IT202100028598A1 - Association of Oleuropein, Berberine and Florizin for the treatment and prevention of metabolic syndrome - Google Patents

Description

TITLE: ?Association of Oleuropein, Berberine and Florizin for the treatment and prevention of metabolic syndrome?

DESCRIPTION

FIELD OF INVENTION

The present invention concerns an association for use in the prevention and treatment of metabolic syndrome based on Oleuropein, Berberine and Florizin.

STATE OF ART

Metabolic Syndrome? one of the incipient pathologies by incidence throughout the world, mainly in the West. The nutraceutical approach to this syndrome, in order to delay the use of pharmacological therapy as much as possible. possible, ? one of the most important research areas? promising at this juncture.

Various substances have in fact shown clinical efficacy in the control of the focal physiological parameters for the development of the syndrome and for its degeneration in diabetic pathology which are: blood triglyceride levels, blood glucose levels, blood lipid levels, abdominal circumference, body weight.

The definition of ?Metabolic Syndrome? refers to a pool of factors and conditions that can lead to the development of pathologies, mainly but not exclusively in the cardiovascular area.

Since 1998, the year in which the WHO (World Health Organization) recognized and defined the syndrome for the first time, many definitions have been given, and many different parameters have been used to diagnose the syndrome. However, the definition most? known and applied in clinical practice? that of the 2010 National Cholesterol Education Program Adult Treatment Panel (ATP) III (see Table 1).

Table 1

From a health risk perspective, a person diagnosed with metabolic syndrome has a two-fold greater risk of developing heart disease and a five-fold greater risk of developing diabetes.

The determining factors for the diagnosis of metabolic syndrome are: presence of abdominal adipose tissue with an abdominal circumference greater than 94 cm in men and 80 cm in women; obesity? (BMI>30); low blood HDL cholesterol levels (less than 40 mg/dl in men and less than 50 mg/dl in women); hypertriglyceridemia (above 250 mg/dl); hypertension (greater than 140 sys., greater than 90 diast.); high blood sugar levels (fasting above 100 mg/dl).

In the presence of at least three of these risk factors, metabolic syndrome is full-blown, and brings with it? a long list of increased risks of developing pathologies (Figure 1). The more and more obvious correlation between obesity? and metabolic syndrome, and in particular with diabetes, gave rise to the neologism Diabesity.

Berberine

Berberis aristata, also known as Indian barberry, is a shrub belonging to the Berberidaceae family, of the Berberis genus, native to the northern Himalayan region. The plant ? widely distributed from the Himalayas to Sri Lanka, Bhutan and the hilly areas of Nepal. It grows at a height of 2000-3000 m and can be grown there. found in some mountain ranges in South India.

Berberine? a plant quaternary ammonium salt, belonging to the group of isoquinoline alkaloids (2,3-methylenedioxy.9,10-dimethoxyprotoberberin chloride). ? characterized by a yellow color and an intense flavour. It has several other pharmacological effects including antimicrobial, antidiabetic, anticancer effects. Its chemical structure has a quaternary basis and is commercially prepared in berberin-chloride or sulfate salt form for clinical applications (

therapeutic potential of an alkaloid found in several medicinal plants. Altern Med Rev 1997;2: 94-103).

Berberine? one of the substances with the most evidence of effectiveness in the control of blood triglycerides in particular, and? for this reason it is widely exploited in nutraceutical preparations aimed at metabolic syndrome.

Berberis extract in traditional Chinese medicine, India and the Middle East was used for its antimicrobial effects and as a treatment for diabetes 2. ancient times of the berries, as a blood purifying agent, is documented by the clay tablets of the Assyrian emperors.

Can Berberine? regulate several metabolic disorders, in particular ? capable of controlling blood glucose levels, regulating blood pressure and lipid levels, also thanks to its promotional action on insulin secretion and improving insulin resistance.

Precisely for these properties, its use is associated with a modulation of the symptoms of metabolic syndrome, characterized precisely by pathophysiological changes based on insulin resistance with relevant clinical manifestations such as: obesity, increased blood sugar, increased lipidemia and hypertension (Figure 2).

Berberine as a hypoglycemic agent

Berberine? proved to be effective in improving glycemic control and lipid profile. In fact, berberine influences glucose metabolism by increasing insulin secretion, stimulating glycolysis, suppressing adipogenesis, inhibiting mitochondrial function, promoting the activation of the AMP-activated kinase pathway, AMPK, and increasing the activity of glucose. glycokinase. ? also able to increase the level of glucose transporters (GLUT4) and the glucagon-like peptide GLP-1 (Derosa, Giuseppe; Maffioli, Pamela; Cicero, Arrigo FG (2012). Berberine on metabolic and cardiovascular risk factors: an analysis from preclinical evidences to clinical trials. Expert Opinion on Biological Therapy, 12(8), 1113?1124).

Can berberine improve the activity? of the AMPK of the pancreatic islets, promoting the activation of cyclic adenosine monophosphate (cAMP) and the enzymatic cascade which, generating calcium influx, determines the degranulation of the vesicles and the release of insulin.

Berberine also inhibits DPP4 (dipeptidyl dipeptidase 4) responsible for the degradation of GLP1 and GIP, incretin peptides responsible for increasing insulin levels. The inhibition action prolongs the activity. of these peptides, improving glucose tolerance.

In culture cells? An insulin-sensitizing action has been observed for berberine, which is why its activity is ? been compared to that of metformin. In a rat model of type 2 diabetes, berberine showed improved activity. reduction in blood glucose and LDL levels compared to metformin (

Berberine: Botanical Occurrence, Traditional Uses, Extraction Methods, and Relevance in Cardiovascular, Metabolic, Hepatic, and Renal Disorders. Frontiers in Pharmacology, 9;

. Berberine on metabolic and cardiovascular risk factors: an analysis from preclinical evidences to clinical trials. Expert Opinion on Biological Therapy, 12(8), 1113?1124).

