Composition based on plant extracts useful for the treatment of dyslipidaemias

Abstract

The present invention relates to a composition containing plant extracts of Berberis aristata DC., Olea Europea L., Trigonella foenum-graecum L., Cynara scolymus L. and a mixture of phytosterols extracted from Helianthus annuus L., and the use thereof in the treatment of dyslipidaemias.

Description

[0001] “COMPOSITION BASED ON PLANT EXTRACTS USEFUL FOR THE

[0002] TREATMENT OF DYSLIPIDAEMIAS” * * *

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to a medicinal or nutraceutical composition comprising extracts of Berberis aristata DC., Olea Europea L., Trigonella foenum- graecum L., Cynara scolymus L. and Helianthus annuus L. The composition is useful in the treatment of dyslipidaemias, particularly hypercholesterolaemias.

[0005] BACKGROUND OF THE INVENTION

[0006] Cardiovascular (CV) diseases are the leading cause of death globally.

[0007] Among CV diseases, the atheroma-based component is the main one and it represents, also in Europe, one of the leading causes of premature death, together with ischaemic heart disease. Deaths under 75 years of age due to these causes are estimated to account for 42% in women and 38% in men (2019 ESC / EAS Guidelines for the management of dyslipidaemias: lipid modification to reduce cardiovascular risk: The Task Force for the management of dyslipidaemias of the European Society of Cardiology (ESC) and European Atherosclerosis Society (EAS) - European Heart Journal, Volume 41, Issue 1, 1 January 2020, Pages 111- 188).

[0008] Atherosclerosis is a condition normally associated with ageing, but it can be accelerated by several risk factors, the main one being hypercholesterolaemia. Specifically, LDL-cholesterol (LDL-C) is positively correlated with the risk of CV events, whereas HDL- cholesterol (HDL-C) is inversely correlated with the same risk.

[0009] At clinical level, there is evidence to support the efficacy of LDL-C reduction in preventing CV events; in particular, it is known that LDL-C is not only a marker of high risk of CV disease, but that it is a direct causal factor. In fact, its reduction, in the long term, leads to a subsequent decrease in CV events (Rees K, Dyakova M, Wilson N, et al. Dietary advice for reducing cardiovascular risk. Cochrane Database Sy st Rev 2013;(12):CD002128). These findings led to the inclusion of the concept of LDL-C reduction as a primary goal in all guidelines on the management of dyslipidaemia, presented by the European Atherosclerosis Society (EAS) and Cardiology Society (ESC) (Catapano AL, Graham I, De Backer G, et al; ESC Scientific Document Group. 2016 ESC / EAS Guidelines for the Management of Dyslipidaemias. Eur Heart J 2016;37:2999-3058).

[0010] The drug class most widely used in the treatment of hypercholesterolaemia, and in particular in the reduction of LDL-C, is statins. Despite clear indications regarding the use of statins by physicians and the supporting bibliography, studies have shown that about half of patients on statin therapy discontinue the treatment 6 months after starting, and only a quarter of patients continue these treatments in the long term. Several adverse effects related to statin use have been reported over the years, and among these the most common seem to be muscle symptoms such as cramps, myalgia or fatigue, which contribute to the decision to stop statin therapies.

[0011] Moreover, patients often refuse drug treatment from the beginning, especially in cases where the cholesterolaemia level is not yet pathological, but moderately high (LDL-C > 115 mg / dL and < 190 mg / dL).

[0012] In this type of patient, treatment with food supplements for cholesterol control and improvement of the lipid profile is preferable, as well as recommended by international guidelines, always in combination with a balanced diet. This should be coupled with advice on a healthy lifestyle, opting, for example, to reduce a sedentary lifestyle in favour of physical exercise, avoiding alcohol, stopping smoking and reducing stress.

[0013] A food supplement is therefore part of a strategy to delay or prevent the need for drug therapy.

