A composition comprising myrtus communis extract and OLEA europea extract for the maintenance and / or in the restoration of physiological blood lipid levels

Abstract

The present invention relates to the use of an association, comprising a hydroalcoholic extract from the leaves of Myrtus communis, a dried extract from the fruits and / or leaves of Olea europea, for the physiological maintenance of blood lipid levels.

Description

[0001] TITLE

[0002] A COMPOSITION COMPRISING MYRTUS COMMUNIS EXTRACT AND OLEA EUROPEA EXTRACT FOR THE MAINTENANCE AND / OR IN THE RESTORATION OF PHYSIOLOGICAL BLOOD LIPID LEVELS

[0003] DESCRIPTION FIELD OF THE INVENTION

[0004] 5 The present patent application relates to an association of two plant extracts, that of myrtle and that of olive, which allows for controlling blood lipid levels. In particular, the association has a cholesterol-lowering function.

[0005] STATE OF THE ART

[0006] For some time, the need has been felt to control lipid levels, particularly total 10 cholesterol, whether in the form of LDL or HDL, through the intake of food supplements.

[0007] For example, for the control of LDL cholesterol levels, the reference "gold standard" is red yeast rice containing monacolins.

[0008] Problem of the known art

[0009] The European Commission is questioning the safety of the use of these ingredients, in particular of monacolin K, which can present the adverse reactions typical of synthetic statins.

[0010] After a long regulatory process, on October 5, 2021, the proposal to amend Regulation (EC) 1925 / 2006 was examined, which provides for:

[0011] 20 • the inclusion of monacolins from red yeast rice among the substances subject to restrictions and among those subject to surveillance (parts B and C of Annex III of the Regulation);

[0012] • the reduction to less than 3 mg of the permitted daily dose of the product.

[0013] In view of the entry into force of the new regulation, operators in the sector are looking 25 for valid alternatives to red yeast rice extract, capable of guaranteeing an equal or superior cholesterol-lowering action compared to that of red yeast rice extract, in addition to solid safety of use.

[0014] SUMMARY OF THE INVENTION

[0015] A first object of the invention is the use of an association comprising- a hydroalcoholic extract from the leaves of Myrtus communis,

[0016] - a dried extract from the fruits and / or leaves of Olea europea,

[0017] for the physiological maintenance of blood lipid levels.

[0018] Said association can also be useful for treating dyslipidemia and / or hypercholesterolemia. A further object of the invention is an oral formulation which comprises

[0019] - said association, being the only active ingredient in the oral formulation, and - suitable excipients and / or diluents,

[0020] or, alternatively,

[0021] - said association, being a first active ingredient, and

[0022] - at least a second lipid-lowering and / or cholesterol-lowering active ingredient, other than said association, and chosen from the group consisting of: plant extracts, such as fenugreek extract; vitamins and / or minerals; berberine; policosanols; phytosterols and plant sterols; and combinations of the foregoing,

[0023] - suitable excipients and / or diluents.

[0024] Advantages of the invention

[0025] The claimed association constitutes a valid alternative to red yeast rice extract.

[0026] The Applicant has demonstrated this property of the association through the development of a cellular experimental model to simulate the condition of hypercholesterolemia and to verify the biochemical effects of the two extracts, analyzed individually and in association, comparing them to the current nutraceutical gold standard, red yeast rice (for brevity also indicated below as " RYR", from the English " Red Yeast Rice").

[0027] As detailed in the Examples section, after determining the maximum non-cytotoxic concentration of the natural extracts in human HepG2 cells, the following targets were evaluated:

[0028] • regulation of the expression levels of the HMG-CoA Reductase, LDL Receptor and PCSK9 genes;

[0029] • modulation of the levels of the PCSK9 protein secreted in the culture medium;

[0030] • evaluation of the modulation of the intracellular catalytic activity of the native HMG-CoA reductase enzyme.The Applicant has investigated the modulation effects of the mRNA levels transcribed from the HMG-CoA Reductase, LDL Receptor and PCSK9 genes in human hepatocytes HepG2 (section 4.2, Figures 3A-3C).

[0031] All treatments did not modulate the gene expression of the LDL receptor (Figure 3B).

[0032] The association of Myrtus and Olea extracts was able to keep the gene expression levels of HMG-CoA reductase unchanged, while Red Yeast Rice more than doubled the gene expression of the target enzyme (Figure 3C).

[0033] Regarding PCSK9, only the association of Myrtus and Olea extracts has been shown to be able to significantly reduce the gene expression levels of the protein (Figure 3 A). This latter data concerning the expression of the PCSK9 protein was confirmed by the statistically significant inhibition of the levels of PCSK9 protein secreted by the association not only with respect to the Red Yeast Rice extract, but also with respect to the two extracts taken individually (section 4.3, table 3).

[0034] The latter data concerning the statistically significant inhibition of the levels of PCSK9 protein secreted by the association is of particular importance since, according to the Applicant, it demonstrates that the plant extracts are able to reduce the levels of protein secreted in the medium, and this means that the effects on the cellular model are able to provide a significant modulation of the different transcriptional and translational processes; this is uncommon evidence in nutraceutics, not even when it comes to in vitro experimental tests.

[0035] Furthermore, although the Myrtus extract inhibited the intracellular catalytic activity of the HMG-CoA reductase enzyme with the same potency as Red Yeast Rice (at the same dose), it is the association of Myrtus and Olea that has inhibited the activity of the enzyme in a superior manner with respect to red yeast rice (section 4.4, Figure 6C).

[0036] DESCRIPTION OF THE FIGURES

[0037] Figures 1A-1D - Cytotoxicity assays performed in HepG2 cells after 24h of treatment with the dose response of Myrtus communis (0 - 1000 pg / ml) (Figure 1A), with the dose response of Olea europea (0 - 1000 pg / ml) (Figure IB), with the combination between the maximum non-cytotoxic concentration of Myrtus communis (62.5 pg / ml) and the maximum non-cytotoxic concentration of Olea europea (62.5 pg / ml) (Figure 1C); with the Red Yeast Rice extract RYR (62.5 pg / ml) (Figure ID). The results are expressed as a percentage of cell viability with respect to the cell viability of cells treated with 0.4% DMSO (0 pg / ml, set at 100% viability) and presented as mean ± SDof three independent experiments (n=3) performed in quadruplicate. **p<0.01; *** p <0.001, evaluated by One-way ANOVA.

[0038] Figure 2 - Scheme of the reverse transcription of RNA into cDNA by reverse transcriptase.

[0039] Figures 3A-3C - Determination of the modulation of the gene expression levels of the molecular targets PCSK9 (Figure 3A), LDL Receptor (Figure 3B) and HMG-CoA Reductase (Figure 3C) in HepG2 cells after 24h treatment with 30 mM glucose+M. C.

[0040] 62.5 pg / ml, 30 mM glucose+O. E. 62.5 pg / ml, 30 mM glucose+M. C. 62.5 pg / ml+O. E.

[0041] 62.5 pg / ml, 30 mM glucose+RYR 62.5 pg / ml, comparing the results with the " Glucose 30 mM" treatment.

[0042] Figure 4 - Evaluation of the modulation of the gene expression levels of the molecular targets PCSK9 (Panel D), LDL Receptor (Panel E) and HMG-CoA Reductase (Panel F) in HepG2 cells after 24h treatment with 30 mM glucose, comparing the results with the NT (Non-Treated) condition. The results are presented as mean value ± SD of three independent experiments (n=3) performed in triplicate. *p<0.05, **p<0.01, evaluated by paired Student's t-Test.

[0043] Figure 5 - Scheme of the phases of the experimental protocol of an ELISA assay: the "capture antibody" interacts with the target protein present in the sample at a specific epitope, then the primary (detection)+biotin antibody interacts with another specific epitope of the target protein and the streptavidin HRP complex binds to the biotin. Finally, the addition of the colorless substrate TMB allows the ELISA assay reaction to develop, leading to the formation of a blue product in the wells of the 96-well plate; the Absorbance data will then be acquired at λ=450 nm.

[0044] Figures 6A-6C - Determination of the inhibition of the catalytic activity of recombinant HMG-CoA Reductase by Pravastatin 1 pM (Figure 6A); induction of the catalytic activity of native intracellular HMG-CoA Reductase of HepG2 after 24h treatment with 30 mM glucose (Figure 6B); evaluation of the inhibition of the catalytic activity of native intracellular HMG-CoA Reductase of HepG2 after 24h treatment with 30 mM glucose+M. C. 62.5 pg / ml, 30 mM glucose+O. E. 62.5 pg / ml, 30 mM glucose+M. C. 62.5 pg / ml+O. E. 62.5 pg / ml, 30 mM glucose+RYR 62.5 pg / ml (Figure 6C). The results are presented as mean value ± SD of three independent experiments (n=3) performed in duplicate. *p<0.05; **p<0.01; ***p<0.001, evaluated by paired Student's t-Test.

[0045] Figure 7 - Representation of the layout of the in vivo study of Example 5.DETAILED DESCRIPTION OF THE INVENTION

[0046] In the following, the invention and its preferred embodiments are described in more detail. It should be noted that, although the structure has been organized into paragraphs and subparagraphs, the information contained in each paragraph or subparagraph is not isolated, and may be combined with that contained in other paragraphs or, more generally, with other information contained in the text of the patent application.

[0047] Association of the invention

[0048] For the purposes of the invention, by association is meant a combination or mixture which comprises or consists of two elements, corresponding to the two plant extracts described below.

[0049] The association comprises or consists of

[0050] - a hydroalcoholic extract from the leaves of Myrtus communis,

[0051] - a dried extract from the fruits and / or leaves of Olea europea,

[0052] and is for use in the treatment of dyslipidemia and / or hypercholesterolemia.

