Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
  • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
  • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
  • A61K31/05—Phenols
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
  • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
  • A61K36/18—Magnoliophyta (angiosperms)
  • A61K36/185—Magnoliopsida (dicotyledons)
  • A61K36/55—Linaceae (Flax family), e.g. Linum
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
  • A61K36/18—Magnoliophyta (angiosperms)
  • A61K36/185—Magnoliopsida (dicotyledons)
  • A61K36/63—Oleaceae (Olive family), e.g. jasmine, lilac or ash tree
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
  • A61K36/18—Magnoliophyta (angiosperms)
  • A61K36/185—Magnoliopsida (dicotyledons)
  • A61K36/70—Polygonaceae (Buckwheat family), e.g. spineflower or dock
  • A61K36/704—Polygonum, e.g. knotweed
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
  • A61K36/18—Magnoliophyta (angiosperms)
  • A61K36/185—Magnoliopsida (dicotyledons)
  • A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
  • A61K36/736—Prunus, e.g. plum, cherry, peach, apricot or almond
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
  • A61K36/18—Magnoliophyta (angiosperms)
  • A61K36/185—Magnoliopsida (dicotyledons)
  • A61K36/75—Rutaceae (Rue family)
  • A61K36/752—Citrus, e.g. lime, orange or lemon
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

• the present invention relates to a polyphenol composition comprising hydroxytyrosol for increasing the bioavailability of hydroxytyrosol and its therapeutic activity in the prevention and treatment of cardiovascular diseases, more specifically, atherosclerosis, cerebrovascular accident, peripheral vascular disease, and coronary disease.
• Cardiovascular diseases are still the main cause of death worldwide and are responsible for about 17.8 million deaths in 2017, which represents a 12.5% increase in mortality over the last decade.
• Atherosclerotic vascular disease (ASVD) or atherosclerosis is one of the most important causes of CVD. It is a chronic vascular inflammatory disease associated with oxidative stress and with endothelial dysfunction, in which it has been suggested that oxidized low-density lipoproteins (oxLDL) play an important role. oxLDLs bind with higher affinity than native LDLs do to several receptors, so their absorption by macrophages is more rapid, which leads to the buildup of cholesterol and to the formation of atherosclerotic plaque.
• ASVD oxidized low-density lipoproteins
• oxLDLs promote endothelial cell activation and dysfunction, vascular smooth muscle cell migration and proliferation, and platelet activation, causing an inflammatory response, endothelial dysfunction; aggravating the atherogenic process.
• concentration of circulating oxLDL is considered one of the most important biomarkers of the progression of atherosclerosis.
• the treatment of atherosclerosis consists of changing the lifestyle to a healthier one. However, if this is not enough, it is usually combined with the administration of drugs which in most cases are targeted at reducing blood pressure and/or cholesterol. In advanced cases, surgical intervention may be necessary to dilate the narrowed artery/arteries. Therefore, there is a need in the state of the art for treatments that allow increasing the benefits of a healthy lifestyle over the development of atherosclerosis.
• the present invention is based in the finding that combinations of hydroxytyrosol (HT) present in olive oil with polyphenols not present naturally in the olive tree ( Olea europaea ) or in olive oil, such as those present in an almond skin extract, in a grapefruit, flax, or polygonum extract, allow increasing the bioavailability of HT after their oral administration in comparison with the administration of HT in the absence of polyphenols.
• the inventors have observed how the administration of a composition comprising HT and almond skin polyphenols (ASP) to subjects with moderate hypercholesterolemia (treated group) reduces serum levels of oxidized low-density lipoproteins (oxLDL), as well as the oxLDL/LDL ratio.
• oxLDL oxidized low-density lipoproteins
• serum levels of oxLDL increase after the treatment, as does the oxLDL/LDL ratio.
• a first aspect of the invention relates to the use of at least one polyphenol that is not hydroxytyrosol and/or that does not occur naturally in the tree Olea europaea ((). europaea ) for increasing the bioavailability of hydroxytyrosol after the oral administration of hydroxytyrosol or for increasing the antiatherosclerotic effect of hydroxytyrosol after its oral administration.
• the invention relates to a polyphenol that is not hydroxytyrosol and/or that does not occur naturally in the tree O. europaea for use in increasing the bioavailability of hydroxytyrosol after the oral administration of hydroxytyrosol or in increasing the antiatherosclerotic activity of hydroxytyrosol after its oral administration.
• the invention relates to hydroxytyrosol for use in the treatment of atherosclerosis, wherein hydroxytyrosol is administered orally in combination with a polyphenol that is not hydroxytyrosol and/or that does not occur naturally in the tree O. europaea.
• the invention in a fourth aspect, relates to a composition or kit of parts comprising hydroxytyrosol and at least one polyphenol, wherein the polyphenol that is not hydroxytyrosol and/or wherein the at least one polyphenol does not occur naturally in the tree O. europaea.
• the invention in a fifth aspect, relates to a method for obtaining the composition of the fourth aspect of the invention, which method comprises contacting a composition comprising hydroxytyrosol with a composition comprising at least one polyphenol that is not hydroxytyrosol and/or that does not occur naturally in the tree (). europaea.
