WO2024110926A1

WO2024110926A1 - Nutraceutical supplement based on polyphenol matrix

Info

Publication number: WO2024110926A1
Application number: PCT/IB2023/061863
Authority: WO
Inventors: Giulio TORELLO; Francesco FRATI
Original assignee: Global Pharmacies Partner In Forma Abbreviata GPP Srl Srl
Current assignee: Global Pharmacies Partner In Forma Abbreviata GPP Srl Srl
Priority date: 2022-11-24
Filing date: 2023-11-24
Publication date: 2024-05-30
Anticipated expiration: 2025-05-24
Also published as: EP4622641A1; IT202200024246A1

Abstract

A polyphenol matrix, and related nutraceutical supplement, comprising or consisting of: - a polyphenol component, from 7.5% to 36% by weight, comprising or consisting of naringin, neohesperidin, and neoeriocitrin; - ellagic acid, from 4.1% to 20% by weight; and - hesperidin, from 3.8% to 20% by weight in addition to any excipients or additives.

Description

Description of the Industrial Invention entitled:

NUTRACEUTICAL SUPPLEMENT BASED ON POLYPHENOL MATRIX

The present invention relates to a nutraceutical supplement based on a polyphenol matrix, which effectively allows for the promotion of beneficial effects in various areas of human and animal health, such as the nervous system, the cardiovascular system through a regulation of lipid metabolism and glucose metabolism and a control of triglyceride levels in the blood, basically obtaining an improvement in metabolic syndrome parameters that allow, for example, to reduce states of inflammation, to control diabetes, to provide a side- effect-free treatment of visceral fat, obtaining an improvement in the lipid profile and hematochemical parameters.

It is known that some nutraceutical supplements have been developed in the prior art, for example, for the treatment of visceral fat and to reduce the amount of blood sugar, decreasing the risks of cardiovascular disease and Alzheimer's disease. Some of such nutraceutical supplements of the prior art are disclosed in documents US20090252796A1, EP2559346A1 and WO2018104131 A1.

Specifically, a solid powder composition, obtained as a bergamot extract made according to the teaching of W02018104131 A1, is currently marketed under the trade name Vazguard® by the Italian company Indena S.p.A., as described on the web page https://www.indena.com/product/vazguard. This composition comprises a polyphenol fraction of bergamot and phospholipids, wherein the flavonoid content extracted from bergamot is standardized to contain an amount ranging from 14 to 18% of total flavanones by HPLC. Said phospholipids appear to be necessary in the solid composition of WO2018104131 A1 because they are introduced for the explicitly described purpose of increasing the low bioavailability of the active substances contained in the bergamot extract.

However, such nutraceutical supplements of the prior art are not sufficiently effective. In addition, they have unfavorable interactions with the active ingredients of many drugs possibly used to treat concomitant diseases, so they cannot be taken at the same time as such drugs.

The purpose of the present invention, therefore, is to obtain a nutraceutical supplement that allows effective treatment of visceral fat and effective improvement of lipid profile and hematochemical parameters, effectively allowing for the promotion of beneficial effects in various areas of human and animal health, such as the nervous system, the cardiovascular system through a regulation of lipid metabolism and glucose metabolism, and a control of blood triglyceride levels.

Further purpose of the present invention is to make such a nutraceutical supplement by drastically reducing, if not eliminating, its interactions with drug active ingredients

It is a specific object of the present invention a polyphenol matrix comprising or consisting of:

- a polyphenol component, from 7.5% to 36% by weight, comprising or consisting of naringin, neohesperidin, and neoeriocitrin;

- ellagic acid, from 4.1% to 20% by weight; and

- hesperidin, from 3.8% to 20% by weight. It is a specific object of the present invention a polyphenol matrix comprising or consisting of:

- ellagic acid, from 4.1% to 20% by weight; and

- hesperidin, from 3.8% to 20% by weight; in addition to any excipients or additives.

According to another aspect of the invention, the ratio polyphenol component : ellagic acid : hesperidin can be equal to

10 : x : y wherein:

- x ranges from 3.6 to 6.7, optionally from 4 to 6, more optionally from 4.5 to 5.5, wherein even more optionally x is equal to 5, and

- y ranges from 1 8 to 6.7, optionally from 2 to 6, more optionally from 2.5 to 3.5 or from 45 to 5.5, wherein even more optionally y is 3 or 5

According to a further aspect of the invention, the polyphenol component may comprise or consist of:

- naringin, from 4% to 16% by weight;

- neohesperidin, from 4% to 16% by weight; and

- neoeriocitrin, from 3.5% to 14% by weight.

- naringin, from 4% to 16% by weight;

- neohesperidin, from 4% to 16% by weight; and

- neoeriocitrin, from 3.5% to 14% by weight; in addition to any other polyphenols.

According to an additional aspect of the invention:

- the polyphenol component can be extracted from bergamot {Citrus x bergamia), optionally d.e. at 38% polyphenols, and/or

- ellagic acid can be extracted from pomegranate, optionally d.e. at 40% ellagic acid, and/or

- hesperidin can be extracted from bitter orange {Citrus aurantium), optionally d.e. at 60% hesperidin.

In other words, according to a preferred aspect of the present invention, the polyphenol matrix of the invention comprises or consists of:

- a polyphenol component in the form of dry extract (d.e.) of bergamot fruit {Citrus x bergamia), preferably is bergamot d.e. titrated at 38% total polyphenols; and/or

- ellagic acid in the form of dry extract (d.e.) of pomegranate fruit {Punica granatum), preferably a pomegranate d.e. titrated at 40% ellagic acid; and/or

- hesperidin in the form of dry extract (d.e.) of bitter orange fruit {Citrus x aurantium), preferably a bitter orange d.e. titrated at 60% hesperidin. According to another aspect of the invention, the polyphenol component may also include:

- brutieridin, from 1% to 4% by weight, and

- melitidin, from 0.5% to 2% by weight.