[0014] For several years, fermented red rice, a dietary supplement containing monacolin K, which is chemically identical to lovastatin, and is active in inhibiting the enzyme hydroxymethylglutaryl-CoA reductase (HMG-CoA), key to endogenous cholesterol production, has been studied and widely used as a natural replacement for statins (A MetaAnalysis of Red Yeast Rice: An Effective and Relatively Safe Alternative Approach for Dyslipidemia, Li Y, Jiang L, Jia Z, Xin W, Yang S, et al. (2014)).

[0015] Intake of fermented red rice has been recommended as an alternative to statin drug therapy, however, it must be produced according to GMP principles, to ensure standard dosages of active ingredients and the absence of health-threatening contaminants, which makes it a more cost- and time-consuming product at industrial level.

[0016] The problem of finding new hypocholesterolemic (cholesterol-lowering) dietary supplements, in particular, those useful in the treatment of borderline patients, statin- intolerant patients or patients who refuse to take statins, remains to this day.

[0017] BACKGROUND ART

[0018] EP3590356 describes formulations containing phytosterols, fermented red rice and hydroxytyrosol in the treatment of hypercholesterolaemias, and a method for producing these formulations.

[0019] EP1983989 describes a combination of botanical extracts with cardiovascular benefits. In particular, the application reports the synergy between berberine and phytostanols in reducing blood cholesterol levels.

[0020] WO20221 25667 describes associations of berberine, phytosterols, silymarin and polydatin, with olive extract as a possible additional component.

[0021] EP3270707 describes compositions containing extracts of apple, green tea, grape and olive and possibly other ingredients, including berberine and phytosterols.

[0022] SUMMARY OF THE INVENTION

[0023] The invention relates to a pharmaceutical or nutraceutical composition comprising an extract of Berberis aristata DC., an extract of Olea Europea L., an extract of Trigonella foenum- grae cum L., an extract of Cynara scolymus L. and a mixture of phytosterols from Elelianthus annuus L.

[0024] The invention further relates to the use of said composition as a medicament, and in particular for use in the treatment of hypercholesterolaemia, or as an adjuvant in statin therapy.

[0025] DETAILED DESCRIPTION OF THE INVENTION

[0026] The inventors have now found a combination of plant extracts that act synergistically in the treatment of dyslipideamias, particularly hypercholesterolaemia.

[0027] The invention relates to a pharmaceutical or nutraceutical composition comprising an extract of Berberis aristata DC., an extract of Olea Europea L., an extract of Trigonella foenum- grae cum L., an extract of Cynara scolymus L. and a mixture of phytosterols from Elelianthus annuus L.

[0028] In a preferred embodiment, the composition of the invention comprises, independently of each other:

[0029] (i) an aqueous extract of bark from branches of Berberis aristata DC., wherein said extract is optionally dried;

[0030] (ii) the water-soluble fraction obtained by the pressing of the fruit of Olea Europea L., wherein said fraction is optionally dried;

[0031] (iii) an extract of Trigonella foenum- graecum L. seeds in a water / ethanol mixture, wherein said extract is optionally dried;

[0032] (iv) an extract of Cynara scolymus L. leaves in a water / ethanol mixture, wherein said extract is optionally dried;

[0033] (v) a mixture of phytosterols from Helianthus annuus L. seeds, wherein said mixture is optionally dried.

[0034] The first ingredient is an aqueous extract of bark from Berberis aristata DC branches containing berberine, which is an alkaloid known to treat hypercholesterolaemia, and is known for its action on the increased membrane expression of a receptor protein capable of internalising LDL.

[0035] The second ingredient is an aqueous extract of Olea Europea L. containing hydroxytyrosol capable of inducing a significant improvement in dyslipidaemia in subjects with high cholesterol (115-190 mg / dL), causing a significant reduction in LDL.

[0036] The third component is an extract in a water and ethanol mixture, preferably in a ratio of 5: 1, of Trigonella foenum- graecum L. seeds, an ingredient known for its ability to improve dyslipidaemias, even in type II diabetic patients.

[0037] The fourth component is an extract in a water and ethanol mixture, preferably in a ratio of 5: 1, of Cynara scolymus L. leaf containing chlorogenic acid, known for its ability to inhibit the enzyme HMGCoA-reductase, thus reducing hypercholesterolaemia.

