DESCRIPTION

The present invention relates to a nutraceutical or pharmaceutical composition effective in the prevention and/or treatment of cardiovascular pathologies in both humans and animals.

Background of the invention

The term "cardiovascular diseases" includes all diseases affecting the heart and blood vessels. The most common are diseases of atherosclerotic origin, in particular, cardiac diseases (e.g., acute myocardial infarction, angina pectoris, cardiomyopathies, heart failure, and arrhythmias), cerebrovascular diseases (e.g., ischemic and hemorrhagic stroke), and peripheral vascular disorders (e.g., peripheral arteriopathy obliterans).

Hypertension

The risk of developing cardiovascular disease increases with increasing blood pressure values. When the maximum (systolic blood pressure) and minimum (diastolic blood pressure) pressure values are equal to or greater than 130/85 mmHg, this is called hypertension.

Hypertension is a condition in which the heart, having to overcome an increase in peripheral resistance, exerts a pressure on the arterial walls that exceeds the reference values. Hypertension can be classified into primary and secondary.

Primary hypertension has no precise etiology: high blood pressure values are the result of a disruption of physiological mechanisms that regulate pressure (autonomic nervous system, plasma mediators, etc.). Secondary hypertension, on the other hand, is the consequence of diseases affecting the kidneys, adrenals, vessels, and heart.

In such a case, treatment of the disease may result in normalization of blood pressure values. The main classes of antihypertensive drugs are: diuretics (thiazide, loop, and potassium-sparing), peripherally acting (β-blockers, a- blockers, and mixed adrenergic antagonists) and centrally acting (ganglioplegics and adrenergic neuron blockers) sympatholytics, vasodilators, calcium channel blockers, ACE inhibitors, and sartans. However, taking these drugs results in several side effects including orthostatic hypotension, cough, headache, cramps, vomiting, nausea, itching, rash, sedation, bradycardia, and libido disturbances.

Dyslipidemias

Dyslipidemias represent a group of pathological conditions characterized by abnormal blood concentrations of lipids, in particular, cholesterol and triglycerides.

One of the most significant risk factors for cardiovascular disease is precisely elevated cholesterolemia . Total cholesterolemia should be less than 200 mg/dL. In particular, an HDL cholesterol value of more than 40 (man) -45 (woman) mg/dL is desirable and LDL cholesterol less than 100 mg/dL in patients at high cardiovascular risk and less than 70 mg/dL in patients who have already had a cardio or cerebrovascular event. High plasma triglyceride concentration (over 150 mg/dL) also contributes to higher LDL cholesterol values.

The main hypolipidemic drugs are: the antilipogenics, which inhibit triglyceride and cholesterol synthesis at the hepatic level (e.g., fibrates and statins); antilipolytics, which inhibit lipolysis at the level of adipose tissue resulting in reduced levels of fatty acids necessary for hepatic triglyceride synthesis (e.g., nicotinic acid and derivatives); and - sequestrants, which stimulate the conversion of cholesterol to bile acids or limit intestinal absorption of fats (e.g., cholestyramine, probucol, and ion exchange resins).

The most common side effects reported by patients on therapy with hypolipidemic drugs, particularly statins, are myalgia, arthralgia, pain in the extremities, muscle spasms, joint swelling, back pain, nasopharyngitis, headache, pharyngolaryngeal pain, epistaxis, constipation, flatulence, dyspepsia, nausea, diarrhea and increased creatinephosphokinase . Atherosclerosis Cholesterol is transported in the blood by specific lipoproteins such as low-density lipoproteins (LDL), which supply peripheral tissues with cholesterol, and high-density lipoproteins (HDL), which, in contrast, remove excess cholesterol from tissues (including blood vessels) and return it to the liver. Free radicals can oxidize LDL lipoproteins, which infiltrate and deposit in the intima of vessels leading to endothelial damage.

The latter triggers a chronic inflammatory process with transformation of monocytes into macrophages, which, after phagocytizing oxidized LDL, turn into foam cells. Foamy cells represent the main center of accumulation of lipids, substances derived from the inflammatory process (fibrinogen, white blood cells, macrophages, etc.) and blood elements (red blood cells, platelets, calcium, etc.) from which the atheromasic plaque (atheroma) originates. Atheromas cause a non-elastic thickening of the vessel wall that narrows or completely occludes the vessel. If atheromas are found in the coronary arteries, they can cause the onset of angina pectoris or heart attack; if, on the other hand, they are found in vessels that carry blood to the brain, they can cause an ischemic stroke.