According to another aspect of the invention, the polyphenol component may also include:

- brutieridin, from 1% to 4% by weight, and

- melitidin, from 0.5% to 2% by weight, also preferably present in the form of dry extract (d.e.) of bergamot fruit (Citrus x bergamia), preferably a bergamot d.e. titrated at 38% total polyphenols.

It is also a specific object of the present invention a nutraceutical supplement based on a polyphenol matrix configured to be taken orally, consisting of a composition comprising or consisting of the polyphenol matrix previously described, and one or more food additives, wherein the nutraceutical supplement is gastroresistant.

According to another aspect of the invention, said one or more food additives may be selected from the group comprising emulsifying additives, thickening additives, diluent or filler additives, anti-caking additives, and stabilizing, gelling and thickening additives.

According to a further aspect of the invention, said one or more food additives may comprise or consist of an emulsifying additive, optionally magnesium salts of fatty acids, from 1% to 36% by weight.

According to an additional aspect of the invention, said one or more food additives may consist of an emulsifying additive, optionally magnesium salts of fatty acids, from 9% to 36% by weight, and the nutraceutical supplement based on a polyphenol matrix may be in the form of a gastroresistant capsule.

According to another aspect of the invention, each gastroresistant capsule can weigh 595 mg and consist of:

- 95 mg of polyphenol component in 250 mg of bergamot (Citrus x bergamia) extract, d.e. at 38% polyphenols, containing 20 mg of naringin, 20 mg of neohesperidin, 17.5 mg of neoeriocitrin, 5 mg of brutieridin and 2.5 mg of melitidin;

- 125 mg of pomegranate fruit d.e. at 40% ellagic acid, containing 50 mg of ellagic acid;

- 125 mg of Citrus x aurantium fruit, d.e. at 60% hesperidin, containing 75 mg of hesperidin;

- magnesium salts of fatty acids, 95 mg.

In other words, according to the previous aspect of the invention, each gastroresistant capsule can weigh 595 mg and consist of:

- 250 mg of dry extract of bergamot fruit (Citrus x bergamia), specifically a bergamot fruit d.e. titrated at 38% total polyphenols corresponding to 95 mg of total polyphenols comprising 20 mg of naringin, 20 mg of neohesperidin, 17.5 mg of neoeriocitrin, 5 mg of brutieridin and 2.5 mg of melitidin;

- 125 mg of dry extract of pomegranate fruit (Punica granatum), specifically a pomegranate fruit d.e. titrated at 40% ellagic acid, corresponding to 50 mg of ellagic acid;

- 125 mg of dry extract of bitter orange fruit (Citrus x aurantium), specifically a bitter orange fruit d e. titrated at 60% hesperidin, corresponding to 75 mg of hesperidin; - magnesium salts of fatty acids, 95 mg.

Said gastroresistant capsule can be prepared according to all techniques known to the expert in the field, using the materials and equipment currently in use.

According to a further aspect of the invention, said one or more food additives may comprise or consist of:

- an emulsifying additive, optionally magnesium salts of fatty acids, from 1 % to 4% by weight,

- a thickening additive, even more optionally carboxymethyl cellulose, from 0.5% to 2% by weight,

- a diluent or filler additive, even more optionally dibasic calcium phosphate, from 6.5% to 25% by weight,

- a stabilizing, gelling and thickening additive, even more optionally microcrystalline cellulose, from 4.1% to 17% by weight, and

- an anti-caking additive, even more optionally silicon dioxide, from 0.8% to 4% by weight, and the nutraceutical supplement based on a polyphenol matrix may be in the form of gastroresistant tablet. According to an additional aspect of the invention, each gastroresistant tablet may weigh 1200 mg and consist of:

- 190 mg of polyphenol component in 500 mg of bergamot (Citrus x bergamia) extract, d.e. at 38% polyphenols, containing 40 mg of naringin, 40 mg of neohesperidin, 35 mg of neoeriocitrin, 10 mg of brutieridin and 5 mg of melitidin;

- 250 mg of pomegranate fruit d.e. at 40% ellagic acid, containing 100 mg of ellagic acid;

- 150 mg of Citrus x aurantium fruit, d.e. at 60% hesperidin, containing 90 mg of hesperidin;

- magnesium salts of fatty acids, 20 mg;

- carboxymethyl cellulose, 10 mg;

- dibasic calcium phosphate, 150 mg;

- microcrystalline cellulose, 100 mg; and

- silicon dioxide, 20 mg.

In other words, according to the previous additional aspect of the invention, each gastroresistant tablet may weigh 1200 mg and consist of:

- 500 mg of dry extract of bergamot (Citrus x bergamia), specifically a bergamot fruit d.e. titrated at 38% total polyphenols corresponding to 190 mg of total polyphenols comprising 40 mg of naringin, 40 mg of neohesperidin, 35 mg of neoeriocitrin, 10 mg of brutieridin and 5 mg of melitidin;

- 250 mg of dry extract of pomegranate fruit (Punica granatum), specifically a pomegranate fruit d.e. titrated at 40% ellagic acid, corresponding to 100 mg of ellagic acid;

- 150 mg of dry extract of bitter orange fruit (Citrus x aurantium), specifically a bitter orange fruit d.e. titrated at 60% hesperidin, corresponding to 90 mg of hesperidin;

- magnesium salts of fatty acids, 20 mg;

- carboxymethyl cellulose, 10 mg;

- dibasic calcium phosphate, 150 mg;

- microcrystalline cellulose, 100 mg; and - silicon dioxide, 20 mg.

Said gastroresistant tablet can be prepared according to all techniques known to the expert in the field, using the materials and equipment currently in use.

It is also a specific object of the present invention the nutraceutical supplement previously described for use in the prevention and/or treatment of at least one of the diseases or conditions selected from the group comprising or consisting of states of inflammation, diabetes, visceral fat, hypercholesterolemia, hypertension, hepatic steatosis, dyslipidemia, obesity, and cardiovascular risks.

The benefits of the present invention are significant.