[0038] The last component substantially consists of a mixture of phytosterols from Helianthus annuus L. seeds, known for their ability to decrease hypercholesterolaemia, according to several clinical studies.

[0039] It was surprisingly found out that, by combining the above-mentioned five ingredients in one formulation, a synergistic cholesterol-lowering effect is achieved. Without being bound to a specific mechanism of action, it was observed that the combination of active ingredients according to the invention acts synergistically at different levels of cholesterol synthesis and metabolism, in particular through inhibition of the enzyme HMG-CoA reductase, inhibition of PCSK9-LDLR binding, and inhibition of the transcription factor SREBP involved in cholesterol biosynthesis.

[0040] According to preferred embodiments of the invention:

[0041] (i) the extract of Berberis aristata DC. has a berberine hydrochloride content of at least 85% by weight, preferably at least 97% by weight on a dry basis; and / or

[0042] (ii) the extract of Olea Europea L. has a total polyphenol content comprised between 6 and 15% by weight, and a hydroxytyrosol content of at least 4% by weight, preferably between 4 and 9% by weight; and / or

[0043] (iii) the extract of Cynara scolymus L. has a chlorogenic acid content of at least 0.5% by weight; and / or

[0044] (iv) the mixture of phytosterols from Helianthus annuus L. has a beta- sitosterol content of at least 40% by weight, preferably comprised between 40 and 50% by weight.

[0045] In a further preferred embodiment, the composition comprises 27 to 33% by weight of extract of Berberis aristata DC., 20 to 26% by weight of extract of Olea Europea L., 2 to 8% by weight of extract of Trigonella foenum- graecum L., 14 to 20% by weight of extract of Cynara scolymus L., and 20 to 26% by weight of mixture of phytosterols from Helianthus annuus L. seeds, wherein said percentages refer to the total weight of the extracts.

[0046] In a particularly preferred embodiment, the composition of the invention contains the following quantities of active ingredients per dosage unit:

[0047] In a preferred embodiment, the composition according to the invention is in a form suitable for oral administration. Preferably, forms suitable for oral administration include tablets, effervescent tablets, powders, granules, capsules, sticks, effervescent sticks, singledose liquid vials and syrups. Capsules are the preferred form of administration and also include modified-release capsules.

[0048] The composition of the present invention may further comprise one or more excipients acceptable for pharmaceutical or food use, in particular chosen from among diluents, filling agents, flow agents, anti-caking agents (anti-agglomerating agents), dispersing agents, thickeners, dissolution promoters, flavour modifiers, sweeteners.

[0049] In the case of the preferred form of administration, namely the capsule, it may have various compositions selected from a list that includes, but is not limited to, hydroxy-propyl- methyl-cellulose (HPMC), hydroxy-propyl-methyl-cellulose and gellan gum, gelatin, plant polysaccharides and any plasticisers, not limited to sorbitol, glycerine, colouring agents, disintegrants and lubricants; preferably HPMC. In the case of filling agents, they are selected from a list comprising, but is not limited to, maltodextrin, maize starch, pre- gelatinised maize starch, microcrystalline cellulose, calcium carbonate, acacia fibre, FOS, inulin, GOS, preferably maltodextrin.

[0050] In the case of anti-agglomerating agents, they are selected from a list comprising, but is not limited to, silicon dioxide, sodium and aluminium acid phosphate, calcium silicate, magnesium silicate, talc, sodium aluminium silicate, potassium aluminium silicate, and preferably silicon dioxide.

[0051] In the case of flow agents, they are selected from a list comprising, but is not limited to, magnesium stearate, cross-linked sodium carboxymethyl cellulose, cross-linked cellulose gum, enzymatically hydrolysed carboxymethyl cellulose, enzymatically hydrolysed cellulose gum, sodium, potassium and calcium salts of fatty acids, magnesium salts of fatty acids, and diglycerides of fatty acids, acetic esters of mono- and diglycerides of fatty acids, lactic esters of mono- and diglycerides of fatty acids, citric esters of mono- and diglycerides of fatty acids, tartaric acid esters of mono- and diglycerides of fatty acids, mono- and diacetyl tartaric acid esters of mono- and diglycerides of fatty acids, mixed acetic and tartaric acid esters of mono- and diglycerides of fatty acids, sucroglycerides, polyglycerol esters of fatty acids, poly glycerol polyricinoleate propane- 1,2-diol esters of fatty acids, sodium stearoyl-2- lactylate, calcium stearoyl-2-lactylate, stearyl tartrate, sorbitan monostearate, sorbitan tristearate, sorbitan monolaurate, sorbitan monooleate, sorbitan monopalmitate, preferably magnesium stearate.