[0053] Preferably, the weight ratio of the amount of the Myrtus communis extract to the amount of the Olea europea extract is ranging between 1:0.5 and 1:2, preferably between 1:0.7 and 1:2, preferably between 1:0.8 and 1:2, preferably between 1:1 and 1:2, preferably between 1:1 and 1:1.5, preferably between 1:1 and 1:1.2.

[0054] It should be noted that said weight ratio impacts the effect of regulating lipid levels, especially cholesterol, in the blood, as well as the synergistic effect between the two extracts.

[0055] Preferably, the association does not comprise: red yeast rice extract; monacolins. For the purposes of the present invention, by extracts are meant, in general, products of plant origin which are obtained from medicinal plants and herbs of various types. The parts of the plant that can be used to obtain these products are multiple: leaves, flowers, fruits (pulp and / or peel), seeds, roots, rhizomes, bark, etc. They can be preparations in liquid form, solid form (dried extracts) or of intermediate consistency (soft extracts), obtained by suitable extraction processes; the latter include the use of appropriate solvents, supercritical extraction, the use of maceration or percolation, or other suitable procedures.Myrtus communis extract

[0056] Pharmacology studies have shown that aqueous and ethanolic extracts of myrtle leaves possess anti-inflammatory, analgesic, antidiabetic, antimicrobial and antiviral, and antioxidant effects, particularly for cardiovascular health [2], Studies on the antioxidant effects have confirmed that hydroalcoholic extracts of myrtle leaves show a greater antioxidant power compared to extracts with ethyl acetate or aqueous solvents, from the same part of the plant. The hydroalcoholic solvent is, in fact, able to extract more polyphenols, in particular galloyl-derivatives, to which the antioxidant effects have been ascribed.

[0057] In addition to the known flavonoids (such as quercetin and myricetin), phenolic acids, tannins and tocopherols, myrtle leaves contain structurally unique non-prenylated oligomeric acylphloroglucinols, such as semimyrtucommulone and myrtucommulone A. These molecules have shown potent protective effects in simplified models of oxidative degradation of cholesterol and LDL, two processes that are known to play a role in cardiovascular and / or dysmetabolic pathologies. These plant phenols qualify as dietary antioxidants with an interesting antiatherogenic potential [3], Preferably, the Myrtus communis extract is a soft, liquid, or dried extract from the leaves of Myrtus communis.

[0058] Soft extracts are preparations of intermediate consistency between liquid extracts and dried extracts. They are obtained by partial evaporation of the liquids or extractive solvents. Preferably, the dry residue is preferably comprised between 20% and 80% by weight, preferably between 30% and 70% by weight, of the total weight of the extract.

[0059] The Myrtus communis extract is a hydroalcoholic extract from the leaves of Myrtus communis, preferably, the ethanol, as an extraction solvent, is in a quantity comprised between 40% and 60% by weight, preferably 50% by weight.

[0060] Preferably, the Myrtus communis extract is a dried hydroalcoholic extract from the leaves of Myrtus communis.

[0061] Preferably, for the dried hydroalcoholic extract from the leaves of Myrtus communis, its dry residue is preferably comprised between 20% and 80% by weight, preferably between 30% and 70% by weight, of the total weight of the extract.

[0062] Preferably, the soft or liquid extract of Myrtus communis can be obtained by extraction according to the following procedure:- the leaves of the plant are prepared, pulverized or in herbal tea cut, and are mixed with a hydroalcoholic solvent, preferably the hydroalcoholic solvent is in a ratio from 1:1 to 1:25, preferably from 1:3 to 1:20, more preferably from 1:6 to 1:15;

[0063] - then, under stirring, it is heated to a temperature in the range from 30°C to 90°C, preferably from 40°C to 80°C, more preferably from 50°C to 70°C, for a period of time from 20 minutes to 240 minutes, preferably from 30 to 180 minutes, more preferably from 40 minutes to 120 minutes, until the crude extract is obtained which comprises a solid phase and a liquid phase;

[0064] - subsequently, the liquid phase of the crude extract is separated from the solid phase to obtain the isolated liquid phase corresponding to the liquid extract; preferably, the isolated liquid phase can be concentrated by completely or partially removing the solvent, for example by evaporation, until a soft extract is obtained.

[0065] Preferably, when the solvent is partially removed, a soft extract is obtained, the dry residue of which is preferably comprised between 20% and 80% by weight, preferably between 30% and 70% by weight, of the total weight of the extract.

[0066] The dried extract of Myrtus communis is obtained preferably from the drying of the soft extract by fluid bed granulation or spray drying with suitable excipients, such as maltodextrins or colloidal silica, to obtain a homogeneous granular or fine powder. Preferably, the Myrtus communis extract has a content in polyphenols, for example in myricetin, comprised between 0.1% and 5%, preferably between 0.5% and 4%, more preferably between 1% and 3%, preferably equal to 2% by weight of the total weight or volume of the extract.

[0067] Preferably, the Myrtus communis extract is in a quantity comprised between 30% and 70% by weight, preferably between 35% and 65% by weight, preferably between 40% and 50% by weight, preferably between 42% and 50% by weight, of the total weight of the association.

[0068] Preferably, the myricetin is in a quantity comprised between 0.05% and 2.5% by weight, preferably between 0.25% and 2% by weight, preferably between 0.5% and 1.5% by weight, preferably between 0.8% and 1.2% by weight, preferably equal to 1% by weight, of the total weight of the association.

[0069] Olea europea extract

[0070] The use of olive derivatives for cardiovascular health in humans is well known, so much so that, in 2011, EFSA approved a health claim for olive oil polyphenols, whichcan boast the protection of LDL particles from oxidative damage for intakes of at least 5 mg of hydroxytyrosol. Furthermore, a recent review of randomized clinical studies has evaluated the effects of olive oil consumption, in particular for its polyphenol content, on blood lipid parameters, concluding that the food is able to improve circulating HDL (with consequent protective effects on cardiovascular health) and to reduce LDL cholesterol levels for high daily consumption [4],

[0071] The plant extract of Olea europea is able to act on the PCSK9 protein, which is involved in the degradation of the LDL receptor. The inhibition of PCSK9 increases the levels of LDL receptor in the liver, increasing the intracellular catabolism of LDL, thus favoring the reduction of circulating levels of LDL cholesterol. The scientific community assumes that this is the mechanism underlying the effectiveness of Berberine in reducing cholesterolemia. A study has evaluated the ability of a phenolic complex derived from olive vegetation waters, titrated in hydroxytyrosol, tyrosol and verbascoside, for its ability to inhibit the expression and protein synthesis of PCSK9 in a cellular model that simulated the condition of hypercholesterolemia [5], Preferably, the Olea europea extract is a dried extract, preferably it is a dried extract obtained from the fruits and / or leaves of Olea europea, preferably from the fruits of Olea europea.

[0072] The Applicant has noted that, compared to a liquid extract of Olea europea, the dried extract of Olea europea is more easily processable from a technological point of view, for example in order to combine it with the Myrtus extract when the latter is in liquid or soft form.

[0073] Preferably, the dried extract of Olea europea can be, for example, obtained by the following preparation: the fruits of Olea europea are minced to facilitate extraction; the prepared plant matrix is subjected to a maceration or dynamic extraction process using a mixture of water and ethyl alcohol as a solvent. The extracted liquid is separated from the residual solid fraction by filtration or centrifugation. The filtrate is then concentrated under vacuum using a rotary evaporator or an industrial thin-film plant, in order to remove the excess solvent and obtain a denser extract. To enrich the content of the secondary metabolites of interest, the extract can be subjected to purification techniques such as ion exchange resins (to selectively retain the phenolic compounds) or industrial-scale liquid chromatography (to further fractionate the extract). The concentrated extract is preferably dried by fluid bed granulation or by spray drying with suitable excipients, such as maltodextrins or colloidal silica, to obtain a homogeneous granular or fine powder.Preferably, the dried extract of Olea europea has a hydroxytyrosol content comprised between 2% and 15% by weight, preferably between 2% and 10% by weight, preferably between 3% and 10% by weight, preferably equal to 5% by weight of the total weight of the extract.

[0074] Preferably, the Olea europea extract is in a quantity comprised between 30% and 70% by weight, preferably between 35% and 65% by weight, preferably between 40% and 60% by weight, preferably between 45% and 58% by weight, of the total weight of the association.

[0075] Preferably, the hydroxytyrosol is in a quantity comprised between 1% and 7.5% by weight, preferably between 1% and 5% by weight, preferably between 1.5% and 5% by weight, preferably between 2% and 3% by weight, preferably equal to 2.5% by weight, of the total weight of the association.

[0076] Physical form of the association of the invention

[0077] Preferably, the association is in liquid or solid physical form.

[0078] According to a first preferred form, when the association is in solid form, the Olea europea extract is in solid form and the Myrtus communis extract is in solid form. In accordance with a second preferred form, when the association is in solid form, the Olea europea extract is in solid form and the Myrtus communisextract is in liquid or soft form. For this second preferred form, the association is obtained by granulating the soft myrtle extract on the dried olive extract; in other words, the soft myrtle extract is sprayed on the dried olive extract by fluid bed granulation.

[0079] Preferably, when the association is in solid form, the association may also comprise additional inert components, in the sense that they do not impact the modulation effect of blood lipid levels and / or the synergy between the two plant extracts.

[0080] Preferably, the additional inert components can be of an organic or inorganic nature; preferably, the additional inert components are solid; preferably, the additional inert components act as adsorbents.

[0081] For example, the additional inert components can be selected from: silica, calcium carbonate, calcium phosphate, maltodextrins, polydextrose, polyols and sugars, raw materials of plant origin, and combinations of the foregoing.

[0082] Preferably, the solid and adsorbent raw materials of plant origin are known to the person skilled in the art; for example, they are selected from the group consisting of:cellulose derivatives, plant fibers, gum arabic or acacia fiber, starches, inulin, and combinations of the foregoing.