In fact, the components of the nutraceutical supplement based on a polyphenol matrix according to the invention, namely neoeriocitrin, naringin and neohesperidin associated with ellagic acid and hesperidin, which are biologically active substances that act as metabolic activators, act synergistically with different mechanisms of action producing significantly amplified effects by acting on the absorption of lipids from the intestine and/or increasing their excretion, enhancing hepatic absorption of cholesterol and limiting its hepatic synthesis, and increasing thermogenesis and lipid metabolism. This results in an enhanced efficacy profile, improved outcome performance, and increased speed of action of the nutraceutical supplement based on a polyphenol matrix according to the invention.

In particular, the nutraceutical supplement based on a polyphenol matrix according to the invention has some beneficial functions on lipid profile.

In fact, it has a cholesterol-lowering effect, behaving like statins and inhibiting the enzyme HMG-CoA reductase. Thus, the nutraceutical supplement based on a polyphenol matrix according to the invention enables improvement of cholesterolemia by raising HDL cholesterol concentrations at the expense of LDL cholesterol, allowing effective treatment of hypertension and prevention of cardiovascular risks.

In addition, the nutraceutical supplement based on a polyphenol matrix according to the invention inhibits the activity of the enzyme acylcoenzyme A-cholesterol acyltransferase (ACAT), reducing the assembly of VLDL and LDL lipoproteins.

Additionally, it acts on the enzyme phosphatidyl phosphohydrolase (PAP), inhibiting hepatic triglyceride synthesis and consequently reducing triglyceride levels in the liver.

The nutraceutical supplement based on a polyphenol matrix according to the invention also reduces serum leptin levels, implying effective treatment of metabolic syndrome and prevention of diabetes.

In addition, it has a hypoglycemic effect by increasing AMP-activated protein kinase (AMPK) activity and glucose uptake in liver and muscle, improving insulin sensitivity and glycidic tolerance and lipoprotein profile through a mechanism that involves, at least in part, precisely AMPK activation.

Additionally, the nutraceutical supplement based on a polyphenol matrix according to the invention reduces lipid accumulation in cells and adipogenesis by inhibiting resistin release

Again, it attenuates plasma markers of metabolic syndrome. In addition, the nutraceutical supplement based on a polyphenol matrix according to the invention is effective in the treatment of hepatic steatosis and dyslipidemia, which are conditions closely associated with obesity.

Additionally, it also has beneficial effects on the oxidative state, the chronic underlying inflammatory pattern, and metabolic changes associated with visceral fat formation.

Again, the nutraceutical supplement based on a polyphenol matrix according to the invention has no side effects, as no hematochemical signs of toxicity were found in tests conducted by the inventors. In particular, the tests showed no incompatibility with other drugs.

Thus, the nutraceutical supplement based on a polyphenol matrix according to the invention can be an effective alternative to drug treatment for both primary prevention and treatment of visceral fat.

The present invention will now be described, by way of illustration and not limitation, according to its preferred embodiments.

In the following description and claims, the words "equal to approximately" mean "equal to" according to usual industrial tolerances.

The nutraceutical supplement according to the invention comprises or consists of the polyphenol matrix comprising or consisting of:

- ellagic acid, from 4.1% to 20% by weight; and

- hesperidin, from 3.8% to 20% by weight.

- ellagic acid, from 4.1% to 20% by weight; and

- hesperidin, from 3.8% to 20% by weight, in addition to any excipients or additives.

In the preferred embodiments of the nutraceutical supplement according to the invention, in the polyphenol matrix, it has that the ratio polyphenol component : ellagic acid : hesperidin is equal to

10 : x : y wherein:

- x ranges from 3.6 to 6.7, optionally from 4 to 6, more optionally from 4.5 to 5.5, wherein even more optionally x is equal to 5, and - y ranges from 1.8 to 6.7, optionally from 2 to 6, more optionally from 2.5 to 3.5 or from 4.5 to 5.5, wherein even more optionally y is 3 or 5.

Advantageously, the polyphenol component comprises or consists of:

- naringin, from 4% to 16% by weight;

- neohesperidin, from 4% to 16% by weight; and

- neoeriocitrin, from 3.5% to 14% by weight.

Even more advantageously, the polyphenol component comprises or consists of:

- naringin, from 4% to 16% by weight;

- neohesperidin, from 4% to 16% by weight; and

Optionally:

- the polyphenol component can be extracted from bergamot (Citrus x bergamia), more optionally d e. at 38% polyphenols, and/or

- ellagic acid can be extracted from pomegranate, more optionally d.e. at 40% ellagic acid, and/or

- hesperidia can be extracted from bitter orange (Citrus aurantium), more optionally d.e. at 60% hesperidin. In other words, optionally:

- the polyphenol component may be in the form of dry extract (d.e.) of bergamot fruit (Citrus x bergamia), preferably it is bergamot d.e. titrated at 38% total polyphenols, and/or

- ellagic acid may be in the form of dry extract (d.e.) of pomegranate fruit (Punica granatum), preferably a pomegranate d.e. titrated at 40% ellagic acid, and/or

- hesperidin can be in the form of dry extract (d.e.) of bitter orange fruit (Citrus x aurantium), preferably a bitter orange d.e. titrated at 60% hesperidin.

It should be kept in mind, however, that in other embodiments of the polyphenol matrix and nutraceutical supplement according to the invention, the polyphenol component may be obtained differently from extraction from bergamot (Citrus x bergamia) and/or ellagic acid may be obtained differently from extraction from pomegranate and/or hesperidin may be obtained differently from extraction from bitter orange (Citrus aurantium).

In other words, according to other aspects of the invention, the polyphenol matrix, ellagic acid and hesperidin according to the invention, and the supplement of which they are a part, may have a different derivation than the specific dry extracts as previously stated, i.e., bergamot fruit d.e., pomegranate fruit d.e. and bitter orange fruit d.e. respectively.

In other embodiments of the polyphenol matrix according to the invention, the polyphenol component may also comprise:

- brutieridin, from 1% to 4% by weight, and

- melitidin, from 0.5% to 2% by weight. In other embodiments of the polyphenol matrix according to the invention, the polyphenol component may also comprise:

- brutieridin, from 1% to 4% by weight, and

- melitidin, from 0.5% to 2% by weight, also preferably present as a dry extract (d.e.) of bergamot fruit (Citrus x bergamia), preferably a bergamot d.e. titrated at 38% total polyphenols.