[0052] The invention also relates to a pharmaceutical or nutraceutical composition as defined herein, for use as a medicament.

[0053] Preferably, the composition of the invention is for use in the treatment of dyslipidaemias, particularly dyslipidaemias characterised by hypercholesterolaemia, in a subject in need thereof.

[0054] Furthermore, the composition of the invention may be conveniently used in combination with hypocholesterolemic (cholesterol-lowering) drugs, i.e., substances and / or preparations capable of reducing levels of circulating cholesterol in a subject, in particular statins.

[0055] Thus, in a further aspect, the invention is directed to the composition as defined herein for use in the treatment of a subject undergoing a pharmacological treatment with hypocholesterolemic (cholesterol-lowering) agents, preferably statins.

[0056] The composition may be administered to a subject at a frequency of at least one administration per day, e.g. one or two or more capsules per day, depending on several factors such as the subject's weight and age, any concomitant treatment with other supplements or drugs, severity of the condition to be treated, the subject's response to the treatment, the subject's intolerance to the active ingredients, and other factors.

[0057] The present invention is illustrated in detail in the examples provided below.

[0058] EXAMPLES

[0059] Example 1. Compound extraction process

[0060] The extract of Berberis aristata DC. was obtained in the following sequential steps:

[0061] Percolation of Berberis Aristata cortex ex ramis with dilute sulphuric acid pH adjustment to 2-3 with concentrated HC1

[0062] “Salting out” with 5% (Kg / L) NaCl

[0063] Obtaining extract of Berberis aristata DC concentrated in crude Berberine hydrochloride Addition of water and heating to 95-100 °C to dissolve pH adjustment to 8.5-9.5 with NaOH solution

[0064] Filtration

[0065] Cooling of the filtrate to 62-67 °C pH adjustment to 2-3 with HC1

[0066] Cooling to room temperature

[0067] Filtration and rinsing

[0068] Drying at 70-80 °C for 8-10 hours

[0069] Grinding and sieving

[0070] Obtaining extract of Berberis aristata extract DC concentrated in Berberine hydrochloride

[0071] The final product is then subjected to granulation by:

[0072] Mixing the extract with pre- gelatinised starch

[0073] Granulation

[0074] Sieving through 20-60 mesh membrane

[0075] Obtaining the Granulated product

[0076] The extract of Trigonella foenum- graecum L. was obtained in the following steps:

[0077] Extraction with water and ethanol (5: 1) of Trigonella foenum- graecum L seeds.

[0078] Concentration of the extract under vacuum

[0079] Crystallisation and precipitation

[0080] Filtration

[0081] Spray drying

[0082] Shredding and sieving to obtain a fine powder. The extract of Cynara scolymus L was obtained in the following sequential steps:

[0083] Grinding of Cynara scolymus L. leaves

[0084] Weighing of compound Cynara scolymus E. and solvent (Water / Ethanol 5:1)

[0085] Extraction using solvent

[0086] Separation of plant residue

[0087] Concentration of the thus-obtained compound

[0088] The product can be further processed as follows:

[0089] Addition of Maltodextrin

[0090] Drying

[0091] Mixing

[0092] Sieving the dried extract (sieve No. 180 mesh) to obtain a fine powder.

[0093] The extract of Olea Europea L. was obtained from the olive oil production process. After the olives are pressed and the oil is extracted, the OOMW (olive oil mill waste) is recovered and subjected to the following steps:

[0094] Filtration

[0095] Concentration of the filtrate to obtain a liquid product

[0096] Foam-drying

[0097] Obtaining the final product of interest

[0098] The mixture of phytosterols from Helianthus annuus L. is extracted from the seeds of the plant by the following steps: grinding of seeds to powder extraction of the saponifiable fraction with potash extraction of the unsaponifiable fraction (phytosterols) with hexane Concentration of the unsaponifiable fraction

[0099] Spray drying

[0100] Shredding and sieving to obtain a fine powder.