[0083] Preferably, the additional inert components are in a quantity < 20% by weight, preferably between 1% and 15% by weight, preferably between 2% and 8% by weight, of the total weight of the association.

[0084] It should be noted that other compounds which have not been expressly indicated herein, but which possess the same function as additional inert components, are in any case within the scope of the invention.

[0085] Use of the association of the invention

[0086] Physiologically, for a subject (human) who is in good health, the total cholesterol is < 200 mg / dl; the fractionated LDL cholesterol up to 100 mg / dl; the fractionated HDL cholesterol is not, however, lower than 50 mg / dl.

[0087] Physiologically, for a subject (human) who is in good health, the blood level of triglycerides is lower than 150 mg / dl.

[0088] Preferably, a subject (human) with altered cholesterol emia (or borderline), for whom, for example, the association or formulation of the invention is intended, is a subject with a total plasma cholesterol value comprised between 200 and 239 mg / dl.

[0089] Preferably, a subject (human) with altered cholesterol emia, for whom, for example, the association or formulation of the invention is intended, is a subject with an LDL cholesterol value < 159 mg / dl, preferably between 130 and 159 mg / dl.

[0090] In humans, a high LDL cholesterol value is preferably comprised between 160 and 189 mg / dL. A very high LDL cholesterol value is > 190 mg / dL [National Cholesterol Education Program (NCEP) “Executive Summary of the Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III)" (Revision_2013), https: / / www.ncbi.nlm.nih.gov / books / NBK542294 / #article-19466.r18].

[0091] For the purposes of the invention, by lipid is meant one selected from the group consisting of total cholesterol, LDL cholesterol, HDL cholesterol, triglyceride, and combinations of the foregoing.

[0092] The association or the oral formulation are for use in the maintenance of physiological lipid levels, for example of total cholesterol and / or LDL.For the purposes of the invention, by "use in the maintenance of physiological lipid levels" in the blood is meant the ability to preserve physiological blood lipid levels. Preferably, the association or the oral formulation are for use in the restoration of physiological lipid levels, for example of total cholesterol and / or LDL.

[0093] In this sense, the association is preferably able to reduce blood lipid levels, for example in a subject with borderline cholesterol emia.

[0094] For the purposes of the invention, by "use in the restoration of physiological lipid levels in the blood" is meant the ability to bring blood lipid levels that have undergone an alteration back to physiological conditions.

[0095] Preferably, the association or the oral formulation are for use in the restoration of physiological blood lipid levels, for example total cholesterol and / or LDL cholesterol and / or triglycerides, in conjunction with a condition selected from the group consisting of: pathologies inducing an alteration of physiological blood lipid levels; excessive alcohol consumption; drug intake.

[0096] Preferably, the pathologies inducing an alteration of physiological blood lipid levels, for example total cholesterol and / or LDL cholesterol and / or triglycerides, are selected from the group consisting of: dysmetabolic diseases, such as, for example, diabetes mellitus; kidney pathologies, such as chronic kidney disease; liver pathologies, such as biliary cirrhosis; thyroid pathologies, such as hypothyroidism; pathologies with a genetic etiology, such as familial hypercholesterolemia; and combinations of the foregoing.

[0097] Preferably, examples of drugs inducing an alteration of physiological blood lipid levels, for example total cholesterol and / or LDL cholesterol and / or triglycerides, are selected from the group consisting of: estrogens; oral contraceptives; corticosteroids; retinoids; thiazide diuretics; cyclosporine; tacrolimus; antivirals used for the treatment of human immunodeficiency virus (HIV) infection and AIDS; and combinations of the foregoing.

[0098] Preferably, the association or the oral formulation are for use in the treatment of dyslipidemia and / or hypercholesterolemia.

[0099] Preferably, the association or the oral formulation are for use in the treatment of dyslipidemia and / or hypercholesterolemia concomitant with or caused by pathologies inducing an alteration of physiological blood lipid levels (for example total cholesterol and / or LDL cholesterol and / or triglycerides) selected from the group consisting of:dysmetabolic diseases, such as, for example, diabetes mellitus; kidney pathologies, such as chronic kidney disease; liver pathologies, such as biliary cirrhosis; thyroid pathologies, such as hypothyroidism; pathologies with a genetic etiology, such as familial hypercholesterolemia; and combinations of the foregoing.

[0100] Preferably, the association or the oral formulation are for use in the prevention of atherosclerotic cardiovascular disease.

[0101] Preferably, the association or the oral formulation are for use in the reduction of altered (or non-physiological) levels of cholesterolemia and / or LDL cholesterol and / or triglycerides to prevent atherosclerotic cardiovascular disease.

[0102] Oral formulation comprising the association of the invention

[0103] Preferably, the oral formulation comprises

[0104] - the association for the use described so far, being the only active ingredient in the oral formulation, and

[0105] - suitable excipients and / or diluents,

[0106] or, alternatively,

[0107] - the association for the use described so far, being a first active ingredient, and - at least a second lipid-lowering and / or cholesterol-lowering active ingredient, other than said association,

[0108] - suitable excipients and / or diluents.

[0109] Preferably, when present, the at least one second lipid-lowering and / or cholesterol-lowering active ingredient is an active that is able to act on lipid metabolism and / or on cholesterol metabolism and / or on triglyceride metabolism.

[0110] When present, the at least one second active ingredient can therefore preferably contribute to enhancing the medical benefit provided by the association of the invention.

[0111] Preferably, the at least one second active ingredient is preferably selected from the group consisting of: lipid-lowering and / or cholesterol-lowering plant extracts, such as fenugreek extract; micronutrients, for example vitamins and / or minerals, such as vitamin Bl and / or coenzyme Q10; berberine; policosanols; phytosterols and plant sterols; and combinations of the foregoing.Said at least one second active ingredient is known to the person skilled in the art; for example, it can be selected from the extracts with a known lipid-lowering and / or cholesterol-lowering function listed in Annex 1 of the Ministerial Decree of August 10, 2018, which regulates the use of plant substances and preparations in food supplements.

[0112] Examples of lipid-lowering and / or cholesterol-lowering plant extracts known to a person skilled in the art belong to a genus of plants selected from the group consisting of: Allium, Camelina, Citrus, Commiphora, Costus, Cynara, Echium, Euterpe, Fucus, Glycine, Ilex, Indigofera, Lespedeza, Linum, Medicago, Moringa, Panax, Paullinia, Plantago, Terminalia, Triticum, Viscum, Acacia, Acer, Amaranthus, Astracantha, Cyclanthera, Chamaecrista, Ipomoea, Trigonella, Malus, and combinations of the foregoing.

[0113] Preferably, the at least one second active ingredient can be selected from the group consisting of: vitamin Bl; coenzyme Q10; fenugreek extract; Allium cepa L.; Camelina sativa (L.) Crantz; Citrus aurantium var. amara L.; Commiphora mukul; Costus speciosus Sm.; Cynara scolymus L.; Echium plantagineum L.; Euterpe oleracea Mart.; Fucus vesiculosus L.; Glycine max (L.) Merr.; Ilex paraguariensis A. St.-Hil.; Indigofera tinctoria L.; Lespedeza capitata Michx.; Linum usitatissimum L.; Medicago sativa L.; Moringa oleifera Lam.; Panax notoginseng, Panax pseudoginseng Wall.; Paullinia cupana Kunth; Plantago indica L.; Plantago ovata Forssk; Terminalia bellerica (Gaertn.) Roxb.; Triticum aestivum L.; Viscum album L.; Acacia catechu (L.f.) Willd.; Acacia decurrens Willd.; Acacia nilotica (L.) Delile; Acacia polyacantha Willd.; Acacia Senegal (L.) Willd.; Acer campestre L.; Amaranthus caudatus L.; Amaranthus cruentus L.; Amaranthus hypochondriacus L.; Astracantha microcephala (Willd.) Podlech; Cyclanthera pedata (L.) Schrad.; Allium sativum L.; Allium ursinum L.; Chamaecrista nomame (Sieber) H. Ohashi; Ipomoea batatas (L.) Poir; Trigonella foenum-graecum L.; Citrus x bergamia Risso & Poit.; Malus domestica Borkh.; berberine; policosanols; phytosterols and plant sterols; and combinations of the foregoing.

[0114] When present, the at least one second active ingredient is preferably present in a quantity comprised between 0.1% and 20% by weight, preferably between 0.2% and 20% by weight, preferably between 0.5% and 16% by weight, preferably between 0.5% and 10% by weight, preferably between 0.5% and 8% by weight, preferably between 0.5% and 5% by weight, preferably between 0.5% and 3% by weight, preferably between 0.5% and 2% by weight, preferably between 0.5% and 1.5% by weight, of the total weight of the formulation. It should be noted that the oralformulation can preferably be used for the same medical purposes indicated above for the association of the invention.

[0115] Preferably, the oral formulation is in solid form.

[0116] Preferably, the oral formulation is in a physical form selected from: powder, granulate, tablet; for example, it is a granulate, or a swallowable tablet, or a capsule.

[0117] Preferably, the suitable excipients and / or diluents are those known in the state of the art and suitable for the preparation of oral formulations, preferably solid formulations. Preferably, the suitable excipients and / or diluents are selected from the group consisting of: bulking agents, thickeners, glidants, lubricants, flow agents, disintegrants, flavoring agents, pH adjusters, sweeteners, gelatin, plasticizers, solvents, silicon derivatives, and combinations of the foregoing.

[0118] Preferably, the suitable excipients and / or diluents are selected from the group consisting of: calcium phosphate; calcium carbonate, microcrystalline cellulose, mono- and di-glycerides of fatty acids, cross-linked sodium carboxymethylcellulose, silica, magnesium stearate, and combinations of the foregoing.

[0119] Preferably, the suitable excipients and / or diluents are in a quantity comprised between 15% and 75% by weight, preferably between 40% and 65%, preferably between 35% and 60% by weight, preferably between 35% and 57% by weight, preferably between 37% and 55% by weight, of the total weight of the formulation.