In particular, the polyphenol matrix according to the invention can be manufactured by any conventional technique, such as mixing individual components.

The inventors carried out some tests, some of which are shown below, that demonstrated the beneficial effects of the polyphenol matrix according to the invention and the nutraceutical supplement based on it.

The polyphenol component of the polyphenol matrix according to the invention, namely naringin, neohesperidin, and neoeriocitrin, exerts benefits in several areas of human health, such as the nervous system, reducing anxiety and mental stress and improving cognitive function and sleep quality; the cardiovascular system, through a regulation of lipid metabolism; and the treatment of inflammation, diabetes, and skin dysfunction such as psoriasis. In addition, this polyphenol component is rich in flavonoids that regulate antioxidant and anti-inflammatory mechanisms effective on human vessel endothelial cells (HUVECs). In particular, such antioxidant and anti-inflammatory mechanisms are also related to the increased activities of superoxide dismutase and catalase and its ability to protect plasma vitamin E, which, in turn, may well attenuate the overproduction of reactive oxygen species in the vascular wall, thereby restoring the imbalanced endothelial function. In other words, this polyphenol component counteracts oxidative stress and suppresses inflammation in endothelial cells, thereby inhibiting plaque formation and improving arterial reactivity.

Another benefit of the flavonoids of the polyphenol matrix component according to the invention is related to their hypoglycemic activity and their activity to counteract atherosclerosis, as well as their effect of significantly reducing serum cholesterol, triglyceride, LDL levels and increasing HDL levels, particularly in human and animal suffering from pure or mixed hyperlipidemia or not associated with hyperglycemia, i.e., metabolic syndrome. In this regard, naringenin is able to significantly increase AMPK activity and glucose uptake in muscle cells and liver. The hypoglycemic activity of naringenin in vivo may be more complex and may depend on multilevel effects on lipid metabolism leading to increased insulin sensitivity and glucose tolerance.

In the embodiments in which the polyphenol component also includes the two flavonoid conjugates brutieridin and melitidin, it is important to note that these have a structure reminiscent of that of the cholesterol-inhibiting drugs statins: like statins, brutieridin and melitidin contain the hydroxyl mevalonate moiety and bind to the active site for endogenous HMG, thereby inhibiting 3-hydroxy-3-methylglutaryl-CoA Reductase (HMGR), i.e. the enzyme that catalyzes the reduction of HMGCoA to mevalonate, thereby counteracting cholesterol biosynthesis and significantly decreasing cholesterol levels and consequently reducing the risk of cardiovascular disease including stroke. It is known that more than 40% of patients eligible for therapeutic treatment with statins are intolerant to them and prone to severe side effects, such as myalgia, myopathy and liver disease: therefore, the polyphenol matrix according to the invention can be reliably used as a safe and alternative hypolipidemic agent.

Additionally, the polyphenol matrix component according to the invention is capable of improving overall biomarkers of hyperlipemia through reducing serum cholesterol, triglycerides, LDL and small dense lipoproteins, as well as increasing HDL and promoting healthy body weight management. In addition, such the polyphenol component also exerts a positive effect in maintaining normal serum glucose concentrations reducing insulin resistance.

Ellagic acid and hesperidin act as metabolic activators of the polyphenol matrix component according to the invention, as well as acting metabolically on fats.

In particular, due to its antioxidant and anti-inflammatory properties, ellagic acid has beneficial body weight regulation and adiposity reduction effects by regulating lipid metabolism and having an activity to counteract various diseases, such as hepatic steatosis, atherosclerosis, type II diabetes, and obesity. In this regard, the positive effects of ellagic acid on body weight depend partly on its ability to inhibit the activity of pancreatic lipase, an enzyme that plays a key role in fat metabolism, and partly on its ability to reduce hunger.

The nutraceutical supplement based on a polyphenol matrix according to the invention, which is configured to be taken orally, consists of a composition comprising or consisting of the polyphenol matrix according to the invention illustrated above and one or more food additives, wherein the nutraceutical supplement is gastroresistant.

Thus, once taken, the nutraceutical supplement is able to pass the gastric barrier, avoiding causing stomach discomfort, e.g., burning, and to be absorbed with maximum nutritional efficacy at the level of the entero- hepatic circulation, i.e., at the duodenal level up to the liver. The presence of a gastroresistant coating, in fact, allows the actives to be released at the level of the anatomical district that allows maximizing their absorption and, therefore, their bioavailability. In particular, tests performed by the inventors have shown that the nutraceutical supplement according to the invention, unlike the nutraceutical supplements of the prior art, has a modest cytochromic interaction, particularly on cytochrome P450, scientifically documented, and consequently shows a reduced interaction with the active ingredients of most of the actives used in chronic diseases such as antihypertensive anti-diabetic drugs and drugs for metabolic syndrome and dyslipidemia. This is, as mentioned, a significant difference from the prior art nutraceutical supplements for which there is insufficient scientific documentation that they do not interact with cytochromes normally used to metabolize drugs taken to treat concomitant diseases.

Advantageously, said one or more food additives of the nutraceutical supplement based on a polyphenol matrix according to the invention are selected from the group comprising emulsifying additives, thickening additives, diluent or filler additives, anti-caking additives, and stabilizing, gelling and thickening additives. Optionally, said one or more food additives of the nutraceutical supplement based on a polyphenol matrix according to the invention comprise or consist of an emulsifying additive, optionally magnesium salts of fatty acids, from 1 % to 36% by weight, more optionally from 1 % to 4% by weight or from 9% to 36% by weight. In the case where said one or more food additives consist of an emulsifying additive, optionally magnesium salts of fatty acids, from 9% to 36% by weight, the nutraceutical supplement based on a polyphenol matrix according to the invention is in the form of a gastroresistant capsule.