[0101] Example 2. Powder mixing process

[0102] To produce 1 kg of composition as an example, the following weighing was carried out:

[0103] • 305.89 g of Berberis aristata DC.

[0104] • 235.29 g of phytosterols

[0105] • 235.29 g of Olea Europea L.

[0106] • 176.47 g of Cynara scolymus L.

[0107] • 47.06 g of Trigonella foenum graecum L.

[0108] The powders were then added one after the other in a mixer, in which they were mixed for 20 minutes by 16 rotations to the right alternating with 16 rotations to the left on an inclined plane.

[0109] Example 3. Anti-cholesterolaemic activity in vitro

[0110] In order to evaluate the anti-cholesterolaemic activity of the composition of plant extracts according to the present invention and reported below, and to verify its synergy with respect to the individual extracts, three specific mechanisms of action related to cholesterol control were investigated.

[0111] Specifically, the composition under study is composed of:

[0112] The plant extracts are as follows:

[0113] Berberis aristata DC., extract - herein also called “Berberine”

[0114] Phytosterols 95% - herein also called “Phytosterols”

[0115] Trigonella foenum graecum L., extract - herein also called “Fenugreek” Cynara Scolymus L., extract - herein also called “Artichoke” Olea europea L., extract - herein also called “Olea europea”

[0116] The individual extracts and the composition were tested for activity in all three models reported below at different concentrations.

[0117] Example 4. Inhibition of HMG-CoA reductase enzyme activity

[0118] For the study, the extracts and the above-reported composition diluted in 100 ml of water were analysed to simulate daily intake of the composition as an anti-cholesterolaemic food supplement. The maximum amount that can be used in a multiwell was then chosen for the study.

[0119] An aliquot of each extract and said composition was added to the reaction mix consisting of NADPH, HMG-CoA and HMG-CoA reductase.

[0120] The reaction was monitored kinetically by measurements every 20 seconds for 10 minutes on a spectrophotometer at 340 nm.

[0121] In parallel, control tests were set up:

[0122] White: reaction mixture without enzyme; then subtracted from all the measurements; Standard enzyme activity: reaction mixture without sample, to verify enzyme activity in the experimental system.

[0123] The final quantities per well are as follows: Berberine 6.5 pg, Phytosterols 5 pg, Fenugreek 1 pg, Artichoke 3.75 pg, Olea Europea 5 pg, Mix (i.e. the above-mentioned composition) 29.5 pg.

[0124] An aliquot of the samples submitted for study was used for the assessment of HMG- CoA reductase enzyme activity by using a commercially available enzyme assay (HMG-CoA Reductase Assay Kit, CS1090, Sigma- Aldrich). The assay is based on spectrophotometric measurement of the decrease in absorbance at 340 nm, which represents the oxidation of NADPH by the catalytic subunit of HMGR in the presence of the substrate HMG-CoA.

[0125] The table shows:

[0126] The activity of HMG-CoA reductase in the presence of each extract and the composition (“MIX”), and the comparison with the standard activity of the enzyme.

[0127] Data are expressed as pmol of NADPH converted by the enzyme HMG-CoA reductase to NADP+ per min per mg of protein. No inhibitory activity was detected by Berberine and Artichoke in this system. Their activity in controlling cholesterol is in fact associated with other biochemical and molecular pathways, as shown in the following tests.

[0128] Example 5. Inhibition of PCSK9-LDLR binding

[0129] PCSK9 (Proprotein convertase subtilisin / kexin type 9) is a protein belonging to the subtilisin family, which acts by binding to the LDLR (LDL cholesterol membrane receptor), accelerating its lysosomal degradation and thereby reducing its receptor density on the surface of hepatocytes. As a result, circulating cholesterol is not internalised. Inhibition of the binding between PCSK9 and the LDL-cholesterol receptor results in reusing of the receptor itself, which absorbs circulating cholesterol intracellularly.