In some cases it not only acts as an insulin-sensitizing agent, but appears to have a direct action on the pancreas. In a study conducted on rats with streptozocin-induced diabetes, administrations of 120 mg/kg/day for 5 weeks were associated with an increase in GLP1 levels in plasma and intestine, an increase in plasma insulin levels and to an increase in the number of ?-pancreatic cells ( . Berberine on metabolic and cardiovascular risk factors: an analysis from preclinical evidences to clinical trials. Expert Opinion on Biological Therapy, 12(8), 1113?1124;

. Effects of berberine on glucose-lipid metabolism, inflammatory factors and insulin resistance in patients with metabolic syndrome. Experimental and Therapeutic Medicine).

Berberine as an anti-hyperlipidemic agent

The first study to investigate the use of Berberine in the treatment of cardiovascular disorders dates back to 1980. This study showed its ability to of berberine to improve cardiac dysfunction in patients with severe congestive heart failure.

The excessive production of lipids, particularly cholesterol, is associated with an increased risk of developing cardiovascular disorders.

According to in vitro and in vivo clinical studies on animals, berberine has proven to be a safe and effective substance in reducing cholesterol levels. In particular, in 32 hypercholesterolemic patients subjected to oral administration of berberine equal to 0.5 g twice a day for 3 months, yes? recorded a reduction in serum cholesterol levels equal to 29%, triglycerides by 35% and LDL cholesterol levels equal to 25% ( Berberine is a novel cholesterol-lowering drug working through a unique mechanism distinct from statins. Nat Med 2004; 10 (12):1344-51). This ability? ? linked to the down-regulation of LDL cholesterol and the up-regulation of LDL receptors. Berberine exerts a double action on the metabolism of these receptors: it increases the expression of the LDLR receptor; inhibits the expression of the proprotein convertase subtilisin/kexin type 9-PCSK9 (deputized for the degradation of LDLR and the reduced metabolism of LDL) through the ubiquitination and degradation of the hepatocyte nuclear growth factor (HNF1?), determining an increase of LDLR (

Berberine decreases PCSK9 expression in HepG2 cells. Atherosclerosis 2008;201(2):266-73).

Furthermore, it inhibits the absorption of cholesterol and promotes its excretion from the blood. In a study conducted on animals with a large quantity of cholesterol accumulated in the liver yes? recorded a significant reduction in total cholesterol, triglyceride and LDL levels in the serum, a reduction in liver cholesterol and an increase in bile cholesterol levels, after treatment with 50/100mg/kg of berberine. These results indicate that the promotion of cholesterol excretion from the liver into the bile, is one of the modes? by which berberine reduces serum lipids (L Effect of Berberine on promoting the excretion of cholesterol in high-fat diet-induced hyperlipidemic hamsters. J Transl Med 2015; 13(1):278).

This ability? ? due to the effect of berberine on the ACAT (cholesterol acyltransferase) enzymes, responsible for the transformation of cholesterol into esters. Does berberine carry out its activity? on ACAT2, reducing the intestinal absorption of cholesterol and reducing its plasma levels (

Berberine: Botanical Occurrence, Traditional Uses, Extraction Methods, and Relevance in Cardiovascular, Metabolic, Hepatic, and Renal Disorders. Frontiers in Pharmacology, 9).

Berberine as an antihypertensive agent

The hypotensive action of Berberine? has been observed in several animal models.

In one study the following were observed: delayed onset and attenuation of severity of hypertension and improvement of hypertension-induced renal damage after administration of berberine in rats with hypertension. Furthermore, berberine can inhibit the activity? of the RAS system and the pro-inflammatory cytokines (Il-6, IL-17, IL-23) which are involved in the pathophysiology of hypertension.

The results of other studies have shown that the antihypertensive action of berberine is due to the inhibition of the ACE enzyme and the direct release of NO, vasodilating in the vascular tissues ( (2017). A review of the effects of Berberis vulgaris and its major component, berberine, in metabolic syndrome. Iranian journal of basic medical sciences, 20(5), 557).

Pharmacokinetics of Berberine

Berberine undergoes intense metabolism after oral administration, by the enzymes CYP3A4 and CYP2D6, resulting in extremely low plasma concentrations. However, its metabolites appear to contribute to the pharmacological effects. The metabolic pathways of berberine are: demethylation, demethylation, reduction, hydroxylation and subsequent in vivo conjugation. Various metabolites are formed in humans, but the main one is? demethyleneberberine-2-Osulfate ( (2017). The metabolism of berberine and its contribution to the pharmacological effects. Drug Metabolism Reviews, (), 1?19) (Figure 3).

In chronic administration (15mg/kg) for 3 months, the levels of berberine and its metabolites in plasma were considerably higher than those recorded in acute administration, probably due to bioaccumulation. It is also necessary to consider the variability? individual.

Berberine absorption

Can Berberine? be easily absorbed both in the intestinal tract of rats and in humans after oral administration, however, its bioavailability? absolute? extremely low (< 1%).

After an oral dose of 400 mg of berberine, in a pilot pharmacokinetic study, the maximum mean plasma concentration found in 20 volunteers was ? was approximately 0.4 ng/mL while the AUC was only 9.18 h*ng/mL. Its distribution in tissues? For? was generally greater than in plasma, 4 hours after administration ( (2017). The metabolism of berberine and its contribution to the pharmacological effects. Drug Metabolism Reviews, (), 1?19). ;;According to the biopharmaceutical classification system, berberine belongs to class 3, having that high solubility? and poor permeability? (partition coefficient: 1.5, permeability coefficient 0.178*10-4 cm/s) (

(2016). Research progress on berberine with a special focus on its oral bioavailability. Phytotherapy).