More optionally, said one or more food additives of the nutraceutical supplement based on a polyphenol matrix according to the invention comprise an emulsifying additive, optionally magnesium salts of fatty acids, from 1% to 4% by weight, and also comprise:

- a stabilizing, gelling, and thickening additive, even more optionally microcrystalline cellulose, from 4.1 % to 17% by weight, and

- an anti-caking additive, even more optionally silicon dioxide, from 0.8% to 4% by weight.

In this case, the nutraceutical supplement based on a polyphenol matrix according to the invention is in the form of a gastroresistant tablet.

The following examples further describe the invention.

Example 1

Polyphenol matrix consisting of:

- pomegranate fruit, d.e.at 40% ellagic acid, containing 100 mg of ellagic acid;

- Citrus x aurantium fruit, d.e. at 60% hesperidin, containing 90 mg of hesperidin.

That is, in other words, a polyphenol matrix consisting of:

- 150 mg of dry extract of bitter orange fruit (Citrus x aurantium), specifically a bitter orange fruit d.e. titrated at 60% hesperidin, corresponding to 90 mg of hesperidin.

The inventors commissioned comparative tests against the solid powder composition comprising polyphenol fraction of bergamot and phospholipids, obtained as bergamot extract made according to the teaching of W02018104131 A1 , marketed under the trade name Vazguard® by the Italian company Indena S.p.A.; these comparative tests were carried out by Merieux Nutrisciences at the ECSIN- ECAMRICERT s.r.l. laboratory An initial test involved the comparative evaluation of intestinal absorption of flavonoids.

1250 mg of the polyphenol matrix according to the invention and 1000 mg of the solid powder composition according to WO2018104131 A1 were subjected to the in vitro digestive process with a final digestion volume of 19 mL. The in vitro digestive process is designed in order to simulate the human digestive process, divided into the oral, gastric and intestinal compartments, and is based on the use of fluids simulating the physiological secretions typical of the digestive process, i.e. saliva, gastric juices, pancreatic juices and bile. The above amount of each formulation was weighed and to this was added 3 mL of saliva, pH 6.8. The solution was kept under constant agitation for 5 min at 37 °C. At the end of this incubation, 6 mL of gastric juice simulating fluid, pH 1.3, was added to the digest, and the pH was adjusted to 2.5. The resulting mix was kept under constant agitation at 37°C for 2 hours. Next, 6 mL of fluid simulating intestinal juices, pH 8 1, 3 mL of bile, pH 8.2, and 1 mL of sodium bicarbonate were added and the pH was adjusted to a value of 6.5. The resulting mix was kept under constant agitation at 37°C for another 2 hours. After a centrifugation step, with 2750 g for 5 min, the pellet, equivalent to the excreted fraction, was separated from the supernatant, corresponding to the bioaccessible fraction that was then used for intestinal absorption experiments.

Intestinal absorption of the flavonoids present in the two formulations was determined using an in vitro human intestinal epithelium model based on the co-culture of human intestinal adenocarcinoma Caco-2 (ATCC, HTB-37TM) and Burkitt's lymphoma-derived cells, Raji (ATCC, CCL-86 TM) organized as a functional monolayer on Transwell® inserts. Transwell® inserts are characterized by two compartments separated by a microporous membrane: apical (or luminal) and basolateral (or serosal). As indicated by des Rieux A. et al in "Transport of nanoparticles across an in vitro model of the human intestinal follicle associated epithelium," Eur J Pharm Sci, 2005, Caco-2 cells were seeded in the apical compartment and, after two weeks of maturation, Raji cells were added to the basolateral compartment. The co-culture thus composed was allowed to mature for another week to allow the Raji-conditioned medium to stimulate the differentiation of a portion of the Caco-2 from enterocytes to microfold cells (M cells). Prior to the absorption experiment, the Raji cells were removed from the basolateral compartment, leaving a human follicle- associated epithelium (FAE) characterized by polarized cells with morphological and functional features typical of enterocytes (i.e., presence of microvilli, occluding junctions (tight junction), and presence of glycoprotein-p (P-gp)) and M cells (i.e., transcytosis process).

The impact of digested formulations on the viability of intestinal epithelium was assessed by dose-response curve. Following the digestion process, the supernatant (bioaccessible fraction), resulting from the digestion of the formulations, was diluted in digestive fluids. The different amounts of the polyphenol matrix according to the invention and the solid powder composition according to WO2018104131 A1 (from 0 to 100 % of the bioaccessible fraction) were added to the apical compartment of the intestinal epithelium in vitro, while Hank's Balanced Salt Solution Buffer (HBSS) supplemented with 1% BSA (Bovine Serum Albumin) was added to the basolateral compartment. As a negative control, digestive fluids in the absence of the active were used. After 3 h of incubation, the viability of intestinal epithelia was assessed by MTS assay. In parallel, the impact of the bioaccessible fractions of the analyzed formulations on the barrier integrity of the intestinal epithelium was assessed by measuring the transepithelial electrical resistance (TEER) of the cell monolayer before treatment, immediately after treatment, and at 24 h after exposure.

At the end of the digestion process, the bioaccessible fraction of the two formulations, diluted according to a factor of 1 :4, was added to the apical compartment of the intestinal epithelium in vitro. Digestive fluids devoid of the active were used as a negative control, while HBSS fortified with 1 % BSA was added to the basolateral compartment. After 3 h of exposure, the basolateral fractions were recovered and their content in total flavonoids and hesperidin determined by HPLC analysis. Results are expressed as percent (%) bioavailability relative to the starting amount of total flavonoids and hesperidin applied in the apical compartment

Regarding intestinal absorption of flavonoids, intestinal epithelia in vitro were exposed for 3 hours to the bioaccessible fraction of the two formulations diluted by a factor of 1 :4 (25%). At the end of exposure, the amount of total flavonoids and hesperidin in the basolateral (serosal) compartment were determined. Absorption experiments show that after 3 h of exposure of the intestinal epithelium, in the basolateral (bioavailable) fraction, the amount of flavonoids absorbed (%) is higher following exposure with the polyphenol matrix according to the invention than the value observed after treatment with solid powder composition according to WO2018104131 A1 (0.45 % ± 0.11 % and 0.28 % ± 0.05 %, respectively). In fact, as shown in the following table, the measured values for hesperidin are between LOD and LOQ for polyphenol matrix according to the invention and lower than LOD for solid powder composition according to W02018104131A1. In addition, naringin is absorbed more in the presence of the polyphenol matrix according to the invention than the solid powder composition according to W02018104131A1 : 0.22 % ± 0.09 % and 0.08 % ± 0.04 %, respectively.