[0130] For the study, the extracts and the composition diluted in 100 ml of water were analysed to simulate daily intake of the composition as an anti-cholesterolaemic food supplement.

[0131] The maximum amount that can be used in a multiwell was then chosen for the study.

[0132] An aliquot of each extract and the composition (Test Inhibitors) was added to the reaction mix consisting of LDLR, PCSK9 and Streptavidin-HRP.

[0133] The reaction was monitored by luminescence spectrophotometry. The assay is based on the spectrophotometric measurement of luminescence mediated by Streptavidin-HRP and resulting from inhibition of LDLR-PCSK9 binding.

[0134] The final quantities per well are as follows: Berberine 65 pg, Phytosterols 50 pg, Fenugreek 10 pg, Artichoke 37.5 pg, Olea Europea 50 pg, Mix (i.e. the above-mentioned composition) 295 pg.

[0135] The results are expressed below as % inhibition of LDLR-PCSK9 binding.

[0136]

[0137] Experimental data show that berberine has a high and significant inhibition of PCSK9- LDLR binding. Fenugreek and olea europea show significant, but reduced inhibition with respect to berberine.

[0138] The mixture of components (namely, the composition according to the present invention) exerts a synergistic effect of inhibiting LDLR-PCSK9 binding.

[0139] Example 6. Inhibition of the transcription factor SREBP involved in cholesterol biosynthesis

[0140] Lipid homeostasis in vertebrate cells is regulated by a family of transcription factors called SREBPs (sterol regulatory elements binding proteins). SREBPs activate the expression of more than 30 genes involved in synthesis of cholesterol and the cellular uptake thereof. SREBP in the inactivated form is fixed in the endoplasmic reticulum and is activated as a consequence of a cellular decrease in cholesterol. In this case, it induces a series of pathways that lead to increased biosynthesis of endogenous cholesterol and production of LDLR for blood cholesterol absorption.

[0141] Among the various transcription factors, some activate cholesterol synthesis by inducing the expression of the enzyme HMG-CoA reductase. An inhibition of SREBP leads to a decrease in endogenous cholesterol biosynthesis.

[0142] For the study, the extracts and the composition diluted in 100 ml of water were analysed to simulate daily intake of the composition as an anti-cholesterolaemic food supplement.

[0143] An aliquot of each extract and the composition was added to the culture medium and diluted 1:20 in order to obtain non-cyto toxic concentrations. Hepatocyte cell cultures (H1000.H15B LOT. HC4-20) were seeded in a 96-well plate and incubated for 72 hours at 37 °C, RH 90%, 5% CO2.

[0144] Culture medium in which each test sample had previously been diluted was added to each well.

[0145] In parallel, control tests were set up:

[0146] Negative CTR: cell cultures not treated with test samples.

[0147] A commercially available kit (SREBP Transcription Factor Assay Kit - aAB133111) is used to assay the transcription factor SREBP in the nuclear extracts and cell lysates.

[0148] This is an ELISA assay using a specific double- stranded DNA (dsDNA) sequence containing the SREBP binding element immobilised in the wells of a 96-well plate. SREBP contained in cell culture extracts binds specifically to the SREBP binding element and is detected by the addition of a specific anti-SREBP primary antibody .

[0149] A secondary antibody is added to provide a sensitive colorimetric reading at 450 nm.

[0150] Tested concentrations of each test product:

[0151] Results - The table shows the colorimetric reading values at 450 nm for each sample and the percentage (%) difference with respect to the negative control.

[0152] All the ingredients under study have SREBP-inhibiting activity. In particular, the composition according to the present invention (“MIX”) has higher activity than the individual ingredients. This means that it is inhibited the active form of the transcription factor SREBP, which is not able to move into the cell nucleus, and thus it does not bind the DNA site and consequently does not activate any biosynthetic pathway.

[0153] Example 7 - Example of capsule according to the present invention

[0154] The composition according to the present invention was formulated as a capsule, according to the following weighting:

[0155] Example 8 - Efficacy of the composition according to the present invention as food supplement.