[0120] Preferably, the association is in a quantity comprised between 10% and 90% by weight, preferably between 20% and 85% by weight, preferably between 25% and 80% by weight, preferably between 35% and 70% by weight, preferably between 40% and 70% by weight, preferably between 40% and 67% by weight, preferably between 40% and 65% by weight, preferably between 42% and 65% by weight, preferably between 44% and 65% by weight, preferably between 44% and 63% by weight, of the total weight of the formulation.

[0121] Preferably, the Olea europea extract is in a quantity comprised between 12% and 42% by weight, preferably between 18% and 38% by weight, preferably between 18% and 35% by weight, preferably between 20% and 32% by weight, of the total weight of the formulation.

[0122] Preferably, the Myrtus communis extract is in a quantity comprised between preferably between 10% and 42% by weight, preferably between 15% and 38% by weight,preferably between 15% and 35% by weight, preferably between 16% and 28% by weight, of the total weight of the formulation.

[0123] Preferably, the oral formulation can be taken 1 to 2 times a day, preferably once a day. Preferably, the oral formulation is a food supplement, or a nutraceutical, or a medicinal product.

[0124] By food supplement is meant 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 normal diet and which are a concentrated source of nutrients, such as vitamins and minerals, or other substances with a nutritional or physiological effect, in particular, but not exclusively, amino acids, essential fatty acids, fibre and extracts of plant origin, both single and multi-compounds, in pre-measured forms.".

[0125] By nutraceutical is meant one or more components or active ingredients of foods that have positive effects on health and the prevention of diseases.

[0126] By medicinal product is meant a pre-packaged pharmaceutical form, produced industrially and authorized on the basis of documentation containing the experimental chemical, biological, pharmaceutical, pharmaco-toxicological and clinical results relating to the drug that is placed on the market with a special name (registered trademark).

[0127] EXAMPLES

[0128] Hereinafter, the Applicant provides examples for illustrative but not limiting purposes.

[0129] Example 1 — Association in dried form of the invention

[0130] Percentage by weight Component referred to the total weight of the association (% w / w) Hydroalcoholic extract of myrtle (Myrtus

[0131] communis L., leaf) in which myricetin is 2% by 42.857% weight of the total volume / weight of the extract

[0132]

[0133] Dried extract of olive (Olea europaea L., fruit) in

[0134] which hydroxytyrosol is 5% by weight of the 51.020%

[0135] total weight of the extract

[0136] Silica 4.082% Calcium carbonate 2.041% Total 100.000%

[0137]

[0138] Example 2 — Swallowable tablet comprising the association of the invention Percentage by Weight weight referred Component in to the total grams weight of the tablet (% w / w) ASSOCIATION IN DRIED FORM COMPRISING THE HYDROALCOHOLIC

[0139] 0.49 61.25% EXTRACT OF MYRTLE LEAVES AND THE DRIED EXTRACT OF OLIVE FRUITS DRIED EXTRACT OF FENUGREEK 0.010 1.25% CALCIUM CARBONATE E 170 0.272 34.00% MICROCRYSTALLINE CELLULOSE (E 460 (i)) 0.008 1.00% CROSS-LINKED SODIUM

[0140] 0.008 1.00% CARBOXYMETHYLCELLULOSE E 468

[0141] SILICON DIOXIDE E 551 0.008 1.00%

[0142]

[0143] MAGNESIUM STEARATE E 470b 0.004 0.50% TOTAL 0.8 100%

[0144]

[0145] Example 3 — Swallowable tablet comprising the association of the invention Percentage by Weight weight referred Component in to the total grams weight of the tablet (% w / w) ASSOCIATION IN DRIED FORM COMPRISING THE HYDROALCOHOLIC

[0146] 0.3528 44.1% EXTRACT OF MYRTLE LEAVES AND THE DRIED EXTRACT OF OLIVE FRUITS DRIED EXTRACT OF FENUGREEK 0.0072 0.9% CALCIUM CARBONATE E 170 0.404 50.50% MONO- AND DI-GLYCERIDES OF FATTY

[0147] 0.008 1.00% ACIDS E 471

[0148] MICROCRYSTALLINE CELLULOSE (E 460 (i)) 0.004 0.50% CROSS-LINKED SODIUM

[0149] 0.012 1.50% CARBOXYMETHYLCELLULOSE E 468

[0150] SILICON DIOXIDE E 551 0.008 1.00% MAGNESIUM STEARATE E 470b 0.004 0.50% TOTAL 0.8 100.00%

[0151]

[0152] Example 4 — In vitro studies

[0153] 4.1. Determination of the maximum non-cytotoxic concentration

[0154] The study relates to the determination of the maximum non-cytotoxic concentration (residual viability >70% after treatment) of the natural extracts obtained from Myrtus communis (M. C.) and Olea europea (O. E.), used individually and in combination, and by the Red Yeast Rice extract (RYR) in human HepG2 cells.

[0155] Methods

[0156] The HepG2 cells were maintained in DMEM+10%FBS+P / S+Glu+2% NEAA medium. Thereafter, cytotoxicity assays were set up using the MTS reagent, a yellow-colored compound that can only be metabolized by living and metabolically active cells, producing a final orange / brown colored compound.

[0157] Results

[0158] The HepG2 cells were seeded in 96-well plates and were then treated for 24h either with a dose response of Myrtus communis extract (0 µg / ml - 1000 µg / ml), or with a dose response of Olea Europea extract (0 µg / ml - 1000 µg / ml), or with the combination of the two natural extracts (62.5 µg / ml+62.5 µg / ml), or with 62.5 µg / ml of Red Yeast Rice Extract (RYR).

[0159] The results indicate that the maximum non-cytotoxic concentration of Myrtus communis (Figure 1A) is equal to 62.5 µg / ml (Residual cell viability: 81.40±2.17%) and that the maximum non-cytotoxic concentration of Olea europea (Figure 1B) is equal to 62.5 µg / ml (Residual cell viability: 81.37±1.81%).

[0160] Furthermore, the combination of the two natural extracts (62.5 µg / ml M. C. + 62.5 µg / ml O. E.) did not produce significant cytotoxic effects, determining a residual cell viability of the HepG2 cells equal to 71.34±1.06% (Figure 1C).

[0161] The experimental results indicate that the treatment of HepG2 cells for 24h with 62.5 µg / ml of red yeast rice extract did not produce a cytotoxic effect in the cells, determining a residual cell viability equal to 86.77±1.21% (Figure 1D).

[0162] % Mean HepG2 Viability Myrtus DS Myrtus Communis Communis 0 µg / ml 100.00 1.00

[0163]

[0164] 15.6 µg / ml 90.70 4.0331.2 µg / ml 88.17 5.1162.5 µg / ml 81.40 2.17125 µg / ml 40.98 5.06250 µg / ml 19.95 3.62500 µg / ml 20.02 3.04100024.13 4.05µg / ml% Mean HepG2 Viability Olea Europea DS Olea Europea0 µg / ml 100.00 1.0015.692.23 7.47µg / ml31.290.08 6.33µg / ml62.581.37 1.81µg / ml125 µg / ml 41.18 10.56250 µg / ml 17.50 1.34500 µg / ml 16.64 2.65100017.18 2.51µg / ml

[0165] % Mean HepG2 Viability MC+OE DS MC+OE Combination Combination

[0166]

[0167] 0 µg / ml 100.00 1.0062.5M.C.+62.5 O.E.µg / ml 71.34 1.06% Mean HepG2 Viability Red yeast rice DS Red yeast rice extract extract0 µg / ml 100.00 1.0062.586.77 1.21µg / ml

[0168]

[0169] Table 1. Means and Standard Deviations (SD) of the residual viability values (%) of HepG2 cells after treatment with the natural extracts of Myrtus communis, Olea europea and Red Yeast Rice.

[0170] 4.2. Determination of the modulation of mRNA levels transcribed from the HMG-CoA Reductase, LDL Receptor and PCSK9 genes in human hepatocytes HepG2

[0171] The study relates to the determination of the modulation of mRNA levels transcribed from the HMG-CoA Reductase, LDL Receptor and PCSK9 genes in human hepatocytes HepG2 by the natural extracts of Myrtus communis (M. C.) [62.5 µg / ml] and Olea europea (O. E.) [62.5 µg / ml], used individually and in combination, and by the Red Yeast Rice extract (RYR) [62.5 µg / ml].

[0172] Methods

[0173] The HepG2 cells were seeded in 6-well plates and were treated for 24h according to the following experimental scheme:

[0174] NT (Non-Treated);

[0175] +Glucose 30 mM;

[0176] +Glucose 30 mM+M. C. 62.5 µg / ml;+Glucose 30 mM+O. E. 62.5 µg / ml;+Glucose 30 mM+ M. C. 62.5 µg / ml+ O. E. 62.5 µg / ml;+Glucose 30mM+ RYR 62.5 pg / ml.

[0177] After the treatment of the HepG2 cells for 24h according to the experimental design described above, the cell lysates were recovered and the RNAs were extracted from the various samples using the " Total RNA Purification Kit" from Norgen Biotek. After having determined the concentration of the RNAs by reading the absorbance at λ=260 nm, 500 ng of each RNA were reverse transcribed into cDNA, exploiting the enzymatic activity of reverse transcriptase (Figure 2).

[0178] The cDNAs were then used as a template for Real-Time quantitative PCR experiments, a technique that allows quantifying the modulation of the expression levels of the target genes, by determining the Ct (" Cycle Threshold") of each gene for each cDNA. These values will then be normalized based on the expression levels of a "housekeeping" gene.