Drugs used to treat atherosclerosis include hypolipidemic agents, antiplatelet agents, anticoagulants, antihypertensives, and antidiabetics.

Angina pectoris

Angina pectoris can occur as a result of in-sufficient oxygenation of cardiac tissue due to transient interruption/slowing of blood flow in the coronary arteries when they are only partially obstructed. It manifests as acute chest pain and often arises in conjunction with increased blood demand (e.g., during physical activity). Angina can be treated with nitroglycerin, possibly combined with a β-blocker and sometimes a calcium antagonist. Antiplatelet agents are used to prevent angina.

Infarction

Acute myocardial infarction can be caused by rupture of an atherosclerotic plaque and subsequent formation of a thrombus, resulting in occlusion of a coronary artery and necrosis of the myocardial tissue supplied by the obstructed artery.

In severe cases, it can complicate to the point of cardiovascular collapse and death. Treatment involves the use of antiplatelet drugs, thrombolytics, β-blockers, ACE inhibitors, analgesics and hypolipidemic drugs.

Stroke

Stroke is a cerebrovascular injury caused by the interruption of blood flow to the brain by occlusion (ischemic stroke) or rupture (hemorrhagic stroke) of a cerebral artery. An artery can become occluded because a clot forms within it (cerebral thrombosis) or because it is reached by clots that have started, for example, from the heart or atherosclerotic plaques in the arteries that carry blood to the brain (cerebral embolism). In the case of hemorrhagic stroke, on the other hand, there is cerebral hemorrhage, often due to excessively high blood pressure resulting in ruptured normal or malformed vessels (aneurysms) or abnormal blood clotting.

In either case, brain cells, deprived of oxygen and nutrients, begin to die resulting in edema that also leads to suffering of brain cells in a large surrounding area. Depending on the part of the brain affected, stroke can result in paralysis of related muscle areas and loss of some functions (speech, vision or memory). Treatment involves the use of antiplatelet drugs, thrombolytics, β-blockers, and ACE inhibitors. Heart failure

Occurs when the heart can no longer pump blood in adequate amounts relative to the body's actual demand, causing insufficient oxygenation of organs and tissues. It may initially involve only the left ventricle but, over time, can affect the entire heart muscle. In therapy, drugs such as ACE inhibitors, β-blockers, diuretics and aldosterone antagonists are used.

Therefore, given the numerous side effects of currently available drugs and the morbidity of the conditions discussed above, there remains a need for effective alternative compositions in the prevention and treatment of cardiovascular disease.

The subject of the present invention is a composition comprising an extract of a plant belonging to the genus Berberis, astaxanthin, and an extract of a plant belonging to the genus Crataegus for the prevention and treatment of cardiovascular diseases.

GLOSSARY

The terms used in this description are as generally understood by the technical expert, except where otherwise indicated.

The term "extract, " in the context of this description, means any product attributable to a plant drug including all products derived from mechanical processing (pulverization, crushing, mixing, and/or other methods) or extractive processing (solvent extraction, distillation, and/or other specific methods) operated on a drug.

DETAILED DESCRIPTION OF THE INVENTION

Several species belong to the genus Berberis of which the main ones are Berberis aristata Berberis vulgaris and Berberis aquifolium.

Numerous components are present in the extracts of these plants but certainly the characteristic marker is the component berberine.

Berberine is an isoquinoline alkaloid compound and is the most widely distributed bioactive component in plants belonging to the genus Berberis, as well as being present in other plant species in the genera Coptis Hydrastis Mahonia Tinospora _r and Xanthorhiza.

Specifically, in the genus Berberis, the distribution of berberine and other alkaloid compounds associated with it may occur predominantly in the rhizome, bark, or petioles of the plant at different concentrations and depending on the species considered.

Berberine is found commercially as a yellow, odorless powder with a bitter aftertaste that is characteristic of alkaloid compounds. For clinical uses it is often used in salt form associated with chloride and sulfate ions and is relatively soluble in water.