• the invention in a sixth aspect, relates to a pharmaceutical composition
• a pharmaceutical composition comprising the composition of the fourth aspect of the invention, or a composition obtained by the method of the fifth aspect of the invention, and a pharmaceutically acceptable excipient.
• the invention relates to a food or nutritional supplement comprising the composition of the fourth aspect of the invention, or a composition obtained by the method of the fifth aspect of the invention, and a nutritionally acceptable excipient.
• the invention relates to the composition or kit of parts of the fourth aspect of the invention, the composition obtained by the method of the fifth aspect of the invention, the pharmaceutical composition of the sixth aspect of the invention, or the food or nutritional supplement of the seventh aspect of the invention, for use in medicine.
• the invention relates to the composition or kit of parts of the fourth aspect of the invention, the composition obtained by the method of the fifth aspect of the invention, the pharmaceutical composition of the sixth aspect of the invention, or the food or nutritional supplement of the seventh aspect of the invention, for use in the treatment of a cardiovascular disease.
• Figure 2 Ratio of oxLDL-cholesterol/LDL-cholesterol of the subjects who took the placebo or the extract at the start, after 4 weeks, and after 8 weeks of treatment. The mean values are represented with dots (dark gray for the control group and light gray for the extract group), and the standard error is represented by means of vertical bars. The different letters indicate significant differences between the times of each group. The asterisk (*) indicates p ⁇ 0.05 between groups at the corresponding time.
• Figure 3 A- Chromatograms at 280 nm resolving olive tree extract polyphenols (bottom chromatogram) and almond extract polyphenols (top chromatogram). B- Chromatograms at 330 nm resolving olive tree extract polyphenols (top chromatogram) and almond extract polyphenols (bottom chromatogram).
• FIG. 1 A- Chromatograms at 280 nm resolving olive tree extract polyphenols (top chromatogram) and flax extract polyphenols (bottom chromatogram). B- Chromatograms at 330 nm resolving olive tree extract polyphenols (top chromatogram) and flax extract polyphenols (bottom chromatogram).
• the inventors have observed that several groups of polyphenols other than HT and not found naturally in the tree O. europaea increase the bioavailability of HT after their oral administration. Furthermore, the administration of a composition comprising HT and almond skin polyphenols to subjects with moderate hypercholesterolemia reduces serum levels of oxLDL.
• the invention relates to the use of at least one polyphenol that is not hydroxytyrosol and/or that does not occur naturally in the tree Olea europaea ( O . europaea ) for increasing the bioavailability of hydroxytyrosol after the oral administration of hydroxytyrosol or for increasing the antiatherosclerotic effect of hydroxytyrosol after its oral administration.
• the first aspect of the invention relates to a method for increasing the bioavailability of hydroxytyrosol after its oral administration or for increasing the antiatherosclerotic effect of hydroxytyrosol after its oral administration comprising the administration of at least one polyphenol that is not hydroxytyrosol and/or that does not occur naturally in the tree Olea europaea (O. europaea).
• the invention relates to a polyphenol that is not hydroxytyrosol and/or that does not occur naturally in the tree O. europaea for use in increasing the bioavailability of hydroxytyrosol after the oral administration of hydroxytyrosol or in increasing the antiatherosclerotic activity of hydroxytyrosol after its oral administration.
• the invention in a third aspect, relates to hydroxytyrosol for use in the treatment of atherosclerosis, wherein hydroxytyrosol is administered orally in combination with a polyphenol that is not hydroxytyrosol and/or that does not occur naturally in the tree O. europaea.
• the third aspect of the invention is defined as a method for the treatment of atherosclerosis in a patient comprising the oral administration to said patient of hydroxytyrosol and wherein hydroxytyrosol is administered orally in combination with a polyphenol that is not hydroxytyrosol and/or that does not occur naturally in the tree O. europaea.
• hydroxytyrosol refers to the polyphenolic compound commonly known to one skilled in the art with said name. In nature, hydroxytyrosol is present in the olive tree (e.g. in the species Olea europaea ) concentrated primarily in the leaves and in the fruit. In a particular embodiment, it refers to the chemical compound with IUPAC name 4-(2-hydroxyethyl)benzene-l,2-diol.
• polyphenol that is not hydroxytyrosol and/or that does not occur naturally in the tree Olea europaea or “polyphenol that is not hydroxytyrosol and/or wherein the at least one polyphenol other than hydroxytyrosol does not occur naturally in the tree O.
• europaea refers to a polyphenol, as defined above, other than hydroxytyrosol (i.e., the characteristics of which do not coincide with those of the product described in the definition of hydroxytyrosol above). Alternatively or additionally, it refers to a polyphenol which is not synthesized by the tree of the species O.
• the polyphenol not occurring naturally in the tree O. europaea is that which is not synthesized by the tree of the species O. europaea as was just described.
• the tree of the species O. europaea is the common olive tree wild variety, i.e., the variety Olea europaea sylvestris.