The bioavailability problems? of berberine are due to: poor absorption (56% of cases); hepatic first pass; poor permeability; Pgp-mediated efflux effect of which ? substrate (P glycoprotein, responsible for the extrusion of some compounds from the absorption stream); self-aggregation.

Self-aggregation can reduce solubility? in the GI tract, limiting oral absorption. Under physiological conditions, berberine is found mainly in the ionized form, so it is It is very easy for it to self-aggregate in the acidic environment of the stomach and in the upper part of the small intestine

(2016). Research progress on berberine with a special focus on its oral bioavailability. Phytotherapy).

Oleuropein

From a phytochemical point of view, given the resistance to damage by pathogens that characterizes the leaves of the olive tree, it is possible. understand that the main constituents of the leaf are the flavonoids which also include Oleuropein, the molecule recognized as mainly responsible for the activities of the leaf. health benefits of the extract (Table 2).

Table 2 (from Goldsmith 2015 Phytochemical properties of olive leaf extracts): Total polyphenols (TPC) are expressed as gallic acid equivalent (GAE)/g of extract, total flavonoids are expressed as rutin equivalent (RE)/g of extract and l ?Oleuropein? expressed as mmol/g of dry extract

Other flavonoids present in quantity? important are rutin and diosmin. Triterpenes are also characterizing, including oleanolic acid.

The olive leaf phytocomplex? one of the most studied by modern phytochemistry, so what? the entire finger print has been determined. The families of compounds present are therefore the following:

Phenols-Flavonoids?Iridoids, monoterpenes: 6-O-oleoeuropein-sucrose, ligustroside, oleuroside, oleoside-7,11-dimethylether, mullein side, oleoside, oleoside-11-methylether, oleuropein, excelsioside, ligustaloside B, morroniside, oleacein;

Phenols-Flavonoids-iridoids?monoterpenes?terpenes, oleoeuropein (6-9%), hydroxytyrosol;

Phenols: tyrosol, hydroxytyrosol, vanillin, vanillic acid, caffeic acid, chlorogenic acids: trans-cinnamic, parahydroxy-benzoic, trans-para-coumaric, orthocoumaric, protocateic, ferulic;

Phenols?Flavonoids: luteolin-7-glucoside, apigenin-7-glucoside diosmetin-7-glucoside, malic acid, tartaric acid, glycolic acid, lactic acid; oleoside, steroleoside, rutin luteolin-4'-glucoside, luteolin, olivine, rutin, apigenin and derivatives, diosmetin;

Terpenes? Triterpenes: oleanolic acid 2%-3%, maslinic acid, betulinic homo-oleestranol, alpha and betaamyrin, uvaol and erythrodiol;

Terpenes? Sesquiterpenes: aromadendrene, eudesmin;

Enzymes: lipase, peroxidase, emulsin;

Carbohydrates: glucose, sucrose, mannitol;

Tannins: Pyrogallic tannin;

Choline;

Alkaloids: cinchonidine, cinchonine;

Lignans: (-)-olivil-4'-glucoside, (+)-acetoxypinoresinol and derivatives, cycloolivil.

Oleuropein as a hypocholesterolemic

Next to the most? note activity? neuroprotective, the olive leaf extract also boasts a neuroprotective activity. particularly marked hypocholesterolemic. The two activities? they are closely linked to each other. In fact, heart disease, hypercholesterolemia and Alzheimer's are united by a very similar pathogenetic mechanism.

Oleuropein has been shown to reduce the oxidation of low-density lipoproteins. (LDL), both in vitro and in vivo (Syed Haris Omar Cardioprotective and neuroprotective roles of oleuropein in olive Saudi Pharmaceutical Journal (2010) 18, 111?121; Effects of the Olive-Derived Polyphenol Oleuropein on Human Health Int. J. Mol Sci.2014, 15, 18508-18524).

The anti-atherosclerotic effect of olive leaves? was suggested in a preclinical study on an animal model which involved the comparison between two groups subjected to a high lipid content diet, with the administration of olive leaf extract in one of the two groups.

The two groups were observed for 6 weeks and the results showed that the group ?without OLE treatment? had higher levels? high triglycerides and LDL cholesterol.

In the same studio? The lipid sedimentation at the level of the vessels (origin of the atherosclerotic plaque) was measured: the results showed how the group not treated with the extract showed a greater thick lipid sedimentation at the level of the aorta, compared to the treated group (The anti-atherosclerotic effect of olive leaf extract is related to suppressed inflammatory response in rabbits with experimental atherosclerosis. Eur J Nutr 2008; 47:235-243).

Oleuropein as hypoglycemic

Olive leaf extract has shown the ability to regulate blood glucose levels attributed in particular to the presence of oleuropein and oleanoic acid firstly, and of luteolin secondly. A preliminary study reports that doses of 16 and 32 mg/kg significantly decreased glycemic values, also leading to greater peripheral glucose absorption in a dose-dependent manner (Omar: Cardioprotective and neuroprotective Saudi Pharmaceutical Journal (2010) 18 , 111?121). The activity luteolin, on the other hand, seems to lead to an inhibitory effect on the increase in postprandial glucose (Syed Haris Omar Cardioprotective and neuroprotective roles of oleuropein in olives Saudi Pharmaceutical Journal (2010) 18, 111?121).