In the past, the remarkable pharmacological properties of berberine have been used to treat various conditions such as diarrhea, hypertension, diabetes mellitus, and carcinogenesis, using this alkaloid compound mainly in Chinese medicine. In recent decades, however, interest in its therapeutic effects has grown significantly, especially in Europe, as scientific studies have shown that berberine can also be used to treat cardiovascular diseases.

In this regard, the cardioprotective action of berberine in patients mainly depends on four different activities such as reduction of blood cholesterol levels, induction of the vasodilation process, anti-platelet aggregation activity and anti-inflammatory activity.

Studies conducted in vitro have shown that the mechanism of berberine underlying the regulation of blood cholesterol levels appears to be different from that of statins. Specifically, berberine has been observed to increase the cellular concentration of messenger RNA of receptors that interact with low-density lipoprotein (LDL).

The over-expression of these receptors occurs at the level of liver cell membranes and enables the removal of more than 70% of circulating LDL by binding these molecules and internalizing them into the cell cytoplasm through processes of endocytosis. It has also been shown that, berberine is able to inhibit the expression of the protein proprotein convertase subtilisin/kexin type 9 (PCSK9).

The latter is responsible for the repression of LDL-binding receptors. Thus, inhibition of PCSK9 by berberine ensures the expression of receptor proteins and consequently a reduction of LDL in the blood.

Such experimental evidence was later also confirmed by clinical trials in which berberine administration reduced total cholesterol and LDL values in patients.

In addition to regulating blood cholesterol levels, berberine is also involved in the process of vasodilation. In this regard, impaired vasodilation of the cardiovascular epithelium is closely related to the phenomenon of epithelial dysfunction, resulting in an increased risk of developing diseases affecting the heart and blood vessels. In fact, epithelial dysfunction in patients with atherosclerosis is one of the main causes of atheromatous plaque formation, thus leading to coronary artery disease.

Scientific studies have shown that berberine is able to induce the expression of the enzyme endothelial nitric oxide synthase (eNOS), thus leading to the production of nitric oxide molecules involved in vasodilation processes in the cardiovascular epithelium. In this case, berberine regulates the expression of AMP-dependent kinase (AMPK), which is able to activate the eNOS enzyme by phosphorylation.

In addition to this, berberine is also able to inhibit the expression of monocyte chemotactic protein (MCP-1) and vascular cell adhesion molecule 1 (VCAM-1) to preserve the functions of the cardiovascular epithelium from monocyte adhesion.

Berberine also exerts its cardioprotective function through anti-platelet aggregation activity. The mechanism behind this probably involves regulation of arachidonic acid metabolism and specifically involves a reduction in blood levels of thromboxane A2, known to be a potent inducer in the process of coagulation and vasoconstriction.

These beneficial effects derived from the use of berberine are then also associated with its anti-inflammatory activity. Indeed, the inflammatory process is a key factor in the development of cardiovascular disease. In this regard, berberine exerts its anti-inflammatory function by reducing the levels of pro-inflammatory cytokines such as IL-6, IL-lp and tumor necrosis factor (TNF-a) and by inhibiting the expression of the enzyme inducible nitric oxide synthase (iNOS) through activation of the enzyme AMPK in macrophages.

All this experimental evidence underscores the multiple cardioprotective effects of berberine making plant extracts belonging to the genus Berberis excellent candidates for the treatment of major cardiovascular risk factors.

Astaxanthin is a carotenoid classified as xanthophyll (red pigment) found in nature mainly in the aquatic environment. In particular, this pigment is produced by certain types of algae and plankton that are the main source of food of animal species such as shrimps, prawns, salmon, trout and lobsters, to which it imparts pink-red color. Astaxanthin is derived mainly from the microalga Haematococcus pluvialis, which produces the carotenoid in an autonomous form when environmental conditions induce oxidative stress. The molecular structure of astaxanthin has an extended form involving a conjugated polyene chain (apolar) at the ends of which is a ring equipped with hydroxyl and ketone groups (polar). This amphipathic structure allows astaxanthin to position itself in the phospholipid bilayer of cell membranes and gives it better biological activity than other antioxidants. Indeed, the polyene chain traps radicals, and the terminal ring can release them both inside and outside the cell membrane.