[0179] Results

[0180] The experimental results obtained in the HepG2 cells treated for 24h with the Myrtus communis extract [62.5 pg / ml], or with the Olea europea extract [62.5 pg / ml], or with the combination of the Myrtus communis extract [62.5 pg / ml]+O / ea europea extract [62.5 pg / ml], or with the Red Yeast Rice Extract RYR [62.5 pg / ml] indicate that, although there is a tendency to decrease the mRNA levels of PCSK9 after treatment with the extracts of Myrtus communis, Olea europea and RYR used individually, only the combination of the two natural extracts Myrtus communis and Olea europea was able to decrease in a statistically significant manner the expression levels (0.417±0.101 Fold; p=0.023) of the PCSK9 gene (Figure 3A).

[0181] It is interesting to note that the decrease in the mRNA levels of PCSK9 induced by the combination of the Myrtus communis and Olea europea extracts, equal to -58.3%, is superior to the sum of the effects induced by the individual treatments (-23.1% for Myrtus communis and -33.2% for Olea europea thus indicating a synergistic biological effect.

[0182] As for the LDL Receptor gene, none of the treatments was able to modulate the expression levels of this molecular target (Figure 3B).

[0183] Furthermore, the treatment with the association of the natural extracts of Myrtus communis and Olea europea did not induce a regulation of the expression levels of the HMG-CoA reductase gene (Figure 3C), while the treatment for 24h with 62.5 pg / ml of RYR induced a statistically significant increase (2.15±0.51 Fold; p=0.009) of the expression levels of the HMG-CoA Reductase gene (Figure 3C), showing an up-regulation effect of endogenous cholesterol biosynthesis consequent to the treatment with this natural extract [Zhou, R. et al., 2022, Cardiology in Review],

[0184] Finally, the experimental results show that the treatment with 30 mM of glucose for 24h did not modulate the mRNA levels of the HMG-CoA Reductase, LDL Receptor and PCSK9 genes, with respect to the NT condition of the HepG2 cells (Figure 4).

[0185] PCSK9 Fold Mean DS

[0186] Glucose 30 mM 1 0.1

[0187] Glucose+MC 0.769 0.039

[0188] Glucose+OE 0.667 0.121

[0189] Glucose+MC+OE 0.417 0.101

[0190] Glucose+RYR 0.707 0.125

[0191] LDL-R Fold Mean DS

[0192] Glucose 30 mM 1 0.1

[0193] Glucose+MC 1.286 0.153

[0194] Glucose+OE 0.761 0.117

[0195] Glucose+MC+OE 0.967 0.082

[0196] Glucose+RYR 0.834 0.235

[0197] HMG-CoA

[0198] Fold Mean DS

[0199] Reductase

[0200] Glucose 30 mM 1 0.1

[0201] Glucose+MC 1.225 0.095

[0202] Glucose+OE 1.064 0.107

[0203] Glucose+MC+OE 1.167 0.162

[0204] Glucose+RYR 2.154 0.510

[0205]

[0206] PCSK9 Fold DS

[0207] Mean

[0208] NT 1 0.1

[0209] Glucose 30 mM 0.996 0.235

[0210] Fold

[0211] LDL-R DS

[0212] Mean

[0213] NT 1 0.1

[0214] Glucose 30 mM 1.070 0.060

[0215] HMG-CoA Fold DS

[0216] Reductase Mean

[0217] NT 1 0.1

[0218] Glucose 30 mM 0.828 0.165

[0219]

[0220] Table 2. Means and Standard Deviations (SD) of the expression values (Fold) of the PCSK9, LDL-R, HMG-CoA Reductase genes in HepG2 cells after treatment with the natural extracts ofMyrtus communis, Olea europea and Red Yeast Rice.

[0221] 4.3. Determination of the modulation of the protein levels of PCSK9 secreted in the culture medium by human hepatocytes HepG2

[0222] The study relates to the determination of the modulation of the protein levels of PCSK9 secreted in the culture medium by human hepatocytes HepG2 after treatment with the natural extracts of Myrtus communis (M. C.) [62.5 µg / ml] and Olea europea (O. E.) [62.5 µg / ml], used individually and in combination, and by the Red Yeast Rice extract (RYR) [62.5 µg / ml].

[0223] Methods

[0224] The HepG2 cells were seeded in 6-well plates and were treated for 24h according to the following experimental scheme:

[0225] NT (Non-Treated);

[0226] +Glucose 30 mM;+Glucose 30 mM+M. C. 62.5 pg / ml;

[0227] +Glucose 30 mM+O. E. 62.5 pg / ml;

[0228] +Glucose 30 mM+M. C. 62.5 pg / ml+ O. E. 62.5 pg / ml;

[0229] +Glucose 30 mM+RYR 62.5 pg / ml.

[0230] After the treatment of the HepG2 cells for 24h according to the experimental design described above, the culture media were recovered from the wells of the 6-well plates, after which a solution of protease inhibitors was added to each sample and the samples thus obtained were used to set up the ELISA assay for the PCSK9 protein (" Human Proprotein Convertase 9 / PCSK9 ELISA Kit, Catalog Number EH384RB, ThermoFisher), as reported in Figure 5.

[0231] Results

[0232] The experimental results obtained in the HepG2 cells treated for 24h with the Myrtus communis extract [62.5 pg / ml], or with the Olea europea extract [62.5 pg / ml], or with the combination of the Myrtus communis extract [62.5 pg / ml]+O / ea europea extract [62.5 pg / ml], or with the Red Yeast Rice extract RYR [62.5 pg / ml] indicate that, although the treatments with the extracts of Myrtus communis and Olea europea used individually are able to reduce the levels of the PCSK9 protein secreted in the culture medium of the HepG2 cells, only the combination of these two natural extracts was able to decrease in a statistically significant manner the protein levels (5.80±2.00 ng versus 37.80±5.29 ng; p=0.009) of the secreted PCSK9 (Table 3).

[0233] PCSK9 Protein ng secreted PCSK9

[0234] NT HepG2 51.13 ± 13.61

[0235] Glucose 30 mM 37.80 ± 5.29

[0236] Glucose+MC 23.80 ± 11.14

[0237] Glucose+OE 19.80 ± 9.17

[0238] Glucose+MC+OE 5.80 ± 2.00 **

[0239] Glucose+RYR 39.13 ± 5.03

[0240]

[0241] Table 3: Values of the PCSK9 protein (ng) secreted in the culture medium of HepG2 cells after 24h treatment with the Myrtus communis extract 62.5 pg ml and the Oleaeuropea extract 62.5 up ml, used individually or in combination (Myrtus communis 62.5 up ml, Olea europea 62.5 up ml and with the Red yeast rice extract RYR 62.5 up ml). The results are presented as mean value ± SI) of three independent experiments (n=3), performed in duplicate. ** p<0.01, when compared with the 30 mM glucose

[0242]

[0243] sample and evaluated by paired Student's t-Test.

[0244] The data obtained indicate that the treatment with the RYR extract did not modulate the levels of the secreted PCSK9 protein (Table 3).

[0245] The experimental results show that the treatment with 30 mM of glucose for 24h did not modulate the protein levels of PCSK9 secreted in the culture medium compared to the NT condition (p=0.144) of the HepG2 cells (Table 3).

[0246] 4.4. Determination of the modulation of the catalytic activity of the HMG-CoA Reductase enzyme using the cellular protein lysates obtained from HepG2 cells The study relates to the determination of the modulation of the catalytic activity of the HMG-CoA Reductase enzyme using the cellular protein lysates obtained from HepG2 cells after treatment with the extracts of Myrtus communis (M. C.) [62.5 pg / ml] and Olea europea (O. E.) [62.5 pg / ml], used individually and in combination, and with the Red Yeast Rice extract (RYR) [62.5 pg / ml].

[0247] Methods

[0248] The HepG2 cells were seeded in 6-well plates and were treated for 24h according to the following experimental scheme:

[0249] NT (Non-Treated);

[0250] +Glucose 30 mM;

[0251] +Glucose 30 mM+M. C. 62.5 µg / ml;+Glucose 30 mM+O. E. 62.5 µg / ml;+Glucose 30 mM+ M. C. 62.5 µg / ml+ O. E. 62.5 µg / ml;

[0252] +Glucose 30 mM+RYR 62.5 pg / ml.

[0253] The experimental model of hypercholesterolemia developed for this research project, which involves the treatment of human hepatocytes with 30 mM glucose for 24h, is based on the scientific evidence indicating that in cells, excess glucose is transformed by the hexokinase enzyme into Glucose 6P which, in turn, is transformed by theGlucose 6P Dehydrogenase (G6PD) enzyme into Glucunolactone+NADPH+H+. Finally, the NADPH thus obtained is used by the HMG-CoA Reductase enzyme to transform HMG-CoA into mevalonate, increasing the catalytic activity of this hypercholesterolemic enzyme [Ruga, S. et al., The Activity of Ten natural Extracts Combined in a Unique Blend to maintain Cholesterol Homeostasis In Vitro Model; 2022, IJMS],

[0254] After the treatment of the HepG2 cells for 24h according to the experimental design described above, the cell lysates were recovered and the cytosols were extracted from the various samples. The concentration of the proteins present in the cytosols of the HepG2 cells was then determined by Bradford assay. Once the volume of each sample necessary to obtain 100 pg of cellular proteins was established, catalytic activity assays of the HMG-CoA Reductase enzyme were set up (using " HMG CoA Reductase Assay Kit CS1090 by Sigma Aldrich") to evaluate the ability of the extracts of Myrtus communis, Olea europea and Red Yeast Rice (RYR) to inhibit the activity of the native intracellular HMG-CoA reductase present in the HepG2 cells.

[0255] Results

[0256] The cytosols of the HepG2 cells treated with the natural extracts provided the native HMG-CoA reductase enzyme for the determination of the residual intracellular catalytic activity after 24h treatment with the extracts of Myrtus communis and Olea europea, used individually or in combination, and with the Red Yeast Rice extract (RYR).

[0257] The experimental assay evaluates the consumption of NADPH (enzymatic co-factor) in the 10 minutes of enzymatic kinetics by the HMG-CoA Reductase enzyme present in the sample, allowing to determine the inhibition of the catalytic activity determined by 24h of treatment with the natural extracts under study.