Table 1

Based on the results obtained, under the experimental conditions used, the polyphenol matrix according to the invention induces 1.6-fold greater flavonoid absorption than the solid powder composition according to W02018104131A1. The amount of hesperidin in the bioavailable fraction is unquantifiable for the latter, while it is between 0.33 mg/L and 1 mg/L for the polyphenol matrix according to the invention. The flavonoid naringin is also absorbed more in the presence of the polyphenol matrix according to the invention, with a ratio to the solid powder composition according to WO2018104131 A1 of 2.75 times.

A second test involved comparative evaluation of intestinal absorption of exogenous cholesterol using the same in vitro model of human intestinal epithelium based on Caco-2 intestinal adenocarcinoma cells (ATCC, HTB-37TM).

The powder solid composition according to WO2018104131 A1 was resuspended at a concentration of 20 mg/mL in lipid-free culture medium, then sonicated for 5 min and centrifuged for 5 min at 2000 g. The polyphenol matrix according to the invention was resuspended at a concentration of 50 mg/mL in lipid-free medium and subjected to the same protocol used for the solid powder composition according to WO2018104131 A1. Lipid-free culture medium was prepared using a delipidized serum, which was obtained via the protocol described by Yang F. et al. in "Egg-Yolk Sphingomyelin and Phosphatidylcholine Attenuate Cholesterol Absorption in Caco-2 Cells," Lipids 2018, 53(2), 217-233 The actual removal of cholesterol from serum was determined using a commercial kit available from the company Sigma-Aldrich.

To assess the impact of the formulations on viability, Caco-2 cell monolayers were incubated for 24 hours with increasing concentrations of the formulations (from 2.5 to 20 mg/mL for the solid powder composition according to WC2018104131A1 and from 0.05 to 50 mg/mL for the polyphenol matrix according to the invention). At the end of incubation, the viability of intestinal epithelial monolayers was assessed by MTS assay, based on the reduction of the MTS tetrazolium compound by viable cells to generate the colored product formazan, which can be quantified by measuring its absorbance at 490 nm using a plate reader (Synergy4, Biotek). Cell viability is expressed as the % ratio of the absorbance of treated cells to the untreated control. Subsequent efficacy experiments were carried out using the highest nontoxic concentration.

To comparatively evaluate the effect of the polyphenol matrix according to the invention and the solid powder composition according to WO2018104131 A1 on intestinal absorption, Caco-2 cell monolayers were treated for 24 h with the highest nontoxic concentration selected for the 2 formulations, and NPC1 L1 receptor expression was determined at the protein level by western blot. Curcumin at a concentration of 250 DM was used as a positive control for the reduction of NPC1L1 expression.

After the 24-hour treatment, Caco-2 cells were detached from the microporous membrane with trypsin, lysed and sonicated. The protein concentration in the cell lysates was determined by Bradford assay. An equal amount of protein from the various samples was subjected to gel electrophoresis under denaturing conditions (SDS-PAGE) to allow separation of the proteins in the sample for analysis. Next, the proteins in the gel were transferred to a nitrocellulose membrane, and the presence of the NPC1 L1 receptor was assessed with specific antibody (Sigma- Aldrich), p-actin expression measured by specific antibody was used as a loading control to normalize NPC1 L1 levels observed in the western blot The specific signals of NPC1 L1 and (3-actin were visualized by a chemiluminescence reaction, and the amount of NPC1 L1 normalized to p-actin was determined by Imaged software. The impact on cell viability of Caco-2 cell monolayer was evaluated after treatment with different concentrations for 24 hours. Plasma cholesterol concentration is regulated at endogenous and exogenous levels. In the endogenous signaling pathway, cholesterol is regulated in the liver and extrahepatic tissues, entering the bloodstream as a component of lipoproteins or excreted through secretion into the bile. The exogenous pathway ensures the supply of cholesterol from food to the intestine. The main player in this pathway is the NPC1 L1 (Niemann-PickC1-Like 1) protein. NPC1L1 is a transmembrane protein located in the apical membrane of enterocytes that acts as a sterol transporter. The impact of formulations in modulating the absorption of exogenous cholesterol was analyzed by evaluating their effect on NPC1L1 expression; Fold-change was calculated compared with the monolayer of untreated Caco-2 (NT) cells As shown in the table below, the polyphenol matrix according to the invention and the solid powder composition according to WO2018104131 A1 similarly decreased the expression levels of NPC1L1 (0.58 ± 0.19 for the latter and 0.53 ± 0.30 for the polyphenol matrix according to the invention). As expected, curcumin 250 pM reduces protein expression of NPC1L1 (0.065 ± 0.002), confirming the responsiveness of the in vitro model used.

Table 2

The results of the comparative test show that both the polyphenol matrix according to the invention and the solid powder composition according to W02018104131 A1 reduce the expression levels of the NPC1 L1 transporter by about half compared with those in untreated cells. The receptor expression values obtained by treating cells with the two formulations are comparable to each other Reduction of the NPC1L1 receptor causes the subsequent reduction of cholesterol uptake exogenously, from food. Although their efficacy in terms of cholesterol uptake can be considered overlapping from the test results, however, the presence of hesperidin in the polyphenol matrix according to the invention versus the solid powder composition according to W02018104131 A1 is a discriminating element that makes the beneficial spectrum in terms of metabolic disorder and presence of comorbidities of the former broader than that of the latter.