Human clinical studies have used astaxanthin administered orally in a dose of 4 mg to 100 mg/day without occurrence of adverse events. Specifically, astaxanthin has been reported to inhibit low-density lipoprotein (LDL) oxidation and increase HDL lipoprotein and adiponectin levels. A study in 24 healthy men shows that astaxanthin consumption, already at a dose of 3.6 mg/day, significantly prolongs LDL oxidation resistance time (LAG time). Another study, conducted in subjects with average hypertriglyceridemia, showed that astaxanthin administered at 12 and 18 mg/day significantly reduced triglyceride levels and increased adiponectin levels, and, administered at 6 and 12 mg/day, produced an increase in HDL lipoprotein. Administering astaxanthin has also been shown to reduce biomarkers of oxidative stress and inflammation. Inhibiting intracellular free radical production in fact is also a way to suppress pro-inflammatory signals. In several studies, moreover, astaxanthin has been shown to suppress the activity and reduce the expression of several mediators of inflammation such as TNF-a, IL-lp, IL-6, inducible nitric oxide synthase (iNOS) and COX-2 in THP-1 macrophages. Inhibition of pro-inflammatory cytokine secretion from macrophages could be one of the mechanisms underlying the beneficial effects of antioxidants on atherosclerosis. Therefore, experimental evidence suggests that astaxanthin exerts a preventive action against atherosclerotic cardiovascular disorder.

To the genus Crataegus belong several species known to be used for health purposes among which the most studied are: Crataegus azarolus, Crataegus laevigata, Crataegus oxyacantha, Crataegus monogyna , Crataegus nigra, Crataegus pentagyna, Crataegus rhipidophylla, and Crataegus curvisepala.

Hawthorn (common name of species belonging to the genus Crataegus) is a perennial plant that is widely distributed in Europe, Asia and North America, growing mainly in temperate climate regions.

The plant has a thorny shrub with petiolate leaves, white flowers grouped in highly fragrant corymbs and small red fruits.

Crataegus oxyacantha and C. monogyna are the most representative species of hawthorn and are known for their many beneficial effects on human health.

In fact, hawthorn has often been used first in Chinese medicine and later in world medicine for the treatment of diarrhea, insomnia, asthma, and digestive problems.

The beneficial action of hawthorn derives mainly from its phenolic component, triterpene compounds, and flavonoids including, in particular, vitexin. All of these molecules are distributed in the plant at different concentrations mainly in the leaves, flowers and fruits, from which dry or hydroalcoholic extracts can be produced. In recent years it has also been observed that hawthorn extracts are useful for treatment of cardiovascular diseases, especially in cases of hypertension and hyperlipidemia.

Clinical studies conducted on patients with moderate hypertension show that taking hawthorn extracts can regulate blood pressure. In fact, there is a reduction in diastolic blood pressure and a potential reduction in systolic blood pressure as well, thus decreasing the risk of occurrence of further cardiovascular complications.

In addition to reducing blood pressure in hypertensive subjects, hawthorn extracts have also been used to regulate blood cholesterol levels in cases of hyperlipidemia.

A clinical study of 49 patients with diabetes and chronic coronary artery disease showed that administration of 400 mg three times a day of hawthorn extracts, in conjunction with taking statins, results in a reduction in total blood cholesterol concentration, and in particular a reduction in low-density lipoprotein (LDL) levels.

Subsequently, hawthorn has also been shown to offer good protection of the endothelium by inhibiting the activity of the enzyme elastase, which is present in neutrophils, and thus ensuring a proper degree of elasticity to the endothelium itself.

This enzyme is in fact involved in the atherosclerotic process, as it degrades molecules of elastin and collagen fibers, compromising the physiological structure of the endothelium.

The cardioprotective action of hawthorn extracts in preserving endothelial morphology and function was then observed through the analysis of its anti-inflammatory activity.

In fact, a clinical study conducted on 88 patients with dyslipidemia showed that, the intake of hawthorn extracts results in the attenuation of the inflammatory process at the endothelial level, resulting in the regulation of tumor necrosis factor-alpha (TNF-a) levels.

In addition, hawthorn extracts also regulate the expression of proteins involved in leukocyte cell adhesion to the endothelium. In particular, a reduction in cellular levels of e-selectin, vascular cell adhesion protein (VCAM-1) and intracellular adhesion molecule (ICAM) was found.