[0258] First, the inhibition by the statin Pravastatin (1 pM) of the recombinant HMG-CoA reductase enzyme provided by the kit was evaluated (Figure 6A). Then, the validity of the experimental model was evaluated, determining the increase in intracellular catalytic activity of the native HMG-CoA Reductase enzyme after treatment of the HepG2 cells for 24h with 30 mM of Glucose (Figure 6B). Finally, the modulation of the enzymatic activity of HMG-CoA Reductase by the extracts of Myrtus communis and Olea Europea, used individually and in combination, and by the RYR extract was evaluated (Figure 6C).The experimental results indicate that the statin Pravastatin inhibited in a statistically significant manner the activity of the recombinant HMG-CoA reductase (11.7±0.6% of residual catalytic activity; p<0.001) (Figure 6A), and that the treatment for 24h with 30 mM glucose induced a significant increase in the catalytic activity of the native intracellular HMG-CoA Reductase (increase of 2.68±0.36 fold; p=0.007), validating the in vitro cellular model of hypercholesterolemia used, represented by human hepatocytes HepG2 treated for 24h with an excess of glucose (Figure 6B).

[0259] Furthermore, the experimental assay showed that the 24h treatments with 62.5 pg / ml oiMyrtus communis extract (12.1±6.0% of residual catalytic activity; p<0.001), with 62.5 pg / ml of Myrtus communis extract in combination with 62.5 pg / ml of Olea europea extract (5.7±3.1% of residual catalytic activity; p<0.001) and with Red Yeast Rice extract (12.7±3.2% of residual catalytic activity; p<0.001) significantly and statistically significantly reduced the enzymatic activity of the native intracellular HMG-CoA Reductase (Figure 6C).

[0260] Finally, the Olea europea extract significantly reduced the enzymatic activity of the native HMG-CoA reductase of the HepG2 (52.3±4.6% of residual catalytic activity; p=0.021), but to a lesser extent compared to treatments with the other natural extracts (Figure 6C).

[0261] Catalytic activity of HMG-CoA

[0262] reductase DS MEDIA (Fold)

[0263] Control 1.000 0.010

[0264] 1 pM

[0265] 0.117 0.006 Pravastatin

[0266] Catalytic activity of HMG-CoA

[0267] reductase DS MEDIA (Fold)

[0268] NT 1.00 0.01

[0269]

[0270] 30 mM Glucose 2.68 0.36

[0271] Catalytic activity of HMG-CoA

[0272] DS

[0273] reductase MEDIA (Fold)

[0274] 30 mM Glucose 1.000 0.010 Glucose+MC 0.120 0.061 Glucose+OE 0.523 0.046 Glucose+MC+OE 0.057 0.031 Glucose+RYR 0.127 0.032

[0275]

[0276] Table 4. Means and Standard Deviations of the values of Residual catalytic activity (Fold) of the HMG-CoA Reductase enzyme in HepG2 cells after treatment with the natural extracts ofMyrtus communis, Olea europea and Red Yeast Rice.

[0277] 4.5. Conclusions

[0278] The experimental results obtained in the HepG2 cells treated with the extracts of Myrtus communis, Olea europea and Red Yeast Rice in an in vitro model of hypercholesterolemia indicate that:

[0279] - the Myrtus communis extract statistically significantly decreases the intracellular catalytic activity of HMG-CoA reductase (12.1±6.0% of residual catalytic activity); - the Olea europea extract statistically significantly decreases the intracellular catalytic activity of HMG-CoA reductase (52.3±4.6% of residual catalytic activity);

[0280] - the association of Myrtus communis and Olea europea extracts statistically significantly decreases the intracellular catalytic activity of HMG-CoA reductase (5.7±3.1% of residual activity); statistically significantly and synergistically decreases the expression levels of the PCSK9 gene (0.417±0.101 Fold versus l±0.1 Fold); statistically significantly decreases the protein levels of secreted PCSK9 (5.80±2.00 ng versus 37.80 ±5.29 ng);

[0281] - the Red Yeast Rice extract statistically significantly decreases the intracellular catalytic activity of HMG-CoA reductase (12.7±3.2% of residual catalytic activity); furthermore, it statistically significantly increases the expression levels of the "cholesterol -lowering" gene HMG-CoA Reductase (2.15±0.51 Fold versus l±0.1 Fold).Example 5 — In vivo study

[0282] The in vivo study is a study on the efficacy of the supplementation of a food supplement based on an olive extract (Olea europaea L., fruit) and a myrtle extract (Myrtus communis L., leaves) for the improvement of lipid metabolism in subjects with slightly altered cholesterol levels. This is a single-center, controlled, randomized, parallel-arm, double-blind clinical study.

[0283] Primary objective

[0284] The primary objective of this clinical study was to evaluate the efficacy of the supplementation of a food supplement based on a mixture of two extracts - the dried extract of olive (Olea europaea L., fruit), and the dried extract of myrtle (Myrtus communis L., leaves; obtained by drying the hydroalcoholic extract from Myrtle leaves) - in maintaining normal blood LDL cholesterol levels in healthy subjects with altered cholesterolemia (total plasma cholesterolemia between 200 and 239 mg / dl and LDL-C cholesterolemia < 159 mg / dl), which the ESC and EAS guidelines indicate as not only a risk factor for atherosclerotic CV disease, but a direct cause of its development.

[0285] This criterion is indicated as a primary endpoint in clinical efficacy studies on hypercholesterolemia, both in the EMA guidelines (https: / / www.ema.europa.eu / en / documents / scientific-guideline / guideline-clinical- investigation-medicinal-products-treatment-lipid-disorders-revision-3_en.pdf), and in the guidelines of the European Food Safety Authority (EFSA)

[0286]

[0287] The method involves the measurement of blood concentration values of LDL cholesterol [Time frame: run-in (tr), baseline (TO), and 90 days (T90) of treatment].

[0288] Secondary objectives

[0289] The proposed study design provides for the evaluation of the following as secondary outcomes:

[0290] - beneficial effect on lipid metabolism. In this case, the method involves the determination of blood levels of: total cholesterol (TC), HDL cholesterol, triglycerides (TG) [Time frame: baseline (TO), and 90 days (T90) of treatment],

[0291] - beneficial effect on glucid metabolism. In this case, the method involves the determination of blood levels of: glycated hemoglobin, glycemia [Time frame: baseline (TO), and 90 days (T90) of treatment].- body weight in terms of reduction of the BMI (Body Mass Index) value and abdominal circumference [Time frame: baseline (TO), and 90 days (T90) of treatment], - hepatic and renal toxicity. In this case, the method involves the determination of the blood concentration of the following biomarkers: alanine transferase (ALT), aspartate transferase (AST), creatinine (CRE) [Time frame: baseline (TO), and 30 days (TR) of treatment and 90 days (T90) of treatment].

[0292] Study design

[0293] A single-center, randomized, controlled, double-blind clinical study with two parallel arms was conducted, with a run-in period of 15 days.

[0294] The treatment time is 3 months with the products under study (food supplement or placebo). This timing was considered adequate by EFSA to evaluate the clinical efficacy of currently used food components or food supplements on hypercholesterolemia.

[0295] Following enrollment, before starting the treatment period, the subjects were subjected to a run-in period of 15 days in which they received no treatment but were required to complete a food diary daily.

[0296] The experimental groups were as follows:

[0297] - GROUP 1 (56 subjects) —> subjects who had to take a capsule of a food supplement consisting of a mixture of olive extracts (Olea europaea L., fruit), and myrtle (Myrtus communis L., leaves);

[0298] - GROUP 2 (56 subjects) —> subjects who had to take the placebo.

[0299] In order to control for some variables that could have constituted biases in the interpretation of the results, the participants were instructed:

[0300] 1) on how to follow, for the entire duration of the study, an isocaloric diet, the specifics of which were reported in the protocol, which had to be recorded through the food diary;

[0301] 2) at each visit, all pharmacological treatments that were taken by the subject were recorded. Each subject from the two experimental groups, before, during and after the administration of the food supplement or placebo, was subjected to blood sampling as reported in the diagram of Figure 7.As indicated in the "schedule of activities" study scheme (Table 5), during the screening visit (TR), the subjects were subjected to the following investigation to see if they met the study participation requirements:

[0302] 1: LDL-C

[0303] 2a: TC

[0304] - eligibility assessment through application of inclusion and exclusion criteria.

[0305] - execution of the HIV test by rapid combined saliva test (IV generation test), which looks for anti-HIV antibodies produced by the individual and parts of the virus (such as the p24 antigen). This test, as reported on the website of the Ministry of Health (https: / / www. salute.gov.it / portale / hiv / dettaglioContenutiHIV.j sp?lingua=italiano&id =185&area=aids&menu=vuoto), can highlight the infection as early as 20 days. This test, the result of which is available in a few minutes, was conducted at the medical office. If the result was doubtful or reactive (positive), the subject was not recruited. - for women of childbearing age: at the first visit, women of childbearing age were subjected to a pregnancy test that measured the levels of beta HCG (human chorionic gonadotropin), the production of which begins at the time of implantation in the uterine wall, i.e., about a week after fertilization. This test, the result of which is available in a few minutes, was conducted at the medical office. If the result was doubtful or positive, the subject was not recruited.

[0306] Subsequently, all enrolled subjects are subjected to the following procedures (as represented in the study layout in Figure 7):

[0307] At TO (i.e., at baseline, following the run-in period - start of treatment):

[0308] 1: LDL-C,

[0309] 2a: TC, HDL-C, TG,

[0310] 2b: HbA1c, glycemia,

[0311] 2c: BMI (body mass index) and abdominal circumference,

[0312] 2d: SGPT, SGOT, creatinine. At T30 (i.e., after 30 days of treatment): 2e: SGPT, SGOT, creatinine.

