage of body fat was obtained from abdominal measurements using the Weltman equation.
    Fasting blood samples were taken to measure total glucose, glycosylated hemoglobin and lipid profile, which includes triglycerides, total cholesterol, good cholesterol (high density lipoprotein) or HDL and bad cholesterol (low lipoprotein density) or LDL. Blood samples were analyzed to measure safety parameters such as hematology,


    electrolytes, creatinine, urea, uric acid, glutamic-pyruvic transaminase, glutamic-oxaloacetic transaminase and C-reactive protein.
    In addition, systolic and diastolic blood pressure and resting heart rate were also measured at baseline, after one month and after two months of study.
    Experimental group (N = 26) Placebo group (N = 20)
    Start Month 1Month StartMonth 1Month 2
    Parameters
    Anthropometric Body Weight 75.12 ±72.76 ±71.08 ±75.16 ± 11.3473.36 ±73.12 ± 11.47 **
    13.62 13.51 ***13.34 ***11.24 ***
    Fat Index 29.20 ±28.26 ±27.59 ±30.23 ± 4.4129.52 ±29.43 ± 4.58 **
    Bodily 4.764.63 ***4.44 ***4.42 ***
    AC1 (cm) 90.87 ±86.59 ±84.39 ±90.93 ± 13.1289.21 ±88.50 ± 11.65 **
    10.81 9.51 ***9.91 ***12.83 ***
    AC2 (cm) 100.5297.10 ±94.71 ±102.32 ± 11.5999.87 ±97.05 ± 10.66 ***
    ± 9.10 8.20 ***8.94 ***11.96 **
    Fold thickness 28.36 ±26.08 ±24.67 ±30.13 ± 5.2428.45 ±27.88 ± 4.69 ***
    triceps 7.576.95 ***7.42 ***4.75 **
    % Body fat 44.36 ±43.53 ±43.04 ±44.67 ± 2.8244.18 ±43.95 ± 2.73 ***
    2.86 2.73 ***2.64 ***2.83 ***
    Vital signs
    Heart rate
    (inch per minute) 79.43 ±74.36 ±71 ±75.75 ± 8.6275.15 ±75.50 ± 11.61
    9.84 9.03 ***8.40 ***11.86
    128.70 ± 118.43 ±109.96 ±126.65 ± 19.06120.70 ± 17.50118.05 ± 15.64 *
    Systolic tension H 12.7413.91 ***9.09 ***
    77.62 ± 72.76 ±67.05 ±78.30 ± 13.1276.10 ± 8.1671.10 ± 11.61 *
    Diastolic tension H 10.9910.54 *9.30 ***
    Parameters
    biochemists
    93.33 ± 16.10 90.44 ± 14.1190.06 ±93.95 ± 12.9694.05 ± 11.1492.63 ± 14.95
    Glucose mg / dl 13.31
    84.83 ± 52.61 76.28 ± 35.2283.17 ±85.63 ± 42.3778.53 ± 27.9588.74 ± 39.23
    Triglycerides mg / dl 39.39
    237.78 ± 215.22 ±207.94 ±229.11 ± 26.13205.53 ±207.53 ± 28.62 **
    Total cholesterol 26.5126.90 *** 25.11 ***24.84 ***
    / dl 60.59 ± 7.2459.23 ± 7.1558.95 ±59.00 ± 7.5456.95 ± 9.02 *57.11 ± 8.58 *
    HDL mg / dl 8.52
    158.52 ± 140.90 ±127.43 ±153.68 ± 24.88132.89 ±132.95 ± 25.39 ***
    LDL mg / dl 25.1723.39 *** 20.83 ***23.18 ***
    0.75 ± 0.11 0.85 ± 0.140.81 ±0.80 ± 0.140.91 ± 0.180.82 ± 0.12
    Creatinine mg / dl 0.12
    33.36 ± 7.96 32.09 ± 8.5232.32 ±32.21 ± 10.5432.37 ± 7.7731.58 ± 7.44
    Urea mg / dl 9.15
    Uric acid mg / dl 4.66 ± 1.014.29 ± 1.094.55 ± 1.184.97 ± 1.534.29 ± 1.034.48 ± 0.96
    22.59 ± 7.39 21.59 ± 9.5321 ± 8.8321.21 ± 6.8221.61 ± 9.6220.05 ± 8.44
    GPT U / I 21.00 ± 5.07 21.59 ± 5.66 21.27 ± 21.11 ± 4.53 21.11 ± 5.51 20.21 ± 5.02
    GOT U / I
    6.85
    Table 4. Anthropometric measurements, initial and after one and two months of study. Intra-group statistical values at the end of the study compared to the values