Finally, it was also observed in this study that hawthorn extract represents a natural remedy that can act synergistically with hypolipidemic drugs used in conventional therapies to decrease plasma cholesterol levels.

Such experimental evidence underscores the cardioprotective action of hawthorn due to its hypotensive, hypolipidemic and anti-inflammatory activity, making it an ideal candidate for the treatment and/or prevention of cardiovascular disease.

The nutraceutical or pharmaceutical composition according to the invention is as defined in the appended claim 1.

Additional features and advantages of the invention are defined in the dependent claims. The claims form an integral part of this description.

A detailed description of some preferred embodiments of the invention is provided below.

As set forth, the nutraceutical or pharmaceutical composition of the present invention comprises an extract of a plant belonging to the genus Berberis, astaxanthin and an extract of a plant belonging to the genus Crataegus for the prevention and treatment of cardiovascular disease.

The words "a plant extract belonging to the genus Berberis" in the context of this description mean that the composition may include an extract chosen from Berberis aristata, Berberis aquifolium, Berberis vulgaris.

The words "an extract of a plant belonging to the genus Crataegus" in the context of this description means that the composition may include at least one extract chosen from Crataegus azarolus, Crataegus laevigata, Crataegus oxyacantha, Crataegus monogyna , Crataegus nigra, Crataegus pentagyna , Crataegus rhipidophylla, Crataegus curvisepala.

The present invention constitutes a prompt and valuable intervention useful for the prevention and/or treatment of cardiovascular disease.

This effect is ascribed to the combined action of its constituent substances. The extract of a plant belonging to the genus Berberis stimulates the production of LDL receptors by improving the lipid profile and also acts simultaneously as a hypoglycemic and anti-inflammatory agent. Astaxanthin due to its unique chemical structure has potent antioxidant activity on LDL and increases HDL cholesterol. The plant extract belonging to the genus Crataegus exerts hypotensive and antiinflammatory effects as well as assisting in the reduction of blood cholesterol levels. The synergistic activity of the above active ingredients can be studied by using in vitro and/or in vivo tests capable of evaluating the activity of the compositions according to the present invention.

In vitro tests on murine or human-derived cells may be useful for evaluating the anti-inflammatory action of the formulation under investigation. Cells are grown in complete DMEM growth medium and kept, for example, at 37°C and in a humid atmosphere in 5% CO ₂ . Following treatment with the composition of the invention, total RNA extracted from the samples is subjected to specific treatments such as RT-PCR to assess expression levels of pro-inflammatory proteins such as IL 1β, TNF-a and iNOS.

An in vitro assay to assess levels of oxidative stress involves treating cells with specific agents such as 2,7- dichloro-dihydrofluorescein acetate. The generated fluorescence can be measured by fluorescence microscopy 5 at 529 nm .

Other methods conventionally known to the branch expert can be used to evaluate the antioxidant activity of the formulation of the present invention. For example, it is possible to study the expression of SOD1 and NOX4 under conditions of LPS- induced stimulation or hyperglycemia before and after treatment with the substances of the present invention. Antioxidant and anti-inflammatory action, relevant to protect the endothelium from damage associated with atheroma formation, can be determined by testing human umbilical vein endothelial cells (HUVEC). Specifically, antioxidant activity can be assessed by inducing oxidative stress in HUVEC cells cultured in the presence of the compounds under investigation by addition of hydrogen peroxide (H2O2). Similarly, antiinflammatory activity can be determined by pre-incubating HUVEC cells with the substances of interest, and subsequently adding tumor necrosis factor-a (TNF-a) in order to induce the release of vascular cell adhesion molecules (VCAM) and E- selectin, the presence of which can be detected by ELISA method.

An additional experimental model useful for the evaluation of the substances under consideration is based on human hepatocarcinoma (HepG2) cells, as it allows to analyze possible changes in the expression of the receptor for low- density lipoprotein (LDL-R). Indeed, induction of LDL-R increases hepatic LDL uptake and, consequently, its plasma reduction. In addition, the same cell line can be used to test for changes in the expression levels of genes involved in the biosynthesis of triglycerides, cholesterol, and very low- density lipoprotein (VLDL), such as DGAT2, APOB100, SREBP1/2, and HMGCR. These determinations can be performed by conventional techniques such as RT-PCR and Western-blotting.