• europaea is from a subspecies selected from the group consisting of: O. europaea europaea, O. europaea cerasiformis, O. europaea cuspidata, O. europaea guanchica, O. europaea laperrinei, and O. europaea maroccana.
• the tree of the species O. europaea is any variety of said subspecies.
• the tree is any organism of one of the taxonomic ranks of O. europaea mentioned.
• the species of O. europaea mentioned in any aspect of this invention refers to the species O. europaea L.
• polyphenol that is not hydroxytyrosol and that optionally does not occur naturally either in the tree Olea europaea is referred to in any aspect of the present invention with the expression “polyphenol that is not hydroxytyrosol”, “polyphenol other than hydroxytyrosol”, or “polyphenol differing from hydroxytyrosol”.
• Methods for determining if a polyphenol occurs naturally in a tree of the species O. europaea , or is synthesized by said tree naturally, as defined above, include obtaining plant extracts from at least one plant product of a tree as described in said definition, and determining the presence of polyphenols in said extract. The absence of said polyphenol in the extracts is indicative that it is not found in said tree nor is it synthesized by said tree.
• Methods for obtaining plant extracts from a plant product are specified below.
• Methods for determining the polyphenols present in a plant extract are specified below.
• the definitions of plant extract and of plant product are also provided below.
• europaea does not occur naturally in the fruits or in the leaves of the tree O. europaea, preferably it does not occur naturally in the leaves of the tree O. europaea.
• methods for determining if a polyphenol occurs naturally in the fruits and/or in the leaves of a tree O. europaea include the previously indicated methods, in which the plant extracts were obtained from the corresponding plant product, i.e., fruits and/or leaves of the tree O. europaea.
• polyphenol refers to the group of chemical substances commonly known to one skilled in the art by said name. It specifically refers to the chemical substances found in plants characterized by the presence of one or several phenol rings and conjugated double bonds. They are found mainly in conjugated forms, associated with carbohydrates bound to -OH groups, although there are also direct bonds of the carbohydrate with the phenol group. It is also common for them to be associated with other compounds, such as carboxylic and organic acids, amines, lipids, and even with other phenol groups.
• Polyphenols suitable for use in the present invention include, without limitation, phenolic acids (hydroxybenzoic acid or hydroxycinnamic acid derivatives), stilbenes, lignans, phenolic alcohols, and flavonoids, the latter constituting the most abundant subclass. Said groups of polyphenols are also used in the present invention for referring to the groups of polyphenols commonly known to one skilled in the art by the corresponding name.
• flavonoids refers to the most abundant subclass of polyphenols in the plant kingdom.
• the chemical structure of flavonoids consists of a diphenylpropane backbone (C6-C3-C6) formed by two aromatic rings bound through three carbon atoms forming an oxygenated heterocycle (C).
• C oxygenated heterocycle
• Flavonoids suitable for use according to the present invention include flavonols, flavones, flavanones, isoflavones, anthocyanidins and flavanols (catechins and proanthocyanidins).
• the most representative flavonols in foods are flavan-3-ols, and these can occur as anthocyanidin monomers (catechin, epicatechin, gallocatechin, and epigallocatechin), as dimers condensed to one another, or oligomers (procyanidins), or they can occur as polymers (proanthocyanidins or condensed tannins).
• Said compounds and groups of compounds are those commonly known to one skilled in the art by the corresponding name.
• Non-limiting examples of flavonoids include: procyanidins, propelargonidins, prodelphinidins, catechin, epicatechin, quercetin, kaempferol, isorhamnetin, rutin, and geni stein.
• lignan refers to phenolic compound dimers, phenylalanine derivatives, and cinnamic alcohols the basic structure of which consists of two C6C3 (n-propylbenzene) units attached by b-b’ bonds.
• lignans suitable for use in the present invention such as simple lignans, complex lignans, or cyclolignans, flavolignans, coumarinolignans, stilbene lignans, or xanthone lignans.
• Said groups of compounds are those commonly known to one skilled in the art by the corresponding name.
• Non-limiting examples of lignans include: secoisolariciresinol diglucoside, secoisolariciresinol, matairesinoside, lariciresinol, pinoresinol, sesamin, schisandrin, deoxyschisandrin, gomisins, pregomisin, sesamolin, sesamol, sesaminol, sesamolinol, or pinoresinol.
• stilbene refers to polyphenolic compounds of formula C14H12, for which there are two isomers: Irans- 1 ,2-diphenylethylene (E-stilbene) and 67 s- 1 ,2-diphenylethylene (Z-stilbene).
• the term stilbene includes hydroxy and alkoxy derivatives of simple stilbene, as well as its heterosides (glycosides) forms and polymers. In a particular embodiment, it refers to the chemical compound with IUPAC name: trans-1,2- diphenylethylene.
• Non-limiting examples of stilbenes include resveratrol, piceatannol, pinosylvin, pterostilbene, rhapontienin, pterostilbene, isorhapontienin, rhapontin, ponticin, piceid, or astringin.