    initials are: * p <0.05; ** p <0.01; *** p <0.001; **** p <0.0001. The data is expressed as mean values ± DS.
    As can be seen in Figure 13, the composition with extracts of Hierbaluisa and Rosella causes a potent increase in the ratio of pAMPK / AMPK in a dose dependent manner that is statistically significant compared to the increase observed in the experiment of control at a dose of 350 µg / mL, while at a dose of 500 µg / mL an increase of approximately 1.5 is observed.
    The anthropometric parameters of both groups do not show appreciable differences at the beginning of the study. The results of the study show a significant improvement in the experimental group compared to the placebo group, after two months of study, particularly in body weight, abdominal circumference and body fat percentage (see table 4).
    The experimental group showed a greater reduction in body weight than the placebo group that is statistically significant with a p <0.01. Both abdominal perimeters AC1 and AC2 were reduced after two months of study, but only the highest abdominal perimeter showed statistically significant differences with the placebo group after one and two months of study. In line with these data, the percentage of body fat decreased in both groups, but in the experimental group it was significantly reduced more than in the placebo group after two months of study, p <0.01. Statistically significant differences were also observed in the measurements of the triceps skin folds (p <0.05) and in the body mass index (p <0.01). EXAMPLE 5. Method of production of a composition with extracts of Hierbaluisa and Rosella according to an aspect of the invention.
    The composition with herbs and rosella extracts is a mixture of two extracts with a very specific active compound composition, that is, at least 10% by weight of anthocyanins in the rosella extract and at least 30% by weight of phenylpropanoids in the extract of Hierbaluisa, which mixed in a weight ratio of 35 to 65, respectively, offer synergistic activity, especially for satiety control.
    The manufacturing process of the extract of Hierbaluisa comprises a hydroalcoholic extraction using alcohol concentrations (ethanol, methanol, propanol, isopropanol or butanol) above 70 and up to 100% by volume in water (Figure 14).
    Hierbaluisa leaves (Lippia citriodora) are analyzed to determine the amount of verbascoside using high pressure liquid chromatography (HPLC). Approximately 330 g of lemongrass leaves are extracted in 4 liters of ethanol diluted in demineralized water (greater than 70% and up to 100% ethanol by volume) maintaining agitation of the liquid in the container, recirculating the evaporated solvent or perching it in nutcha filter at a temperature of 40 to 80 ° C for 2 hours. After two hours, the solution is filtered to separate the leaves and


    other particles The filtered solution is distilled under vacuum at a temperature of 60 to 80 ° C to obtain a concentrated aqueous solution containing 15 to 30% solids. This concentrated solution is spray-dried at a temperature of 70-90 ° C to obtain a dry powder extract containing not less than 30% by weight of phenylpropanoids in total. The yield is 19 to 21% by weight.
    To produce the rosella extract, the flowers of the plant are selected and analyzed to obtain the anthocyanin content by high pressure liquid chromatography (HPLC) and ultraviolet light detection. 320 grams of flowers are taken and extracted with 3.2 liters of ethanol diluted in 50% demineralized water by volume, maintaining the agitation of the liquid in the reactor, and recirculating the evaporated solvent or percolating in a nucha filter at a temperature between 40 and 60 ° C for 2 hours. After two hours of extraction, the solution is filtered with a 0.5 micron pore filter to separate the flowers. The filtered solution is distilled under vacuum at a temperature between 50 and 70 ° C and the concentrate obtained is diluted to 620 liters.
    The product is subjected to a purification step by chromatography on a polymeric adsorption / desorption resin suitable for polyphenols. The eluate constitutes the purified extract in liquid form. The purified liquid is concentrated to a content of 30 to 50% by weight solids. The concentrated solution is spray-dried at a temperature between 70 and 80 ° C to obtain a dry powder extract containing no less than 10% anthocyanins. The yield is 5 to 7% by weight.
    After the production of luisa and rosella herb extracts separately, both are screened and mixed and pass through a metal detection system before being packaged, as shown in the scheme of Figure 15.