In other words, and as previously indicated in the description of the present application, the demonstration of the ability of the polyphenol matrix of the present invention to reduce the expression levels of the NPC1 L1 transporter in an amount comparable to that determined by the composition according to the document of the prior art, should be coupled with the fact that the other components (hesperidin and ellagic acid) peculiar to the matrix of the present invention also provide other benefits, which complement those of the polyphenol component, because they act as its metabolic activators and contribute to the determination that the matrix of the invention does not affect cytochrome P450 activities.

Example 2

Nutraceutical supplement in gastroresistant tablets, each weighing 1200 mg and consisting of:

- 190 mg of polyphenol component in 500 mg of bergamot {Citrus x bergamia) extract, d.e. at 38% polyphenols, containing 40 mg of naringin, 40 mg of neohesperidin, 35 mg of neoeriocitrin, 10 mg of brutieridin and 5 mg of melitidin;

- 250 mg of pomegranate fruit, d.e. at 40% ellagic acid, containing 100 mg of ellagic acid;

- magnesium salts of fatty acids, 20 mg;

- carboxymethyl cellulose, 10 mg;

- dibasic calcium phosphate, 150 mg;

- microcrystalline cellulose, 100 mg; and

- silicon dioxide, 20 mg.

Example 3

Nutraceutical supplement in gastroresistant capsules, each weighing 595 mg and consisting of:

- 95 mg of polyphenol component in 250 mg of bergamot {Citrus x bergamia) extract, d.e. at 38% polyphenols, containing 20 mg of naringin, 20 mg of neohesperidin, 17.5 mg of neoeriocitrin, 5 mg of brutieridin and 2.5 mg of melitidin;

- 125 mg of pomegranate fruit, d.e. at 40% ellagic acid, containing 50 mg of ellagic acid;

- magnesium salts of fatty acids, 95 mg.

Example 4

- 500 mg of dry extract of bergamot {Citrus x bergamia), specifically a bergamot fruit d.e. titrated at 38% total polyphenols corresponding to 190 mg of total polyphenols comprising 40 mg of naringin, 40 mg of neohesperidin, 35 mg of neoeriocitrin, 10 mg of brutieridin and 5 mg of melitidin;

- 250 mg of dry extract of pomegranate fruit {Punica granatum), specifically a pomegranate fruit d.e. titrated at 40% ellagic acid, corresponding to 100 mg of ellagic acid;

- 150 mg of dry extract of bitter orange fruit {Citrus x aurantium), specifically a bitter orange fruit d.e. titrated at 60% hesperidin, corresponding to 90 mg of hesperidin;

-magnesium salts of fatty acids, 20 mg;

-carboxymethyl cellulose, 10 mg; -dibasic calcium phosphate, 150 mg;

-microcrystalline cellulose, 100 mg; and

-silicon dioxide, 20 mg.

Example 5

- 250 mg of dry extract of bergamot fruit {Citrus x bergamia), specifically a bergamot fruit d.e. titrated at 38% total polyphenols corresponding to 95 mg of total polyphenols comprising 20 mg of naringin, 20 mg of neohesperidin, 17.5 mg of neoeriocitrin, 5 mg of brutieridin and 2.5 mg of melitidin ;

- 125 mg of dry extract of pomegranate fruit {Punica granatum), specifically a pomegranate fruit d.e. titrated at 40% ellagic acid, corresponding to 50 mg of ellagic acid;

- 125 mg of dry extract of bitter orange fruit (Citrus x aurantium), specifically a bitter orange fruit d e. titrated at 60% hesperidin, corresponding to 75 mg of hesperidin;

- magnesium salts of fatty acids, 95 mg.

In this regard, since the tablets in Examples 2 and 4 and the capsules in Examples 3 and 5 are gastroresistant, the results of the tests conducted on the polyphenol matrix in Example 1 are also valid for them.

In the foregoing, preferred embodiments have been described and variants of the present invention have been suggested, but it is to be understood that experts in the field may make modifications and changes without thereby departing from the relevant scope of protection as defined by the appended claims.

Example 6

Determination of flavonoid content in starting materials and polyphenol mixture according to the present invention.

Procedure: Glycosidic flavonoid content is determined by reversed-phase high-performance liquid chromatography (RP-HPLC) using a diode array detector (DAD). The sample is extracted in solvent in a 40 mL vial, a known amount of sample is weighed and dissolved in:

- 10mL of MeOH 80% for the determination of flavonoids expressed as naringin, or

- 10 mL of DMSO for the determination of flavonoids expressed as hesperidin.

The sample is placed in an ultrasonic bath for 15 minutes and then filtered into vials with 0.22 pm PVDF filters (in the case of MeOH 80%) or 0.2 pm PTFE filters (in the case of DMSO).

If necessary to make reading by the instrument possible, appropriate dilutions are made.

Operating conditions: injection volume: 5pL column: C18 4.6x250mm 5pm column temperature: 35°C flow rate: 1mL/minute

A: 280nm Gradient elution with: Phase A: water + 0.1% HCOOH; Phase B: acetonitrile

Standard reference materials

Naringin: CAS 10236-47-2, Sigma-Aldrich - 71162 - lot BCBM4171V.

Hesperidin: CAS 520-26-3, Sigma-Aldrich - PHR1794 - lot LRAC5710.

The quantification of flavonoid assay (g/100g) as naringin or hesperidin is done by the external standard method results provided by HPLC-DAD software, using the following formula: (g/100g) = (C_TS x V)/(w x 10) where: is the concentration of flavonoid (naringin Na, hesperidin He, neohesperin neoHe, neoeriocitrin neoEr, brutieridin Bru, melitidin Mel) expressed as naringin or hesperidin in the sample (g/100g);

CTS is the concentration of flavonoid (naringin, hesperidin, neohesperin, neoeriocitrin, brutieridin, melitidin) in the test solution (mg/L), provided by the HPLC-DAD software;

V is the volume of the test solution (L); w is the weight of the sample weighed to prepare the test solution (g);

10 is the conversion factor to g/100g.