• phenolic acid or “phenolcarboxylic acid” refers to phenolic compounds containing a phenol ring and an organic function of carboxylic acid (C6-C1 backbone).
• phenolic acids include 3,4-dihydroxybenzoic acid, parahydroxybenzoic acid, vanillic acid, caffeic acid, paracoumaric acid, ferulic acid, syringic acid, and sinapic acid.
• phenolic alcohol refers to phenolic compounds comprising the alcohol function and the phenol function.
• Non-limiting examples of phenolic alcohols include coniferyl alcohol, sinpyl alcohol, or p-coumaryl alcohol.
• the hydroxytyrosol for use according to the invention or the polyphenols used in the present invention are found as part of a plant extract.
• plant extract refers to the term commonly known by one skilled in the art. It refers to a composition comprising compounds, ingredients, or substances, which have been extracted from a product, or tissue of a plant organism, a tree, or a plant, by means of methods commonly known to one skilled in the art. Non-limiting examples of said methods include the expression in which the plant product is introduced in a hydraulic press and squeezed, the distillation or the incision in the plant product of interest followed by the extraction of a sample of the content or composition of said plant product. Another common method for obtaining plant extracts consists of treating the product or tissue from which the plant extract is to be obtained with solvents. Non-limiting examples of solvents include water, ethanol, hydroalcohol, ethyl acetate, CO2, methanol, acetone, acetic acid, or hexane.
• the plant extract is obtained from fresh or dry plant product or tissue of interest with a solvent at a temperature of about 40 to 100°C for 1-10 hours.
• the solvent is aqueous, preferably water.
• the solvent is a hydroalcohol preferably having an alcohol content between 10 and 96%, wherein the alcohol is preferably ethanol or methanol.
• the process can also include several extraction phases for extracting the plant product with the same or different solvent, with the same or different alcohol content, combining the extracts obtained to continue the process.
• the process also includes several concentration steps by means of chromatography using polymeric adsorbent resins. The obtained extract is then treated with activated carbon and then removed. Finally, and alternatively, a drying step for drying the purified extract is performed.
• the method for obtaining the plant extract comprises: (i) extracting the fresh or dry plant product or tissue of interest with a solvent at a temperature of about 40 to 100°C for 1-10 hours (ii) treating the extract obtained in step (i) with activated carbon and then removing the activated carbon.
• the method for obtaining the plant extract comprises:
• step (iii) treating the extract obtained in step (i) with activated carbon and then removing the activated carbon.
• step (i) of any of the preceding methods includes several extraction phases for extracting the plant product with the initial solvent or a different solvent, combining the extracts obtained to continue the process. If the solvent is a hydroalcohol, the alcohol content in these extraction phases is equal to or different from the initial solvent.
• any of the previously described methods include a drying step for drying the extract.
• the plant extract of one or several products of O. europaea described in any aspect of the present invention is obtained by means of the method for obtaining plant extracts with solvents described in any of the preceding embodiments, wherein the solvent is aqueous, preferably water.
• the plant extract of one or several products oiPrunus dulcis described in any aspect of the present invention is obtained by means of the method for obtaining plant extracts with solvents described in any of the preceding embodiments, wherein the solvent is aqueous, preferably water.
• the plant extract of one or several products of Citrus paradisi described in any aspect of the present invention is obtained by means of the method for obtaining plant extracts with solvents described in any of the preceding embodiments, wherein the solvent is a hydroalcohol.
• the plant extract of one or several products of Linum usitatissimum described in any aspect of the present invention is obtained by means of the method for obtaining plant extracts with solvents described in any of the preceding embodiments, wherein the solvent is a hydroalcohol.
• the plant extract of one or several products of Polygonum cuspidatum described in any aspect of the present invention is obtained by means of the method for obtaining plant extracts with solvents described in any of the preceding embodiments, wherein the solvent is a hydroalcohol.
• activate charcoal refers to the term well known to an expert in the field. It is a form of carbon that is processed to have augmented adsorption properties. It is mostly processed to have small pores, of low volume which increases the surface area available for adsorption or chemical reaction.
• the term “product of a plant organism” or “plant product” refers to any part of a plant organism, including a tissue of the plant organism, a tissue of the reproductive organs of the plant organism, a tissue of the non-reproductive organs of the plant organism, the leaves, the stem, the roots, the fruits, a tissue of the fruits, the exocarp of the fruit, the mesocarp of the fruit, the endocarp of the fruit, the skin of the fruit, the shell of the fruit, the pod of the fruit, the seed of the fruit, or the pod of the plant organism.
• the plant product refers to the total of any of the parts of a plant organism indicated just above. In another particular embodiment, it refers to a portion of any of the parts of the plant organism indicated just above.
• Methods for determining the hydroxytyrosol content in the plant extract or the polyphenol content in the plant extract are commonly known to one skilled in the art. Said methods include any method that allows determining the amount and type of a compound in a composition, such as mass spectrometry methods.