One of the most significant? a clinical study investigating the effectiveness of olive leaf extract in improving glucose homeostasis in adult patients with type II diabetes. I study ? was operated by administering 500 mg of olive leaf extract in tablets to a group, vs placebo; the treatment ? lasted for 14 weeks.

The parameters evaluated were plasma insulin and glycosylated hemoglobin (HbA1c) values. The results showed that subjects treated with olive leaf extract showed a significant reduction in glycosylated hemoglobin and fasting insulin levels. These results lead to the olive leaf extract being among the most promising natural adjuvants of antidiabetic therapy.

Oleuropein as a vasodilator and antihypertensive

In 1991 some researchers demonstrated that the main person responsible for the activity? hypotensive effect of olive leaf extract appears to be oleuropein (

Vasodilator effect of olive leaf. Planta Med 1991; 57:417-419; Blood pressure. lowering effect of olives is mediated through calcium channel blockade. Int J Food Sci Nutr 2005; 56:613-620).

The hypothesized mechanism of action sees olive leaf extract suppressing L-type calcium channels both directly and indirectly resulting in the induction of vasodilation.

A recent preclinical study conducted on human volunteers confirmed these assumptions: ? In fact, the actual relationship between the intake of olive leaf extract and the lowering of diastolic and systolic blood pressure, with an increase in blood NO levels, was highlighted for the first time. ? interestingly, the study reports these results in all volunteers subjected to the test (Effects of the Olive-Derived Polyphenol Oleuropein on Human Health Int. J. Mol. Sci. 2014, 15, 18508-18524; doi:10.3390/ijms151018508).

In a human clinical study? The reduction in blood pressure after taking oleuropein (OLE) was evaluated in 40 pairs of borderline hypertensive homozygous twins. The twins of each pair were divided and assigned to one control group or two treatment groups. The two groups were treated with 500 and 1,000 mg of OLE per day for eight weeks, respectively. Body weight, heart rate, blood pressure, blood glucose, and blood lipids were measured at two-week intervals. Blood pressure values decreased in both groups but with an average difference in decrease in systolic pressure of up to 6 mmHg in the group treated with 500 mg of OLE compared to the control group, and up to 13 mmHg difference between the group 500 mg and the 1,000 mg one after six weeks (Martin de Bock Olive (Olea europaea L.) Leaf Polyphenols Improve Insulin Sensitivity in Middle-Aged Overweight Men: A Randomized, Placebo-Controlled, Crossover Trial; Food supplementation with an olive (Olea europaea L.) leaf extract reduces blood pressure in borderline hypertensive monozygotic twins. Phytother Res 2008; 22:1239-1242).

Olive leaf polyphenols were shown to inhibit platelet function in vitro in the blood of 11 healthy, male, non-smoking volunteers. Furthermore, the polyphenols of this phytocomplex (at increasing concentrations of oleuropein) effect a significant dose-dependent suppression of the release of platelet-ATP and platelet aggregation (Singh I, Mok M, Christensen AM, et al. The effect of polyphenols in olive leaves on platelet function. Nutr Metab Cardiovasc Dis 2008; 18:127-132).

Absorption of oleuropein

Oleuropein (Ole) ? the main constituent of the secoiridoid family in olive leaves. Hydroxytyrosol (HT) is the primary metabolite of Ole/OleA, and shares some of the properties? biological processes described above, but has a greater capacity? antioxidant.

The possibility? whether these biophenols can exert their biological effects depends on the probability? to reach key molecular targets in human tissues at a sufficient dose, which depends on their metabolism and bioavailability. However, data on the metabolism of oleuropein from EVOO (Extra Virgin Olive Oil) or olive leaves in humans are scarce, and results on the level and form found in plasma, and/or excreted in urine, are often conflicting. (

Olive oil phenolics are dosedependently absorbed in humans. FEBS Lett. 2000, 468, 159?160;

Capillary gas chromatography-mass spectrometry quantitative determination of hydroxytyrosol and tyrosol in human urine after olive oil intake. Anal. Biochem. 2001, 294, 63?72; Vissers, M.N.; Zock, P.L.; Roodenburg, A.J.C.; Leenen, R.; Katan, M.B. Olive Oil Phenols Are Absorbed in Humans. J. Nutr.

2002, 132, 409?417). This discrepancy can be explained by the fact that bioavailability? of oleuropein? influenced by various factors, such as the route of administration, genotype, age, sex, interaction with food and by the different extraction processes and analytical methods used (

. A novel bioanalytical method based on UHPLCHRMS/MS for the quantification of oleuropein in human serum. Application to a pharmacokinetic study: Quantification of oleuropein in human serum. Biomed. Chromatogr. 2016, 30, 2016?2023).

A recent human study demonstrated that after oral ingestion, Ole? resistant to gastric acid pH conditions, and is rapidly absorbed (55-60%) in the intestinal tract, reaching the maximum concentration in the blood (23-30 min, depending on the preparation, liquid vs. capsule) before the conjugated metabolites of HT, glucuronidates and sulphates (at 64-93 min ), which constituted 96-99% of Ole metabolites detected in plasma and urine after intake (de Bock, M.; Thorstensen, E.B.; Derraik, J.G.B.; Henderson, H.V.; Hofman, P.L.; Cutfield, W.S. Human absorption and metabolism of oleuropein and hydroxytyrosol ingested as olive (Olea europaea L.) leaf extract. Mol. Nutr. Food Res.2013, 57, 2079?2085).

These data suggest a potential complete metabolization of Ole to HT and other degradation products. In fact, the in vivo efficacy of these must be confirmed. The main criticism of in vitro studies using these molecules is? that the doses used are higher concentrations (?mol/L?mmol/L) than the metabolite concentrations measured in plasma, which are only at the nmol/L concentration (

Polyphenols and human health: A perspective. Crit. Rev. Food Sci. Nutr.2011, 51, 524?546).