[0313] At T90 (i.e., after 90 days of treatment):

[0314] 1: LDL-C,a: TC, HDL-C, TG,

[0315] b: HbA1c, glycemia,

[0316] c: BMI (body mass index) and abdominal circumference,

[0317] d: SGPT, SGOT, creatinine.

[0318] VI V21st Day

[0319] V3 V490th Day Screening Baseline

[0320] 30th End of Visit number (Times) visit - Start Start of

[0321] Day treatment of Run-In treatment

[0322] (T30) (T90) (TR) (TO)

[0323] Information sheet and

[0324] X

[0325] informed consent

[0326] Eligibility check

[0327] (inclusion / exclusion X

[0328] criteria)

[0329] Execution of rapid

[0330] combined HIV test on X

[0331] saliva

[0332] Execution of pregnancy

[0333] X

[0334] test

[0335] Anamnesis X

[0336] Demographic data X

[0337] Distribution of X (quantity

[0338] treatments equal to three

[0339]

[0340] months of

[0341] intake)

[0342] LDL-C X X X TC, HDL-C, TG, body

[0343] X (only TC

[0344] weight and abdominal X X and LDL-C)

[0345] circumference

[0346] Glycated hemoglobin,

[0347] X X

[0348] glycemia

[0349] SGPT, SGOT, creatinine X X X Questions related to

[0350] disorders and any X X adverse events

[0351] Evaluation of

[0352] compliance with X X treatment

[0353] Delivery of food diary X

[0354] Collection of food diary X Concomitant drugs X X X X

[0355]

[0356] Table 5. Study scheme (schedule of activities).

[0357] Dietary regimen

[0358] All enrolled subjects were instructed and educated so that they could follow, for the entire duration of the study, an isocaloric diet, which favored the consumption of fruit, vegetables, carbohydrates from whole grains (fiber), low-fat dairy products, fish, white meat, vegetable oils and which provided for a substantial decrease in red meat, animal fats, salt, simple sugars and alcohol.The dietary regimen to which the dietary indications provided to the participants were inspired was that of the " Dietary Approaches to Stop Hypertension" (DASH) diet. The DASH diet, developed and tested for the first time in 1997, is promoted by the National Heart, Lung, and Blood Institute of the National Institutes of Health (NIH) of the United States, in order to help prevent, or improve, high blood pressure (arterial hypertension) (https: / / www.issalute.it / index.php / la-salute-dalla-a-alla-z-menu / d / dieta-dash#conclusioni).

[0359] In addition to promoting blood pressure control, it has been shown that this dietary model improves insulin resistance, hyperlipidemia and overweight / obesity conditions. In terms of macronutrient composition, the nutritional objectives of the DASH dietary model are as follows: total fat: 27% of calories; saturated fat: 6% of calories; protein: 18% of calories; carbohydrates: 55% of calories; cholesterol: 150 mg; sodium: 2,300 mg; potassium: 4,700 mg; calcium: 1,250 mg; magnesium: 500 mg; fiber: 30 g.

[0360] The evaluation of compliance with the diet was performed through the use of a food diary, which was delivered at TR and collected at T90. The subject had to bring the food diary to each control visit, so that the experimenter could evaluate adherence to the diet and, if necessary, instruct the non-compliant subject more adequately.

[0361] The compilation of the food diary did not have to be performed by the subject every day of the treatment period, but had to be done, for each week, on two working days and one holiday, at the discretion of the subject.

[0362] Inclusion criteria

[0363] Subjects of both sexes were included in the study:

[0364] - with an age between 18 and 70 years;

[0365] - able to understand and sign the informed consent;

[0366] - negative HIV test;

[0367] - negative pregnancy test;

[0368] - borderline total cholesterol values between 200 and 239 mg / dl (Italian National Institute of Health definition);

[0369] LDL cholesterol values < 159 mg / dl (https: / / www.humanitas-care.it / malattie / colesterolo / ) [slightly altered LDL cholesterol between 100 and 159 mg / dl (NCEP Guidelines): optimal: less than 100 mg / dL; near optimal / above optimal: 100 to 129 mg / dL; borderline high: 130 to 159 mg / dL; high: 160 to 189 mg / dL; veryhigh: greater than 190 mg / dL - National Cholesterol Education Program (NCEP) Guidelines “Executive Summary of the Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III)" (Revision_2013), https: / / www.ncbi.nlm.nih.gov / books / NBK542294 / #article-19466.r18];

[0370] who were not taking and who would not take any type of drug for the entire period of the study.

[0371] Exclusion criteria

[0372] The following subjects were excluded from the study:

[0373] - with an age < 18 and > 70 years;

[0374] - subjects exposed to a high risk of cardiovascular events based on 8 risk factors (sex, age, diabetes, smoking habit, systolic blood pressure, total cholesterolemia, HDL-cholesterolemia and anti-hypertensive treatment) in accordance with the parameters of the Cuore project of the Italian National Institute of Health (http: / / www.cuore.iss.it / sopra / calc-rischio.asp);

[0375] - in pharmacological therapy for cholesterolemia and / or hyperglycemia, even at low doses;

[0376] - intake of supplements for the control of cholesterol, glycemia and metabolic syndrome, in the two weeks preceding recruitment;

[0377] - pregnant women, suspected pregnancy or planned pregnancy;

[0378] - breastfeeding women;

[0379] - blood donors in the three months preceding recruitment;

[0380] - non-self-sufficient subjects;

[0381] - subjects who did not show a propensity for collaboration;

[0382] - subjects who had difficulty going to the reference facility at the scheduled times; - subjects who were not considered suitable by the experimenting physician due to the presence of other pathologies considered incompatible with enrollment and requiring pharmacological treatments;

[0383] - subjects with acquired immunodeficiency from HIV;

[0384] - subjects with known allergies to the ingredients of the product under study;- subjects who consume alcohol in quantities greater than 30g / day for men and 20g / day for women;

[0385] - subjects who made use of drugs of abuse;

[0386] - subjects who took drugs.

[0387] Sample size -power analysis

[0388] The study planned to recruit 112 (56 subjects per group).

[0389] As indicated in Table 6, the Power Analysis determined a sample size of 106 subjects in total, to ensure a power of 95% and a significance of 0.05, with a medium effect size (f = 0.25). Estimating a drop out of about 5%, calculated on the basis of an estimate of the subjects who had to leave the study if the need to take certain pharmacological treatments arose, the number of subjects to be enrolled was brought to 112.

[0390] Randomization was performed at the time of enrollment according to a specific randomization table generated by a specific software (see the dedicated section). Primary end

[0391] Effect of product administration on LDL-C levels.

[0392] point

[0393] Randomized, double-blind, placebo-controlled study with two arms: placebo treatment and treatment with the invention's Experimental

[0394] supplement of a single group of subjects with cholesterolemia design

[0395] measurement (primary outcome LDL-C) at the beginning of the experiment (TO) and after three months of treatment (T90).

[0396] Objective of To identify the sample size (optimal number of patients) for the the analysis study in question.

[0397] The most suitable statistical analysis for this type of study was a random intercept linear mixed model (LMM), in which the LDL- C value is the dependent variable, the measurement (TO and T90), the treatment (placebo and supplement) and their interaction are the fixed factors, while the patient's identity was included as a Statistical

[0398] random factor to control for individual variability in the response analysis

[0399] to treatments.

[0400] The Power Analysis (PWA) was performed on the interaction, as it allows to evaluate whether the differences between the placebo- treated group and the supplement-treated group changed differently in the two measurements. The estimates of the sample

[0401]

[0402] size were therefore calibrated to identify a significant effect of the interaction between treatment and measurement.

[0403] To this end, a repeated measures analysis of variance model with within-subject and between-subject effects was used (F test ANOVA repeated measures, within-between effects).

[0404] The factors used in the analysis were:

[0405] - between-subject effect: treatment (factor with 2 levels: placebo and supplement),

[0406] - within-subject effect: measurement (factor with 2 levels: TO and T90).

[0407] A balanced design was assumed (the same number of data for each level of the factor).

[0408] In two single-arm pilot studies, for the study of the effects of some formulations similar to the invention's supplement, a reduction in the LDL-C value from 139±19 to 104±21 mmol / L in a first case and from 121±34 to 113±34 mmol / L in a second case was observed. From these data, effect size estimates corresponding to f=0.85 and f=0.12 were obtained which, according to the Power definitions proposed by Cohen (Cohen, (1969). Statistical power Analysis analysis for the behavioural sciences. New York: Academic Press), corresponded to a large effect in the first case and a medium-small effect in the second.

[0409] Therefore, for the power analysis of this clinical study, three effect size values were chosen corresponding to small effects (f=0.10), medium effects (f=0.25) and large effects (f=0.40).

[0410] The sample size calculations were performed with three power values 1-P equal to 0.80, 0.95 and 0.99 and a significance level a=0.05.

[0411]

[0412] Table 6. Description of the Power Analysis for the estimation of the total sample size. The following table (Table 7) reports the sample sizes for the different hypotheses of power and effect size.

[0413] (1-β)=0.99 (1-β)=0.95 (1-β)=0.80

[0414] Effect size

[0415] a=0.05 a=0.05 a=0.05

[0416]

[0417] Large (f=0.40) 60 44 28

[0418] Medium (f=0.25) 150 106 66

[0419] Small (f=0.10) 922 652 396

[0420]

[0421] Table 7. Results of the Power Analysis.

[0422] Duration of the study

[0423] The total duration of the study was about 4.5 months, specifically 1 month for the enrollment of the subjects, 3 months of carrying out the study considering the timing for the last enrolled subject, and 15 days of run-in. After that, the analysis of the results followed. The study began after a positive opinion from the Ethics Committee, and ended after the last subject had been enrolled and the final analyses had been performed.