• mass spectrometry high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS), high-performance liquid chromatography (HPLC) coupled to an ultraviolet absorbance detector, gas chromatography coupled to mass spectrometry (GC-MS), liquid chromatography coupled to mass spectrometry (LC-MS), direct infusion mass spectrometry or Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), capillary electrophoresis coupled to mass spectrometry (CE-MS), ultra-high performance liquid chromatography coupled to mass spectrometry (UHPLC-MS), supercritical fluid chromatography coupled to mass spectrometry (SFC-MS), flow injection analysis with mass spectrometry (FIA-MS), including quadrupole mass spectrometry, any sequentially coupled mass spectrometry, such as MS-MS or MS-MS-MS, inductively coupled plasma mass spectrometry (ICPMS), pyrolysis-mass spectrometry (Py
• the determination of the levels of hydroxytyrosol in a sample is carried out by means of any of the aforementioned chromatographic techniques using a calibration line obtained by means of a hydroxytyrosol pattern of a known concentration.
• the determination of the polyphenol content in a plant extract is carried out using enzymatic methods and, more preferably, the method based on the reagent Folin-Ciocalteu (Singleton VL etal. , 1999, Methods in enzimology , vol. 299: 152-78).
• bioavailability refers to the fraction and the rate at which the administered dose of a compound reaches its therapeutic target (cell receptors, channels, carriers), which implies reaching the tissue on which it acts. It is considered equivalent to the levels reached in the systemic circulation of a subject who has ingested or who has been administered said compound. This is why, in practice, bioavailability is determined by the percentage of compound occurring in plasma after its administration.
• bioavailability of hydroxytyrosol refers to the bioavailability as defined above, wherein the compound is hydroxytyrosol.
• bioavailability of hydroxytyrosol after its oral administration refers to the percentage of hydroxytyrosol in systemic circulation, or in plasma, once hydroxytyrosol has been administered orally to a subject.
• bioavailability of hydroxytyrosol after its oral administration refers to the percentage of hydroxytyrosol in systemic circulation, or in plasma, once hydroxytyrosol has been administered orally to a subject.
• bioavailability of hydroxytyrosol after its oral administration refers to the percentage of hydroxytyrosol in systemic circulation, or in plasma, once hydroxytyrosol has been administered orally to a subject.
• methods which allow determining the bioavailability of hydroxytyrosol after its oral administration comprise determining the percentage of hydroxytyrosol in serum of a subject who has been administered hydroxytyrosol, or determining the stability of the hydroxytyrosol in the digestive tract of a subject who has been administered hydroxytyrosol orally.
• methods for determining the percentage of hydroxytyrosol in serum of a subject include any of the methods indicated above for determining the presence of polyphenols in a plant extract, applied to a serum sample from said subject.
• methods which allow determining the stability of hydroxytyrosol in the digestive tract include the method described in the section B on materials and methods and in Example 3 of the application.
• the step for detecting the content of hydroxytyrosol in each of the compositions of the method of the application may consist of any of the methods indicated above for detecting polyphenols in a plant extract (wherein the polyphenol is hydroxytyrosol, and the plant extract is substituted with the corresponding composition of the method of the application in which hydroxytyrosol has been incubated).
• the expression “increasing the bioavailability of hydroxytyrosol after its oral administration” refers to increasing by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200% the bioavailability of hydroxytyrosol in a subject with respect to a reference value.
• said reference value is the bioavailability of hydroxytyrosol after the oral administration of hydroxytyrosol without a polyphenol that is not hydroxytyrosol and that does not occur naturally in the tree O. europaea,, as said polyphenol is defined and described above and in the different embodiments of the present invention.
• said reference value is the bioavailability of hydroxytyrosol after the oral administration of a compound the active ingredient of which is only hydroxytyrosol.
• Said compound may consist of a composition consisting of hydroxytyrosol and at least one pharmaceutically acceptable excipient, wherein said excipient can be any of those indicated in the aspect of the invention relating to the pharmaceutical composition of the invention.
• Methods for determining the bioavailability of hydroxytyrosol such as those which have been described just above, allow determining if the bioavailability of hydroxytyrosol is higher or lower, and at what percentage, with respect to a reference value. As one skilled in the art will understand, said methods also allow measuring the bioavailability of hydroxytyrosol defined as a reference value and therefore determining the reference value indicated in the present definition.
• ASVD refers to the cardiovascular disease characterized by chronic inflammation, the accumulation of lipids, modifications of vascular cells in the arterial wall, and the formation of atherosclerotic plaque or atheroma. Said processes trigger a narrowing of the arterial lumen.
• Oxidized low-density lipoproteins (oxLDL) are considered to play a key role in the initiation and progression of atherogenesis. oxLDLs bind with a higher affinity than native LDL to several receptors, so their absorption by macrophages is more rapid, which leads to the buildup of cholesterol and the formation of atherosclerotic plaque, or atheroma.
• oxLDL promotes endothelial cell activation and dysfunction, vascular smooth muscle cell migration and proliferation, and platelet activation, causing an inflammatory response, endothelial dysfunction; aggravating the atherogenic process.