Therefore, delivery systems have been developed based on the esterification/lipophilization and encapsulation of phenolic compounds, or using the creation of liposomes and/or nanoparticles of bioactive compounds, to increase their bioavailability. and bioaccessibility? (

Esterification of Quercetin Increases Its Transport Across Human Caco-2 Cells: Synthesize quercetin ester to improve bioavailability, J. Food Sci. 2016, 81, H1825?H1832; Pagnussatt, F.A.; de Lima, V.R.; Dora, C.L.; Costa, J.A.V.; Putaux, J.-L.; Badiale-Furlong, E. Assessment of the encapsulation effect of phenolic compounds from Spirulina sp. LEB-18 on their antifusarium activities. Food Chem.

2016, 211, 616?623; Gibis, M.; Ruedt, C.; Weiss, J. In vitro release of grape-seed polyphenols encapsulated from uncoated and chitosan-coated liposomes. Food Res.

Int. 2016, 88, 105?113; Gleeson, J.P.; Ryan, S.M.; Brayden, D.J. Oral delivery strategies for nutraceuticals: Delivery vehicles and absorption enhancers. Trends Food Sci. Technol.2016, 53, 90?101).

Much attention recently? was given to the intestinal microbiota, considered an ?organ? metabolic that has an impact on the nutrition of the host and can influence bioavailability? and bioaccessibility? of phenolic compounds through biotransformation into other active substances, which have interesting health benefits with active in intestinal diseases (N

Impact of Gut Microbiota Composition on Onset and Progression of Chronic Non-Communicable Diseases.Nutrients 2019, 11, 1073).

Mosele and collaborators (

M.-J. Faecal microbial metabolism of olive oil phenolic compounds: In vitro and in vivo approaches. Mol. Nutr. Food Res. 2014, 58, 1809? 1819), in an in vitro model experiment, they observed that Ole ? was rapidly deglycosylated during 6 hours of incubation with human fecal microbiota, to oleA; the latter? been degraded into elenolic acid and HT by the activity? microbial esterase, until it disappeared after 48 hours. On the contrary, the HT ? increased steadily during the same fermentation period.

This data ? was confirmed by the same authors in an in vivo study, which showed that after the intake of olive oil rich in phenols for three weeks, the concentration of free HT ? was significantly increased in the feces of all study participants. Other research has shown that the conversion of Ole to HT ? was performed by lactic acid bacteria, in particular by Lactobacillus plantarum (

Impact of Gut Microbiota Composition on Onset and Progression of Chronic Non-Communicable Diseases.Nutrients 2019, 11, 1073).

Florizine

Activities? of Florizin on diabetes and body weight

Florizin in particular has aroused the interest of research in the diabetes field following some studies on animals which suggest its potential. of the molecule in reducing blood glucose levels (Masumoto S, Akimoto Y, Oike H, Kobori M. Dietary phloridzin reduces blood glucose levels and reverses Sglt1 expression in the small intestine in streptozotocin-induced diabetic mice. J Agric Food Chem 2009;57 (11):4651-46; Phloridzin improves hyperglycemia but not hepatic insulin resistance in a transgenic mouse model of type 2 diabetes. Diabetes 2004;53(11):2901-2909; Experimental study on the treatment of diabetes by phloridzin in rats. J Tongji Med Univ 1998;18(2):105-7, 118; Starke A, Grundy S, McGarry JD, Unger RH. Correction of hyperglycemia with phloridzin restores the glucagon response to glucose in insulindeficient dogs: implications for human diabetes. Proc Natl Acad Sci USA 1985;82(5):1544-1546).

In particular, it seems that Florizin promotes renal glucose excretion, both in a healthy animal model and in an animal model with induced type 2 diabetes. In the diabetic animal model, this activity? of the molecule translates into an improvement in the glycemic profile.

Other effect recorded in the study? the reduction of fat mass, following phlorizin supplementation (Phloridzin improves hyperglycemia but not hepatic insulin resistance in a transgenic mouse model of type 2 diabetes. Diabetes 2004;53(11):2901-2909). The effect of phlorizin on obesity, particularly in the presence of metabolic syndrome and diabetes? subject of some recent studies; in particular, yes? highlighted that treatment with the molecule reduces visceral and total white adipose tissue (WAT), the weight and size of adipocytes with an improvement in blood and liver lipid profiles. The mechanism of action by which phlorizin affects hepatic lipid levels appears to involve the inhibition of hepatic lithogenesis, the synthesis of endogenous cholesterol.

Other effects recorded were the decrease in inflammation, glycemia, insulin, glucagon and insulin resistance (Starke A, Grundy S, McGarry JD, Unger RH. Correction of hyperglycemia with phloridzin restores the glucagon response to glucose in insulin-deficient dogs: implications for human diabetes. Proc Natl Acad Sci USA 1985;82(5):1544-1546; Su-Kyung Shin Phlorizin Supplementation Attenuates Obesity, Inflammation, and Hyperglycemia in Diet-Induced Obese Mice Fed a High-Fat Diet, Nutrients 2016, 8, 92; doi:10.3390/nu8020092).

Phlorizine absorption

Does phlorizin have bioavailability? very low due to its clear lipophilicity (Wang Y et al., Enhanced oral bioavailability and bioefficacy of phloretin using mixed polymerid modified self-nanoemulsions, Food Science and Nutrition 2019).

Problem of the prior art

There is a need to have new formulations in the prevention and treatment of metabolic syndrome which include the presence of all three active ingredients Oleuropein, Berberine and Florizin, capable of reducing the absorption problems of these substances at the same time.