Using the previously described procedure, the following were tested, with extraction in both MeOH80% and DMSO:

- the polyphenol fraction of the dry extract of bergamot (Citrus x bergamia) (d.e. Ber);

- the dry extract of pomegranate (Punica granatum) (d.e. Mel);

- the dry extract of bitter orange (Citrus x aurantium) (d.e. Ar);

- the polyphenol mixture of the invention as described in Example 1 (MIX).

Results: The following table (Table 3) shows the values obtained from sample analysis, expressed as g/100g.

/ not integrable due to interference between peaks; n.d. not detected

Table 3

Conclusions: The work carried out involved a chemical characterization of the profile of flavonoids present in each raw material and in the finished product, which consists of the mixture of the 3 raw materials, i.e., dry extract of bergamot (d.e. Ber), dry extract of pomegranate (d.e. Mel) and dry extract of bitter orange (d.e. Ar). The study showed that the profile of the mixture according to the present invention is compatible with the profile of the raw materials, there is no interference or loss of actives in the mixture. In addition, the profile of the mixture in terms of flavonoids is more complete than the profile of the individual raw materials, consequently the intake of the mixture ensures a more complete supply in terms of flavonoids than the individual raw materials.

Claims

1 . A polyphenol matrix comprising or consisting of:

- ellagic acid, from 4.1% to 20% by weight; and

2. The polyphenol matrix according to claim 1 , wherein the ratio polyphenol component : ellagic acid : hesperidin is equal to

10 : x : y wherein:

- y ranges from 1.8 to 6.7, optionally from 2 to 6, more optionally from 2.5 to 3.5 or from 4.5 to 5.5, wherein even more optionally y is 3 or 5.

3. The polyphenol matrix according to claim 1 or 2, wherein the polyphenol component comprises or consists of:

- naringin, from 4% to 16% by weight;

- neohesperidin, from 4% to 16% by weight; and

- neoeriocitrin, from 3.5% to 14% by weight, in addition to any other polyphenols.

4. The polyphenol matrix according to any one of the preceding claims, wherein:

- the polyphenol component is in the form of dry extract (d.e.) of bergamot fruit (Citrus x bergamia), preferably it is a bergamot d.e. titrated at 38% total polyphenols, and/or

- ellagic acid is in the form of dry extract (d.e.) of pomegranate fruit (Punica granatum), preferably a pomegranate d.e. titrated at 40% ellagic acid and/or

- hesperidin is in the form of dry extract (d.e.) of bitter orange fruit (Citrus x aurantium), preferably a bitter orange d.e. titrated at 60% hesperidin.

5. The polyphenol matrix according to any one of the preceding claims, wherein the polyphenol component also comprises:

- brutieridin, from 1% to 4% by weight, and

- melitidin, from 0.5% to 2% by weight, preferably, said brutieridin and said melitidin are present in the form of dry extract (d.e ) of bergamot fruit (Citrus x bergamia), preferably a bergamot d e. titrated at 38% total polyphenols.

6. A nutraceutical supplement based on a polyphenol matrix configured to be taken orally, consisting of a composition comprising or consisting of the polyphenol matrix according to any one of claims 1 to 5 and one or more food additives, wherein the nutraceutical supplement is gastroresistant.

7. The nutraceutical supplement based on a polyphenol matrix according to claim 6, wherein said one or more food additives are selected from the group comprising emulsifying additives, thickening additives, diluent or filler additives, anti-caking additives, and and stabilizing, gelling and thickening additives.

8. The nutraceutical supplement based on a polyphenol matrix according to claim 7, wherein said one or more food additives comprise or consist of an emulsifying additive, optionally magnesium salts of fatty acids, from 1% to 36% by weight.

9. The nutraceutical supplement based on a polyphenol matrix according to claim 7, wherein said one or more food additives consist of an emulsifying additive, optionally magnesium salts of fatty acids, from 9% to 36% by weight, and the nutraceutical supplement based on a polyphenol matrix is in the form of a gastroresistant capsule.

10 The nutraceutical supplement based on a polyphenol matrix according to claim 9, wherein each gastroresistant capsule weighs 595 mg and consists of:

- 125 mg of dry extract of pomegranate fruit (Punica granaturri), specifically a pomegranate fruit d.e. titrated at 40% ellagic acid, corresponding to 50 mg of ellagic acid;

- 125 mg of dry extract of bitter orange fruit (Citrus x aurantium), specifically a bitter orange fruit d.e. titrated at 60% hesperidin, corresponding to 75 mg of hesperidin;

- magnesium salts of fatty acids, 95 mg.

11. The nutraceutical supplement based on a polyphenol matrix according to claim 7, wherein said one or more food additives comprise or consist of:

- a stabilizing, gelling, and thickening additive, even more optionally microcrystalline cellulose, from 4.1% to 17% by weight, and,

- an anti-caking additive, even more optionally silicon dioxide, from 0.8% to 4% by weight, and the nutraceutical supplement based on a polyphenol matrix is in the form of a gastroresistant tablet.

12. The nutraceutical supplement based on a polyphenol matrix according to claim 11 , wherein each gastroresistant tablet weighs 1200 mg and consists of: - 500 mg of dry extract of bergamot (Citrus x bergamia), specifically a bergamot fruit d.e. titrated at 38% total polyphenols corresponding to 190 mg of total polyphenols comprising 40 mg of naringin, 40 mg of neohesperidin, 35 mg of neoeriocitrin, 10 mg of brutieridin and 5 mg of melitidin;

- 250 mg of dry extract of pomegranate fruit (Punica granatum'j, specifically a pomegranate fruit d.e. titrated at 40% ellagic acid, corresponding to 100 mg of ellagic acid;

- magnesium salts of fatty acids, 20 mg;

- carboxymethyl cellulose, 10 mg; - dibasic calcium phosphate, 150 mg;

- microcrystalline cellulose, 100 mg; and

- silicon dioxide, 20 mg.

13 The nutraceutical supplement according to any one of claims 6 to 12 for use in the prevention and/or treatment of at least one of the diseases or conditions selected from the group comprising or consisting of states of inflammation, diabetes, visceral fat, hypercholesterolemia, hypertension, hepatic steatosis, dyslipidemia, obesity, and cardiovascular risks.