• concentration of circulating oxLDL is considered one of the most important biomarkers of the progression of atherosclerosis.
• the expression “antiatherosclerotic effect of hydroxytyrosol” refers to the activity of hydroxytyrosol once administered to a subject, which helps to prevent, cur, reverse, or treat atherosclerosis.
• Suitable indicators of the antiatherosclerotic effect of hydroxytyrosol include the reduction in the concentration of serum oxLDL, or the reduction of the serum oxLDL/LDL-cholesterol ratio, once administered to a subject, preferably orally.
• LDL low- density lipoproteins or LDLs.
• LDLs are one of the 5 main groups of lipoproteins which transport fat molecules in the body in the extracellular medium (ULDs, ultra-low-density lipoproteins; VLDL, very low-density lipoprotein; IDL intermediate-density lipoprotein; HDL, high-density lipoprotein; and LDL as it is here defined). Most of the cholesterol is transported in the blood of a subject along with proteins, forming LDLs.
• ox-LDL refers to LDL lipoproteins as defined above, which are oxidized.
• Oxidized LDL is a general term for LDL lipoproteins with structural components modified by oxidation.
• both the lipid parts and the protein parts of LDL can be oxidized.
• lipids oxidized into LDL can also derive from dietary oxidized lipids.
• ox-LDL is associated with the development of atherosclerosis.
• ox-LDL/LDL-cholesterol or “ox-LDL/LDL” refers to the ratio between the concentration in a specific ox-LDL medium as defined above and the concentration of LDL as defined above in said medium.
• the expression “increasing the antiatherosclerotic effect of hydroxytyrosol” refers to increasing by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 275%, at least 300%, at least 350%, at least 400% any of the effects indicated in the particular embodiments above in the definition of atherosclerotic effect with respect to a reference value.
• the reference value is the value of the parameter changed by the administration of hydroxytyrosol indicated in the corresponding preceding embodiment in the definition of atherosclerotic effect, upon administration of hydroxytyrosol without a polyphenol that is not hydroxy tyro sol and that does not occur naturally in the tree O. europaea, as said polyphenol is defined and described above and in the different embodiments of the present invention.
• the hydroxytyrosol administered for determining the reference value consists of a composition the active compound of which is only hydroxytyrosol, specifically comprising only hydroxytyrosol and optionally a pharmaceutically acceptable excipient, as defined in the aspect of the invention relating to the pharmaceutical composition of the invention.
• the expression “increasing the antiatherosclerotic effect of hydroxytyrosol” refers to decreasing by at least 10%, at least 20%, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 275%, at least 300%, at least 350%, at least 400%, preferably by at least 20% the serum oxLDL concentration with respect to a reference value.
• said decrease with respect to the reference value is of at least 50%. In another particular embodiment, it is of at least 70%.
• said reference value is the serum oxLDL concentration after the oral administration of hydroxytyrosol without a polyphenol that is not hydroxytyrosol and that does not occur naturally in the tree O. europaea, as said polyphenol is defined and described above and in the different embodiments of the present invention.
• the hydroxytyrosol administered for determining the reference value consists of a composition the active compound of which is only hydroxytyrosol, specifically comprising only hydroxytyrosol and optionally a pharmaceutically acceptable excipient, as defined in the aspect of the invention relating to the pharmaceutical composition of the invention.
• Methods for determining said decrease include the methods described in section A- on materials and methods (particularly in the “Analysis of biochemical parameters” section) of the application for determining the serum oxLDL concentration and comparing the value obtained with the reference value.
• the expression “increasing the antiatherosclerotic effect of hydroxytyrosol” refers to decreasing by at least 10%, at least 20%, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 275%, at least 300%, at least 350%, at least 400%, preferably by at least 25% the serum oxLDL/LDL- cholesterol ratio with respect to a reference value.
• said decrease with respect to the reference value is of at least 30%. In another particular embodiment, it is of at least 50%.
• said reference value is the serum oxLD/LDL- cholesterol ratio after the oral administration of hydroxytyrosol without a polyphenol that is not hydroxytyrosol and that does not occur naturally in the tree O. europaea, as said polyphenol is defined and described above and in the different embodiments of the present invention.
• the hydroxytyrosol administered for determining the reference value consists of a composition the active compound of which is only hydroxytyrosol, specifically comprising only hydroxytyrosol and optionally a pharmaceutically acceptable excipient, as defined in the aspect of the invention relating to the pharmaceutical composition of the invention.
• Methods for determining said decrease include the methods for determining the serum oxLDL concentration described in section A- on materials and methods (particularly in the “Analysis of biochemical parameters” section) of the application, and methods for determining the serum LDL concentration commonly known to one person skilled in the art.
• Non-limiting examples of said methods include the direct method (G.D.
• the at least one polyphenol that is not hydroxytyrosol and/or that does not occur naturally in Olea europaea is administered in combination with hydroxytyrosol, i.e., as part of one and the same composition.
• the at least one polyphenol that is not hydroxytyrosol as such and/or that does not occur naturally in Olea europaea is administered sequentially with respect to hydroxytyrosol.