In fact, the aforementioned active ingredients present considerable limitations in terms of poor bioaccessibility. and/or bioavailability? which could counteract and/or reduce the effectiveness of this association.

SUMMARY OF THE INVENTION

Object of the present invention? an association for use in the prevention and treatment of metabolic syndrome comprising (or alternatively consisting of) the following active ingredients: Oleuropein, Berberine and Florizin.

Preferably, the aforementioned association for use can? be contained in an oral formulation, which can? include additional active ingredients in addition to those mentioned above together with excipients.

Preferably, the aforementioned oral formulations contain enteric absorption promoters, consisting of a mixture comprising: triglycerides of medium and/or short chain fatty acids (generally indicated with the acronym "Medium Chain Triglycerides" and with the acronym MCT), lecithin, polysorbate 80.

The Applicant has in fact found that the aforementioned absorption promoters simultaneously solve all three different problems of absorption of these specific active substances contained in the association for use according to the present invention.

Therefore, a further advantage of the invention is? the creation of an innovative delivery system, which promotes the absorption of the aforementioned triptych of active substances, exploiting a mixture of absorption promoters? triglycerides of medium and/or short chain fatty acids, lecithin, polysorbate 80 -, preferably also in association with beta-pinene and olive leaf extract containing flavonoids.

Furthermore, the present association for use advantageously includes substances of preferably natural origin.

DESCRIPTION OF THE FIGURES

Figure 1 ? Graphic representation of the mechanisms linked to the onset of cardiometabolic risk and metabolic syndrome;

Figure 2 ? Diagram about the effects of Berberine;

Figure 3 ? Main metabolites of Berberine.

DETAILED DESCRIPTION OF THE INVENTION

For the purposes of this invention, for ?metabolic syndrome? does it mean a pathological state (or disease) characterized by: arterial hypertension, impaired fasting blood sugar or insulin resistance, dyslipidemia, obesity? abdominal (or marked waist circumference due to excess abdominal fat).

For ?use in the prevention and treatment of metabolic syndrome? it is intended, on the one hand and specifically referring to the expression "for use in prevention", to intervene to reduce morbidity, mortality, or the effects due to certain risk factors with the aim of: avoiding the pathological state; control the spread of the disease. On the other hand, specifically the expression "for use in treatment" means fighting the disease, suppressing the causal agent of the disease; remove the symptoms; interfere with the mechanisms of onset of the disease.

Association for the use according to the invention

For the purposes of the present invention, the expressions ?consists of? or ?composed of? are to be understood exclusively, that is? defining the invention on the basis of the characteristics/elements expressly indicated in the claim and excluding the presence of all that? that does not ? been mentioned. On the contrary, the expressions ?containing? or ?comprehensive? they must be understood in an inclusive way, that is? define the invention on the basis of the characteristics/elements expressly indicated in the claim, without excluding the presence of other elements not expressly mentioned.

The association for the use according to the invention? preferably made up of the following active ingredients: Oleuropein, Berberine and Florizin.

Preferably, the active Oleuropein is in a quantity between 3.0% and 15% by weight, more? preferably between 6.0% and 9.0% by weight, according to a particularly preferred embodiment equal to about 8% by weight on the total weight of the combination.

The active Oleuropein can be obtain preferably from the dry extract of olive or olive leaves (preferably titrated in Oleuropein) in a percentage between 7% and 20% by weight, preferably equal to 15% by weight on the total weight of the extract.

The active Berberina in the association? present as is or in the form of at least one of its pharmaceutically acceptable salts, preferably ? Berberine hydrochloride (or BERC).

Berberine or at least one of its pharmaceutically acceptable salts? present in a quantity between 20% and 70% by weight, more? preferably between 58% and 68% by weight, even more? preferably between 60% and 64% by weight, according to a particularly preferred solution equal to approximately 63% by weight on the total weight of the combination.

The active Berberine or at least one of its pharmaceutically acceptable salts can be used. obtain preferably from the dry extract of Berberis aristata, preferably titrated in Berberine in a percentage between 15% and 95%.

Preferably, the active Florizine is in a quantity between 15% and 25% by weight, preferably between 17% and 23% by weight, preferably equal to about 21% by weight on the total weight of the combination.

The active Florizina can be obtain preferably from the dry extract of Malus domestica L., preferably titrated in Phlorizin from 40% to 65% by weight, preferably 50% by weight on the total weight of the extract.

Oral formulation comprising the association for the use of the invention. The association for the use according to the present invention is preferably contained in an oral formulation in combination with further active ingredients in addition to those contained in said association with suitable excipients and/or diluents.

Alternatively, the association for use according to the invention is preferably contained in the oral formulation, in which said association is preferably the sole active ingredient in combination with suitable excipients and/or diluents.

According to another solution, the oral formulation comprising the association for use can also contain the active chromium picolinate, preferably in quantities such that the NRV (Nutritional Reference Value) is 100%.

In particular, when the oral formulation comprising the association for the use of the invention also includes chromium picolinate, the quantity of the latter is? in a percentage between 0.030% and 0.10% by weight, preferably between 0.050% and 0.090% by weight, preferably equal to 0.080% by weight by weight on the total weight of the combination.

? I know that chromium? an important trace element for the human organism with a central function in glucose metabolism. This trace element? in fact, it is capable of increasing the Glucose Tolerance Factor (GTF) and its deficiency leads to imbalances in the metabolism of sugar, fats and proteins which manifest themselves clinically with hyperglycemia, obesity, decreased sensitivity tissue resistance to insulin, tendency to atherosclerosis and lower resistance to infections.