In order to test the ability of the so-stances of interest to inhibit the process of differentiation of monocytes into macrophages, a preliminary condition for the onset of atherosclerosis, an in vitro assay on monocytic cell line U937 can be employed. In this case, cells can be treated with macrophage colony-stimulating factor (M-CSF) and/or oxidized LDL in order to induce differentiation. Simultaneously, any substances or associations to be evaluated are added to test their effect on cellular levels of mRNA and proteins involved in the differentiation process. The inhibition of monocyte differentiation into macrophages indicates the protective activity of the substances on the maintenance of physiological endothelial morphology.

To assess cholesterol uptake, an in vitro assay can be conducted on a murine or human cell line. Treated and untreated cells are incubated with labeled cholesterol and subsequently washed to remove the excess. At the end of the assay, the radioactivity of the solubilized cells is measured. Using the latter test, it could be appreciated that the composition of the present invention can result in a reduction of cholesterol uptake in the range of 1 to 80 % compared with individual ingredients. The efficacy of the present invention can also be evaluated by an in vivo test on experimental animals in line with European Community and Ministry of Health guidelines. The test is conducted on Apoe mice that, after a period of acclimation, are divided into groups and subjected to the treatment. At time zero and at set intervals until the end of the treatment period, plasma samples are taken and total cholesterol, LDL cholesterol and HDL cholesterol levels are measured.

At the end of the treatment period, ex vivo tests may be performed. Therefore, mice are sacrificed and liver, intestinal and arterial tissue samples are taken. The harvested and lysed tissues are subjected to western blot to assess the levels of certain proteins of interest in particular, on the liver tissue the levels of HMG-5 CoA reductase and squalene monoxidase can be assessed, on the arterial tissue the levels of pro-inflammatory proteins such as IL-1β, TNF-a, TLR2, TLR4 and MAP kinase can be assessed, and on the intestinal tissue the total function of the tissue of interest can be assessed by assessing the levels of splicing proteins such as occludin or JAM-1.

Any of the in vitro or in vivo assays described herein can be used to evaluate the synergistic activity of the compounding referred to in the present invention.

As indicated above, the nutraceutical or pharmaceutical composition of the present invention is included within a pharmaceutical product or a dietary supplement, which is formulated in a suitable dosage form, the composition and preparation of which is within the capabilities of the person skilled in the art.

In a preferred embodiment form, the extract of a plant belonging to the genus Berberis in the nutraceutical or pharmaceutical composition of the invention is present in an amount between 1 mg and 7000 mg, preferably between 10 mg and 5000 mg, even more preferably between 20 mg and 3500 mg per single dosage unit.

Astaxanthin in the nutraceutical or pharmaceutical composition of the invention is present in an amount between 0.01 mg and 1500 mg, preferably between 0.05 mg and 800 mg, even more preferably between 0.1 mg and 500 mg per single posological unit.

The extract of a plant belonging to the genus Crataegus in the nutraceutical or pharmaceutical composition of the invention is present in an amount between 1 mg and 5000 mg, preferably between 5 mg and 3000 mg, even more preferably between 10 mg and 1500 mg per single posological unit.

All the above preferred forms of embodiment are combinable with each other.

The pharmaceutical product or dietary supplement, which comprises the pharmaceutical or nutraceutical composition of the invention, is formulated in a preferably oral pharmaceutical form, which may be solid, semisolid or liquid. Examples include a powder, an orosoluble powder, a granulate, a hard capsule, a soft-gel capsule, a tablet, a sachet, a solution, a syrup, a suspension or an emulsion.

Non-limiting examples of nutraceutical or pharmaceutical compositions that are the subject of the present invention are provided below. As indicated above, such nutraceutical or pharmaceutical compositions are formulated as pharmaceuticals or dietary supplements and administered in a suitable oral dosage form.

The following examples are provided for illustrative purposes only and not limiting the scope of the invention as defined by the appended claims.

EXAMPLES

EXAMPLE 1

Tablet

EXAMPLE 2

Tablet

EXAMPLE 3

Capsule

EXAMPLE 4

Capsule