• the at least one polyphenol that is not hydroxytyrosol as such and/or that does not occur naturally in Olea europaea is administered separately with respect to the hydroxytyrosol.
• the hydroxytyrosol is part of a plant extract.
• the expression “plant extract” has been defined above.
• the plant extract comprising hydroxytyrosol is obtained from any plant product of the tree O. europaea, wherein the plant product is any of those indicated in the definition of “plant product” indicated above.
• the plant extract of which the hydroxytyrosol is part is an extract of the leaf or of the fruit, preferably the leaf, of the tree O. europaea.
• the expression “is part of a plant extract” refers to a specific compound being comprised in a plant extract, wherein the term “comprised”, “comprises”, indicates that the plant extract must contain said compound, but it may optionally contain additional compounds or ingredients.
• the plant extract comprising hydroxytyrosol has been obtained from at least two plant products of the tree O. europaea , selected from those indicated in the definition of plant product, wherein each of said products is the same or different.
• each of said products is obtained from a different tree of O. europaea , wherein each tree is preferably of a different subspecies, more preferably wherein each tree is of a different variety.
• the tree of the species O. europaea mentioned in any definition or particular embodiment of any aspect of the present invention is any variety of the tree of the spice O. europaea.
• it is the common olive tree wild variety, i.e., the variety Olea Europaea sylvestris.
• it is a tree of a subspecies selected from the group consisting of: O. europaea europaea, O. europaea cerasiformis, O. europaea cuspidata, O. europaea guanchica, O. europaea laperrinei, and O. europaea maroccana.
• it is any variety of said subspecies.
• the trees of the species O. europaea mentioned in any definition or particular embodiment of any aspect of the present invention are each of any variety of the tree of the spice O. europaea.
• at least one is the common olive tree wild variety, i.e., the variety Olea Europaea sylvestris.
• at least one is a tree of a subspecies selected from the group consisting of: O. europaea europaea, O. europaea cerasiformis, O. europaea cuspidata, O. europaea guanchica, O. europaea laperrinei, and O. europaea maroccana.
• the at least one is of any variety of said subspecies.
• the plant extract comprising hydroxytyrosol contains 1-100% (w/w), 1-98% (w/w), 1-95% (w/w), 1-90% (w/w), 3-90% (w/w), 5-90% (w/w), 7-90% (w/w), 8-90% (w/w), 9-90% (w/w), 10-90% (w/w), 10-85% (w/w), 10-80%(w/w), 10-70% (w/w), 10-60% (w/w), 10-50% (w/w), 10-40% (w/w), 10-30% (w/w), 10-25% (w/w), 10-20% (w/w) of hydroxytyrosol.
• the plant extract comprising hydroxytyrosol contains 1% (w/w), 2% (w/w), 3% (w/w), 4% (w/w), 5% (w/w), 6% (w/w), 7% (w/w), 8% (w/w), 9% (w/w), 10% (w/w), 11% (w/w), 12% (w/w), 13% (w/w), 14% (w/w), 15% (w/w), 16% (w/w), 17% (w/w), 18% (w/w), 19% (w/w), 20% (w/w), 22% (w/w), 25% (w/w), 27% (w/w), 30% (w/w), 35% (w/w), 40% (w/w), 50% (w/w), 55% (w/w), 60% (w/w), 65% (w/w), 70% (w/w), 75% (w/w), 80% (w/w), 90% (w/w), 95% (w/w), 9
• the plant extract comprising hydroxytyrosol contains 10-90%, preferably 10% (w/w) of hydroxytyrosol.
• the at least one polyphenol is selected from the group of polyphenols consisting of flavonoids, lignans, and stilbenes.
• flavonoids As used in the present invention, the terms “flavonoids”, “lignans” and “stilbenes” refer to the polyphenols commonly known by said name to one skilled in the art. Specifically, they refer to polyphenols as defined above.
• the at least one polyphenol other than hydroxytyrosol of the group of flavonoids is selected from the group consisting of procyanidins, propelargonidins, prodelphinidins, catechin, epicatechin, quercetin, kaempferol, isorhamnetin, naringin, naringenin, genistein, and rutin.
• procyanidins As used in the present invention, the terms “procyanidins”, “propelargonidins”, “prodelphinidins”, “catechin”, “epicatechin”, “quercetin”, “kaempferol”, “isorhamnetin”, “naringin”, “naringenin”, “genistein”, and “rutin” refer to the corresponding term as understood by one skilled in the art.
• procyanidins refers to the group of proanthocyanidins constituted exclusively of (epi)catechin.
• propelargonidins refers to proanthocyanidins containing an (epi)afzelechin unit together with (epi)catechin units.
• prodelphinidins refers to proanthocyanidins containing at least one (epi)gallocatechin unit together with (epi)catechin units.
• proanthocyanidin or “condensed tannins” refers to flavan-3-ol oligomers and polymers widely distributed in the plant kingdom.