[0156] - Study design and setting

[0157] A comparative, randomised, parallel-group clinical trial (RCT) was carried out to evaluate the efficacy of the composition according to the present invention on lipid and metabolic parameters in subjects falling within the “Eligibility Criteria” reported below.

[0158] - Eligibility Criteria

[0159] Subjects who at the screening visit were aligned to the below eligibility criteria, and did not fall the cases of the exclusion criteria (see below), were recruited for the study, after signing a privacy form and informed consent.

[0160] In particular, 36 patients aged between 18 and 75 years, of both sexes, were enrolled with the following criteria:

[0161] - LDL-C > 115 mg / dL and < 190 mg / dL; - Triglycerides (Tg) > 150 mg / dL;

[0162] - Fasting Plasma Glucose (FPG) > 100 and < 125 mg / dL;

[0163] - Waist circumference: males (M) > 102 cm, females (F) > 88 cm;

[0164] - Diastolic blood pressure (DBP) > 90 mmHg and systolic blood pressure (SBP) > 140 mmHg or on treatment.

[0165] The exclusion criteria were:

[0166] - subjects treated in the last 2 months with hypolipidemic drugs, hypoglycaemic drugs, anorectic drugs, psychotropic drugs, diuretics, beta-blockers, biologicals, steroids, immunosuppressants, other food supplements;

[0167] - subjects with Tg > 400 mg / dL;

[0168] - subjects with uncontrolled hypertension;

[0169] - subjects with cardiovascular diseases;

[0170] - obese subjects (body mass index (BMI) > 30 kg / m2);

[0171] - diabetic subjects;

[0172] - subjects with impaired thyroid function;

[0173] - subjects with impaired liver or kidney function;

[0174] - subjects with neoplasms, chronic inflammatory bowel disease, malabsorption syndromes, psychiatric illnesses, cirrhosis of the liver, pancreatitis, HIV;

[0175] - subjects who abuse alcohol and / or drugs;

[0176] - pregnant or breastfeeding women; - concomitant participation in other clinical trials;

[0177] - subjects who have not signed informed consent.

[0178] - Treatment groups

[0179] The selected subjects were randomly divided into the following two treatment groups:

[0180] - Group 1: one capsule according to Example 7, per day, to be taken after dinner;

[0181] - Group 2: two capsules according to Example 7, per day, to be taken after dinner.

[0182] In the event of difficulty in taking the capsule(s), a glass of still water was used to accompany the swallowing of the food supplement.

[0183] Once recruited for the study, the patients began a “run-in” period of 15 days prior to the start of the study, in which they followed only a predetermined low-lipid diet (the same diet was maintained throughout the entire treatment period).

[0184] At the end of the “run-in” period, the study began with the baseline visit (TO), in which a blood sample was collected for blood chemistry tests, a doctor's visit was carried out to confirm that each subject was eligible for participation in the clinical study, and the above- mentioned capsule(s) was(were) given to the patients according to the above treatment groups.

[0185] The follow-up visits took place after 30 (Tl) - 60 (T2) - 90 (T3) days, in which the patients underwent clinical evaluation and blood-chemical tests.

[0186] The food supplement was delivered at each visit (starting from TO) in an adequate quantity to cover the following month of treatment (30 or 60 tablets); unused capsules and empty blister packs have been recalled at the next visit in order to verify patients' adherence to treatment. - Statistic analysis

[0187] The data (raw or variations) were subjected to paired bilateral Student's t test (in the case of intragroup statistical analysis) or to two- sample identical variance (in the case of intergroup statistical analysis).

[0188] Intragroup statistical analysis (pairwise, vs. baseline) was performed on the raw data. Intergroup statistical analysis (pairwise) was carried out on the variations. The statistical analysis was performed using a Microsoft Excel for enterprise spreadsheet (build 17029.20068) and is reported as follows: * p < 0.05, ** p < 0.01, *** p < 0.001).

[0189] - Results

[0190] All 36 subjects recruited for the study completed the 90-day treatment.

[0191] The obtained results have been summarized in the following Table 1 (Group 1) and Table 2 (Group 2).