Without wanting to be bound to any theory, the Applicant has chosen the picolinate form of chromium for this oral formulation since it is Yes ? revealed to be the only one with consistent results in the diabetes field compared to other forms of chromium (Horvath EM, et al.,?Antidiabetogenic effects of chromium mitigate hyperinsulinemia-induced cellular insulin resistance via correction of plasma membrane cholesterol imbalance.?, Mol Endocrinol.22( 4), Apr; 2008; Jain SK, Rains JL, Croad JL.: Effect of chromium niacinate and chromium picolinate supplementation on lipid peroxidation, TNF-alpha, IL-6, CRP, glycated hemoglobin, triglycerides, and cholesterol levels in blood of streptozotocin-treated diabetic rats, Free Radic Biol Med.). Furthermore, chromium picolinate is considered the most? bioavailable. The Applicant considers that chromium picolinate can assist or strengthen the effect induced by the association of the three active ingredients according to the invention.

For purely descriptive and non-limiting purposes, excipients and/or diluents known in the state of the art and suitable or suitable for the preparation of formulations, preferably solid formulations (powders, tablets and/or capsules), are: bulking agents, glidating agents, lubricating agents, flow agents, disintegrating agents, flavoring agents, pH correcting agents, sweetening agents. Examples of suitable excipients are silica or any other excipient, known to those skilled in the art, which favors the compression of powders or granules to obtain tablets.

The oral formulation comprising the association for the use of the invention preferably includes the following excipients as enteric absorption promoters, a mixture of triglycerides of medium and/or short chain fatty acids, lecithin, polysorbate 80. In other words, the mixture of: triglycerides of medium and/or short chain fatty acids, lecithin, polysorbate 80 acts as a promoter of enteric absorption.

For ?short-chain fatty acids? means acids chosen from the group consisting of: acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, caproic acid, lactic acid, succinic acid and combinations of the previous ones.

For ?medium chain fatty acids? we mean medium chain triglycerides or MCTs (Medium Chain Triglycerides), i.e. saturated fatty acids with a number of carbon atoms preferably between 6 and 12. Examples of medium chain fatty acids are the acids preferably chosen from the group consisting of: acid caprylic acid, caprinic acid, lauric acid and combinations of the above.

Polysorbate 80? polyoxyethylene sorbitan monooleate (E433).

According to an embodiment of the invention, the oral formulation, comprising the association for the use of the invention, includes the absorption promoters: triglycerides of medium and/or short chain fatty acids, lecithin, polysorbate 80, in which the them a total quantity? preferably between 3.5% and 10% by weight, more? preferably between 4.5% and 8.5% by weight, even more? preferably between 5.5% and 6.5% by weight, and according to a particularly preferred embodiment equal to about 5.2% by weight on the total weight of the combination.

According to an alternative embodiment of the invention, the oral formulation, comprising the association for the use of the invention, includes the absorption promoters: triglycerides of medium and/or short chain fatty acids, lecithin, polysorbate 80, in which these are in a weight ratio (medium and/or short chain fatty acid triglycerides/lecithin/polysorbate 80) preferably equal to 2:2:1.

Do absorption promoters preferably increase solubility? of the association's assets for the use of the invention, thus constituting an innovative delivery system or technology. In fact, absorption promoters, as amphiphilic components, allow the following effects to be obtained: formation of micelles (or micellization) including the active ingredients, once in contact with physiological fluids; consequent increase in contact surface; increase in speed? of dissolution; increase in affinity for the mucous membranes.

The aforementioned mixture of absorption promoters, which is included in the oral formulation, containing in turn the association for use according to the invention, can preferably also include beta-pinene.

Beta-pinene? preferably present in an amount between 0.50% and 3.0% by weight, preferably between 1.5% and 2.5% by weight, preferably ? approximately equal to 1% by weight of the total weight of the association.

The Applicant has also found that the terpene beta-pinene, ? capable of promoting bioavailability? of the assets of the association for the use of the invention; in particular, beta-pinene? capable of inhibiting the Pgp pump.

The aforementioned mixture of absorption promoters, which is included in the oral formulation, containing in turn the association for use according to the invention, preferably also includes olive leaf extract. The Applicant has in fact found that the olive leaf extract, like the extracts of the Citrus genus, have an inhibitory interaction towards the cytochromes of the CYP450 family.

The Applicant also believes that the olive leaf extract, including flavonoids, is able to inhibit the cytochromes of the CYP-450 family by acting as a promoter of absorption, but also as a further active ingredient included in the oral formulation.

Preferably, olive leaf extract is in an amount between 1.0% and 3.5% by weight, preferably between 1.5% and 2.5% by weight, preferably ? approximately equal to 2.0% by weight of the total weight of the association.

The oral formulation, including the association for use according to the invention, is preferably in solid oral form chosen from tablet, capsule, powder, granules, pi? preferably? in tablet form.

The methods for obtaining oral formulations, for example tablets, are known to those skilled in the art based on knowledge of general pharmaceutical technologies.

The oral formulation including the association for use according to the invention? in the form of a nutraceutical product. For ?nutraceutical product? means, pursuant to art. 2 of Reg. (EU) n. 178/2002, ?any processed, partially processed or unprocessed substance or product intended to be ingested by human beings, or which is reasonably expected to be ingested by human beings?. In other words, by nutraceutical we mean one or more? components or active ingredients of foods that have positive effects on health, prevention and treatment of diseases.

The oral formulation comprising the association for use according to the invention can also be in the form of a supplement or AFMS (Food for Special Medical Purposes).