    权利要求:
    Claims (21)
    [1]
    one. Composition for use in modulating the food intake of an individual by reducing appetite and / or increasing satiety through a modification of the levels of peptides related to appetite, characterized in that said composition comprises an extract with at minus 5% by weight of anthocyanins and an extract with at least 15% by weight of phenylpropanoids.
    [2]
    2. Composition according to claim 1 wherein the extract with at least 5% by weight of anthocyanins comprises an extract with about 10% by weight of anthocyanins.
    [3]
    3. Composition according to claim 1 or 2 wherein the extract with at least 15% by weight of phenylpropanoids comprises an extract with about 30% of phenylpropanoids.
    [4]
    Four. Composition according to any preceding claim in which the extract with anthocyanins comprises a rosella extract (Hibiscus sabdariffa).
    [5]
    5. Composition according to any preceding claim in which the extract with phenylpropanoids comprises an extract of Hierbaluisa (Lippia citriodora).
    [6]
    6. Composition according to claim 5 when it depends on claim 4, wherein the composition comprises a minimum of 50% by weight of the herb extract (Lippia citriodora) and a minimum of 25% by weight of the rosella extract (Hibiscus sabdariffa).
    [7]
    7. Composition according to claim 6, wherein the composition comprises about 65% by weight of the herb extract (Lippia citriodora) and about 35% by weight of the rosella extract (Hibiscus sabdariffa)
    [8]
    8. Composition according to claim 7 administered in a dose of 250 to 1000 mg daily.
    [9]
    9. Composition according to claim 8 wherein the dose administered is 500 milligrams daily.

    [10]
    10. Composition for use in reducing the weight of an individual through a modification in lipid metabolism mediated by an activation of the AMP-activated protein kinase (AMPK) energy sensors of the individual which results in a reduction in the percentage of body fat, characterized in that said composition comprises an extract with at least 5% by weight of anthocyanins and an extract with at least 15% by weight of phenylpropanoids.
    [11]
    11. Composition according to claim 10 wherein the extract with at least 5% by weight of anthocyanins comprises an extract with about 10% by weight of anthocyanins.
    [12]
    12. Composition according to claim 10 or 11 wherein the extract with at least 15% by weight of phenylpropanoids comprises an extract with about 30% of phenylpropanoids.
    [13]
    13. Composition according to any claim from 10 to 12 wherein the extract with anthocyanins comprises a rosella extract (Hibiscus sabdariffa).
    [14]
    14. Composition according to any claim from 10 to 13 wherein the extract with phenylpropanoids comprises a herb extract (Lippia citriodora).
    [15]
    15. Composition according to claim 14 when it depends on claim 13 wherein the composition comprises a minimum of 50% by weight of the herb extract (Lippia citriodora) and a minimum of 25% by weight of the rosella extract (Hibiscus sabdariffa) .
    [16]
    16. Composition according to claim 15 wherein the composition comprises about 65% by weight of the herb extract (Lippia citriodora) and about 35% by weight of the rosella extract (Hibiscus sabdariffa)
    [17]
    17. Composition according to claim 16 administered in a dose of 250 to 1000 mg daily.
    [18]
    18. Composition according to claim 17 wherein the dose administered is 500 milligrams daily.

    [19]
    19. Method of production of a composition comprising an extract with at least 5% by weight of anthocyanins and an extract with at least 15% by weight of phenylpropanoids characterized in that it comprises a hydroalcoholic herb extract (Lippia citriodora) and an extraction
    5 hydroalcoholic rosella (Hibiscus sabdariffa).
    [20]
    20. A method according to claim 19 wherein the hydroalcoholic extraction of lemongrass is carried out using an alcohol solution in water with an alcohol content greater than 70% and up to 100% by volume during
    two hours at a temperature between 40 and 80 ° C.
    [21]
    21. A method according to claim 20 wherein the alcohol content is about 70% by volume.

    Figure 1
    Figure 2

    Figure 3
    Figure 4
    Figure 5

    Figure 6
    Figure 7
    Figure 8

    Figure 9
    Figure 10a

    Figure 10b
    Figure 10c

    Figure 10d
    Figure 11
    Figure 12

    Figure 13
    Figure 14

    Figure 15
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    US20200268823A1|2020-08-27|
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    ES2661579B2|2018-10-25|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2013004856A1|2011-07-01|2013-01-10|Monteloeder, S.L.|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|
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