[0192] The significance of the data compared to the baseline value (TO) was calculated for each of the following analysed parameters: Total Cholesterol (TC, mg / dL), LDL-Cholesterol (LDL, mg / dL), HDL-Cholesterol (HDL, mg / dL), Triglycerides (TG, mg / dL), Glycemia (“Easting Plasma Glucose”, EPG, mg / dL), Insulinemia (“Fasting plasma insulinemia”, FPI, mg / dL) and HOMA-index (“Homeostasis Model Assessment”, obtained according to the following math formula: (FPG x FPI) / 405)). Table 1: Results of Group 1 (1 capsule / day).

[0193] Table 2: Results of Group 2 (2 capsule / day).

[0194] Surprisingly, the treatment with a capsule (Group 1, Table 1), as well as two capsules (Group 2, Table 2), resulted in a significant reduction of cholesterol, LDL, triglycerides, glycemia and insulinemia (***p<0.001) compared to the baseline (TO) value, at all three times analysed (namely, Tl, T2 and T3).

Claims

CLAIMS1. A pharmaceutical or nutraceutical composition for oral administration comprising an extract of Berberis aristata DC., an extract of Olea Europea L., an extract of Trigonella fo enum- grae cum L., an extract of Cynara scolymus L. and a mixture of phytosterols from Helianthus annuus L.

2. The composition according to claim 1 comprising, independently of each other:(i) an aqueous extract of bark from branches of Berberis aristata DC., wherein said extract is optionally dried;(ii) the water-soluble fraction obtained by the pressing of the fruit of Olea Europea L., wherein said fraction is optionally dried;(iii) an extract of Trigonella foenum- graecum L. seeds in a water / ethanol mixture, preferably in a 5:1 ratio, wherein said extract is optionally dried;(iv) an extract of Cynara scolymus L. leaves in a water / ethanol mixture, preferably in a 5:1 ratio, wherein said extract is optionally dried;(v) a mixture of phytosterols from Helianthus annuus L. seeds, wherein said mixture is optionally dried.

3. The composition according to claim 1 or 2, wherein:(i) the extract of Berberis aristata DC. has a berberine hydrochloride content of at least 85% by weight, preferably at least 97% by weight on a dry basis;(ii) the extract of Olea Europea L. has a total polyphenol content comprised between 6 and 15% by weight and a hydroxytyrosol content of at least 4% by weight, preferably between 4 and 9% by weight;(iii) the extract of Cynara scolymus L. has a chlorogenic acid content of at least 0.5%by weight;(iv) the mixture of phytosterols from Helianthus annuus L. has a beta- sitosterol content of at least 40% by weight, preferably comprised between 40 and 50% by weight.

4. The composition according to claims 1 to 3, comprising 27 to 33% by weight of extract of Berberis aristata DC., 20 to 26% by weight of extract of Olea Europea L., 2 to 8% by weight of extract of Trigonellafoenum-graecum L., 14 to 20% by weight of extract of Cynara scolymus L. and 20 to 26% by weight of mixture of phytosterols from Helianthus annuus L. seeds, wherein said percentages refer to the total weight of the extracts.

5. The composition according to claims 1 to 4, wherein said composition is in a form suitable for oral administration preferably chosen from capsules, tablets, effervescent tablets, powders, granules, sticks, effervescent sticks, single-dose liquid vials and syrups, preferably in capsule or modified-release capsule form.

6. The composition according to claim 5, further comprising one or more excipients acceptable for pharmaceutical or food use.

7. The composition according to claim 6, where said excipients are chosen from diluents, filling agents, anti- agglomerating agents, dispersing agents, thickeners, flow agents, dissolution promoters, flavour modifiers, sweeteners.

8. The composition according to claims 1 to7, for use as a medicament.

9. The composition according to claim 8, for use in the treatment of dyslipidaemias, preferably dyslipidaemias characterised by hypercholesterolaemia, in a subject in need thereof.

10. The composition for use according to claims 8-9, wherein said subject is undergoing a pharmacological treatment with cholesterol-lowering agents, preferably statins.

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