By food supplement we mean a formulation that falls within the definition underlying Directive 2002/46/EC and subsequent amendments. In this legislation, food supplements are defined precisely as: ?food products intended to supplement the common diet and which constitute a concentrated source of nutrients, such as vitamins and minerals, or other substances having a nutritional or physiological effect, in particular , but not exclusively, amino acids, essential fatty acids, fibers and extracts of vegetal origin, both single-compound and multi-compound, in pre-dosed forms?.

Food for Special Medical Purposes (or AFMS) means food products expressly processed or formulated for the dietary management of patients with specific nutritional needs, including infants, to be used "under medical supervision". AFMS are intended for the complete or partial feeding of patients who have a capacity limited or impaired in taking, digesting, absorbing, metabolizing or eliminating common foods or certain nutrients contained in them, or who have specific nutritional needs determined by clinical conditions. AFMS differ from other foods and from food supplements themselves due to some characteristics, such as: they require medical supervision; can they replace? totally or partially? normal nutrition; they are indicated for specific pathological conditions. As regards the composition, AFMS are regulated by Regulation 609/13.

Process of preparing the oral formulation including the association for the use of the invention

The aforementioned oral formulation, including the association for the use of the invention, is prepared with a process comprising the following stages:

a) mix Oleuropein, Berberine and Florizin, and possibly chromium picolinate, preferably chromium picolinate in quantities such that it constitutes 100% of daily NRV, to obtain a first mixture;

b) possibly add the olive leaf extract to the first mixture, obtained from the previous step a), to obtain a second mixture;

c) adding the lecithin to the first mixture, coming from the previous stage a), or possibly to the second mixture, coming from the previous stage b), to obtain a third mixture;

d) wetting the third mixture, coming from the previous stage c), with triglycerides of short and/or medium chain fatty acids, polysorbate 80, and possibly beta-pinene, to obtain a fourth mixture;

e) let the fourth mixture, coming from the previous stage d), dry, preferably at room temperature;

f) add excipients and/or diluents suitable to obtain the oral formulation.

EXAMPLES

Below the Applicant reports examples of implementation of the invention, for purely illustrative and non-limiting purposes.

Example 1? Oral formulation comprising the association for use according to the invention in tablet form

Example 2? Oral formulation comprising the association for use according to the invention in tablet form

Example 3? Production process of the oral formulation comprising the association according to the invention in tablet form

Crucial for the effectiveness of the invention and the delivery technology of the invention? the production process of the oral formulation which involves the following phases:

a) Mixing of the powders of the active ingredients of the Oleuropein, Berberine and Florizin association, preferably present in powder form, and possibly chromium picolinate, to obtain phase A;

b) Mixing the olive leaf extract with phase A to obtain phase AB;

c) Mixing of the lecithin with the AB phase powders;

d) Wetting of the powders of phase AB and lecithin obtained from the previous phase (c), with triglycerides of medium and/or short chain fatty acids, polysorbate 80 and betapinene;

e) Drying of the powder obtained in the previous phase (d) at room temperature,

f) Addition of silica to the dried powder of the previous phase (e);

g) Addition of the excipients necessary for the formation of tablets to the powder of the previous phase (f) to obtain the oral formulation in tablet form.

Claims (13)

1. Association for use in the prevention and treatment of metabolic syndrome comprising the following active ingredients: Oleuropein, Berberine and Florizin. 2. Association for use according to claim 1, consisting of the active ingredients: Oleuropein, Berberine and Florizin. 3. Association for use according to any of claims 1 or 2, in which Oleuropein is? in a quantity between 3.0% and 15% by weight of the total weight of the association. 4. Association for use according to any of claims 1 to 3, in which Berberine or at least one pharmaceutically acceptable salt thereof is in a quantity between 20% and 70% by weight of the total weight of the association. 5. Association for use according to any of claims 1 to 4, in which Florizina is? in a quantity between 15% and 25% by weight of the total weight of the association. 6. Association for use according to any of claims 1 to 5, wherein said association is? contained in an oral formulation in combination with additional active ingredients in addition to those contained in said association with suitable excipients and/or diluents. 7. Oral association for use according to claim 6, wherein the oral formulation includes chromium picolinate, preferably in quantities such that the chromium constitutes 100% of the Nutritional Reference Value. 8. Association for use according to any of claims 1 to 5, wherein said association is? contained in an oral formulation, containing said association as the sole active ingredient in combination with suitable excipients and/or diluents. 9. Association for use according to any one of claims 6 to 8, wherein said oral formulation comprises the following excipients as enteric absorption promoters, a mixture of: triglycerides of medium and/or short chain fatty acids, lecithin, polysorbate 80. 10. Combination for use according to claim 9, wherein said mixture of absorption promoters also includes beta-pinene. 11. Combination for use according to any one of claims 9 to 10, wherein said mixture of absorption promoters also includes olive leaf extract. 12. Association for use according to any of claims 6 to 11, wherein said oral formulation is? in the form of tablet, capsule, powder, granules, preferably ? in tablet form. 13. Combination for use according to any one of claims 6 to 12, wherein said oral formulation is prepared with a process comprising the following steps: a) mix Oleuropein, Berberine and Florizin and possibly chromium picolinate to obtain a first mixture; b) possibly add the olive leaf extract to the first mixture, obtained from the previous step a), to obtain a second mixture; c) adding the lecithin to the first mixture, coming from the previous stage a), or possibly to the second mixture, coming from the previous stage b), to obtain a third mixture; d) wetting the third mixture, coming from the previous stage c), with triglycerides of medium chain fatty acids, polysorbate 80, and possibly beta-pinene, to obtain a fourth mixture; e) let the fourth mixture dry, coming from the previous stage d); f) add excipients and/or diluents suitable to obtain the oral formulation.