[0424] Subjects could, at any time, withdraw from the study without giving any reason. Subjects had to be excluded from the study if the following cases occurred:

[0425] - withdrawal of consent by the subject;

[0426] - when a clinically relevant exclusion criterion occurred that could influence the subject's health;

[0427] - for women of childbearing age, if a pregnancy occurred;

[0428] - non-compliance of the subject with the protocol;

[0429] - decision of the principal investigator.

[0430] Treatments in the study

[0431] Food supplement

[0432] Hereinafter, the nutritional information of the food supplement based on Myrtle and Olive extracts in capsule form is reported.

[0433] Ingredients: mix of botanicals: olive (Olea europaea L., fruit) dried extract, myrtle (Myrtus communis L., leaves) dried extract (obtained by drying the hydroalcoholic extract from Myrtle leaves) (the weight ratio between the amount of the myrtle extract and that of the olive extract is about 1:1.14); maltodextrins; vegetable capsule (hydroxy-propyl-methylcellulose, colorant: calcium carbonate); bulking agents:calcium phosphates, cellulose; anti-caking agents: magnesium salts of fatty acids, silicon dioxide; colorant: iron oxides and hydroxides.

[0434] Format: 90 capsules of 550 mg.

[0435] Total content: 47.9 g.

[0436] Directions for use: take one capsule a day to be swallowed whole with a glass of water. Warnings: Keep out of reach of children under 3 years of age; Do not exceed the recommended daily dose; Supplements are not intended as a substitute for a varied and balanced diet and a healthy lifestyle.

[0437] Placebo

[0438] The placebo consists of inert excipients and is equal, in shape, weight, color and packaging to the active treatment, so as to ensure blinding. Ingredients: maltodextrins, vegetable capsule (hydroxy-propyl-methylcellulose, colorant: calcium carbonate), bulking agents: calcium phosphates, cellulose; anti-caking agents: magnesium salts of fatty acids, silicon dioxide; colorant: iron oxides and hydroxides;

[0439] Format: 90 capsules of 550 mg.

[0440] Total content: 52.8 g.

[0441] Directions for use: take one capsule a day to be swallowed whole with a glass of water. Warnings: Keep out of reach of children under 3 years of age; Do not exceed the recommended daily dose; Supplements are not intended as a substitute for a varied and balanced diet and a healthy lifestyle.

[0442] Methods

[0443] The recruited subjects took 1 capsule a day of food supplement or placebo for a duration of three months, depending on the group they belonged to. Before being included in the study, the subjects deemed suitable by the experimenter were given: 1) the information sheet, with clear information regarding the clinical study, the objectives and the methods of implementation;

[0444] 2) the informed consent, which was filled out and signed by the subjects and countersigned by the experimenter in duplicate;

[0445] 3) the information and manifestation of consent to the processing of personal data; 4) the food diary.In order to ensure the maintenance of an adequate sample size until the end of the study, it was planned to enroll about 5% more subjects than indicated by the Power Analysis (total 112 subjects).

[0446] The subjects, recruited and randomized, took 1 capsule a day of food supplement (TREATMENT) or placebo for a duration of three months, depending on the group they belonged to:

[0447] - GROUP 1 (56 subjects): subjects who had to take a capsule of the food supplement based on olive (Olea europaea L., fruit), and myrtle (Myrtus communis L., leaves); - GROUP 2 (56 subjects): subjects who had to take the placebo.

[0448] The recruited subjects went to the diagnostic center to perform the analyses provided for by the protocol (according to the times reported in Table 5). It was specified that said samples were kept for the objectives of the study and, after this period of time, they were destroyed.

[0449] Randomization

[0450] The randomization sequence was generated by a statistician using the STATA 16 software (Stata Statistical Software: Release 16. College Station, TX: StataCorp LLC), and the subjects were assigned to each of the two treatment groups (food supplement (TREATMENT), and PLACEBO) in a random and unpredictable manner through simple randomization (allocation ratio 1:1).

[0451] Blinding

[0452] The study was double-blind as all enrolled subjects remained unaware of the type of treatment they were subjected to, and the assignment to the treatment groups was not known to the experimenter, the study proponent or any other person involved in the conduct of the study, except in case of emergency. The two treatments were therefore made unrecognizable as the packaging was identical, and the dosage forms were the same in color, shape, weight and taste.

[0453] Data collection

[0454] Data collection was carried out through specific forms (Case Reporting Form - CRF) divided into two main sections. A first section related to personal data, the subject's anamnesis, the intake of any concomitant drugs, and the treatment group, and had to be compiled at the time of enrollment. Instead, the second section had to be compiled with the results of the analyses performed on the blood samples taken as reported in Figure 7. Any adverse effects were evaluated through a specially prepared formaccording to that used by the Italian National Institute of Health, Ministry of Health, for the reporting of suspected adverse reactions that could occur after the intake of food supplements.

[0455] Statistical analysis of the collected data

[0456] For each of the measurements in the two experimental groups, a descriptive analysis was first performed with the calculation of the mean, median, standard deviation, min-max range and 95% confidence interval for all the response variables taken into consideration. The subject's identity was used as a random factor to take into account any differences attributable exclusively to the subject's subjectivity and to eliminate them from the model, increasing its power and precision. For each response variable, an independent analysis was conducted.

[0457] BIBLIOGRAPHY

[0458] 1. A. Gholamhoseinian, B. Shahouzchi and F. Sharifi-Far, 2010. Inhibitory Activity of Some Plant Methanol Extracts on 3 -Hydroxy-3 -Methylglutaryl Coenzyme a Reductase. International Journal of Pharmacology, 6: 705-711.

[0459] 2. Alipour G, Dashti S, Hosseinzadeh H. Review of pharmacological effects of Myrtus communis L. and its active constituents [published correction appears in Phytother Res. 2021 Aug;35(8):4626. doi: 10.1002 / ptr.6993], Phytother Res. 2014;28(8): 1125- 1136. doi:10.1002 / ptr.5122

[0460] 3. Rosa A, Melis MP, Deiana M, et al. Protective effect of the oligomeric acylphloroglucinols from Myrtus communis on cholesterol and human low density lipoprotein oxidation. Chem Phys Lipids. 2008;155(1):16-23. doi: 10.1016 / j.chemphyslip.2008.04.005

[0461] 4. Zupo R, Castellana F, Crupi P, et al. Olive Oil Polyphenols Improve HDL Cholesterol and Promote Maintenance of Lipid Metabolism: A Systematic Review and Meta-Analysis of Randomized Controlled Trials. Metabolites. 2023; 13( 12): 1187. Published 2023 Dec 6. doi:10.3390 / metabo13121187.

[0462] 5. Bartolomei M, Bollati C, Li J, Arnoldi A, Lammi C. Assessment of the Cholesterol- Lowering Effect of MO MAST®: Biochemical and Cellular Studies. Nutrients. 2022; 14(3):493. https: / / doi.org / 10.3390 / nul4030493.

[0463] 6. Zhou R, Stouffer GA, Frishman WH. Cholesterol Paradigm and Beyond in Atherosclerotic Cardiovascular Disease: Cholesterol, Sterol Regulatory Element- Binding Protein, Inflammation, and Vascular Cell Mobilization in Vasculopathy. Cardiol Rev. 2022;30(5):267-273.

[0464] 7. Ruga S, Galla R, Penna C, Molinari C, Uberti F. The Activity of Ten Natural Extracts Combined in a Unique Blend to Maintain Cholesterol Homeostasis-In Vitro Model. Int J Mol Sci. 2022;23(7):3805. Published 2022 Mar 30.

Claims

CLAIMS1. Use of an association comprising-a hydroalcoholic extract from the leaves of Myrtus communis,- a dried extract from the fruits and / or leaves of Olea europea,for the maintenance of physiological blood lipid levels.

2. Use of the association according to claim 1, wherein the Myrtus communis extract comprises myricetin, the myricetin being in an amount ranging between 0.05% and 2.5% by weight of the total weight of the association.

3. Use of the association according to any one of claims 1 to 2, wherein the Olea europea extract comprises hydroxytyrosol, the hydroxytyrosol being in an amount ranging between 1% and 7.5% by weight of the total weight of the association.

4. Use of the association according to any one of claims 1 to 3, wherein the weight ratio of the amount of the Myrtus communis extract to the amount of the Olea europea extract is ranging between 1:0.5 and 1:2.

5. Use of the association according to any one of claims 1 to 4, wherein the association is in a liquid or solid form.

6. Association according to any one of claims 1 to 5, for use in the treatment of dyslipidemia and / or hypercholesterolemia.

7. Association for use according to claim 6, wherein the association is for use in the restoration of physiological blood lipid levels in conjunction with a condition selected from the group consisting of:- pathologies inducing an alteration of physiological blood lipid levels, selected, in turn, from the group consisting of: dysmetabolic diseases, such as diabetes mellitus; kidney pathologies, such as chronic kidney disease; liver pathologies, such as biliary cirrhosis; thyroid pathologies, such as hypothyroidism; pathologies with a genetic etiology, such as familial hypercholesterolemia and combinations of the foregoing; - excessive alcohol consumption;- drug intake;- and combinations of the foregoing.

8. Oral formulation comprising- the association according to any one of claims 1 to 7, being the only active ingredient in the oral formulation, and- suitable excipients and / or diluents,or- the association according to any one of claims 1 to 7, being a first active ingredient, and- at least a second lipid-lowering and / or cholesterol-lowering active ingredient, other than said association, and selected from the group consisting of: plant extracts, such as fenugreek extract; vitamins and / or minerals; berberine; policosanols; phytosterols and plant sterols; and combinations of the foregoing, - suitable excipients and / or diluents.

9. Oral formulation according to claim 8, wherein the association is contained in the formulation in an amount ranging between 10% and 90% by weight.

10. Oral formulation according to claim 8 or 9, in a physical form selected from: powder, granulate, tablet, capsule.

11. Oral formulation according to any one of claims 8 to 10, being a food supplement, or a nutraceutical, or a medicinal product.

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