• the most abundant flavan-3-ol units are (+)-afzelechin, (+)-catechin, and (+)-gallocatechin (2R:3S forms) and their diastereoisomers (-)-epiafzelechin, (-)epi catechin, and (-)-epigallocatechin (2R:3R forms), respectively.
• type B procyanidins/propelargonidins/prodelphinidins the flavan-3-ol units are bound by C-4 carbon of the upper unit and C-6 or C-8 carbon of the lower unit, C4-C8 isomers being more abundant than C4-C6 isomers.
• type A procyanidins have an ether type bond between the C-2 carbon of the upper unit and the hydroxyl group of the C-7 carbon of the lower unit (Porter L.J. (1988) Flavans and proanthocyanidins. In The flavonoids (Harbome J.B., Ed.) Chapman and Hall, New York, pp. 21-62).
• the polyphenol other than hydroxytyrosol of the group of flavonoids referred to in any aspect of the present invention can be any polyphenol belonging to the group of proanthocyanidins.
• it is any polyphenol of the group of procyanidins, preferably it is a type B procyanidin.
• it is a procyanidin selected from the group consisting of type B1 procyanidin, type B2 procyanidin, type B3 procyanidin, type B4 procyanidin, type B5 procyanidin, type B6 procyanidin, type B7 procyanidin.
• it is a procyanidin of type Cl.
• it is a procyanidin of type A, preferably selected from the group consisting of procyanidin Al, procyanidin A2.
• it is any polyphenol of the group of propelargonidins, preferably type A propelargonidins.
• it is a type A propelargonidin, more preferably it is a type B propelargonidin.
• it is any polyphenol of the group of prodelphinidins; in a particular embodiment, preferably type A prodelphinidins.
• procyanidin Bl or “type B1 procyanidin” refers to the compound with IUPAC name: (2R,3S)-2-(3,4-dihydroxyphenyl)- 8-[(2R,3R,4R)-2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-3,4-dihydro-2H-chromen-4-yl]- 3,4-dihydro-2H-chromene-3,5,7-triol.
• procyanidin B2 or “type B2 procyanidin” refers to the compound with IUPAC name (2R,3R)-2-(3,4-dihydroxyphenyl)-8-[(2R,3R,4R)- 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-3,4-dihydro-2H-chromen-4-yl]-3,4-dihydro-2H- chromene-3,5,7-triol.
• procyanidin B3 or “type B3 procyanidin” refers to the compound with IUPAC name (2R,3S)-2-(3,4-dihydroxyphenyl)-8-[(2R,3S,4S)- 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-3,4-dihydro-2H-chromen-4-yl]-3,4-dihydro-2H- chromene-3,5,7-triol.
• procyanidin B4 or “type B3 procyanidin” refers to the compound with IUPAC name (2R,3R)-2-(3,4-dihydroxyphenyl)-8-[(2R,3S,4S)- 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-3,4-dihydro-2H-chromen-4-yl]-3,4-dihydro-2H- chromene-3,5,7-triol.
• procyanidin B5 or “type B5 procyanidin” refers to the compound with IUPAC name (2R,3R)-2-(3,4-dihydroxyphenyl)-6-[(2R,3R,4S)- 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-3,4-dihydro-2H-chromen-4-yl]-3,4-dihydro-2H- chromene-3,5,7-triol.
• procyanidin B6 or “type B6 procyanidin” refers to the compound with IUPAC name (2R,3S)-2-(3,4-dihydroxyphenyl)-6-[(2R,3S,4R)- 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-3,4-dihydro-2H-chromen-4-yl]-3,4-dihydro-2H- chromene-3,5,7-triol.
• procyanidin B7 refers to the compound with name (2R,3S)-2-(3,4-dihydroxyphenyl)-6-[(2R,3R,4S)-2-(3,4- dihydroxyphenyl)-3,5,7-trihydroxy-3,4-dihydro-2H-chromen-4-yl]-3,4-dihydro-2H- chromene-3,5,7-triol.
• procyanidin Cl or “type Cl procyanidin” refers to the compound with IUPAC name (2R,3R,4S)-2-(3,4-dihydroxyphenyl)-4-[(2R,3R)- 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-3,4-dihydro-2H-chromen-8-yl]-8-[(2R,3R,4R)-2- (3,4-dihydroxyphenyl)-3,5,7-trihydroxy-3,4-dihydro-2H-chromen-4-yl]-3,4-dihydro-2H- chromene-3,5,7-triol.
• procyanidin Al or “type A1 procyanidin” refers to the compound with IUPAC name: (lR,5R,6S,13S,21R)-5,13-bis(3,4- dihydroxyphenyl)-4, 12, 14-trioxapentacyclo[l 1.7.1.02, 11.03,8.015, 20]henicosa- 2( 11 ),3(8),9, 15, 17, 19-hexaene-6,9, 17,19,21 -pentol .
• procyanidin A2 or “type A2 procyanidin” refers to the compound with IUPAC name (lR,5R,6R,13S,21R)-5,13-bis(3,4- dihydroxyphenyl)-4, 12, 14-trioxapentacyclo[l 1.7.1.02, 11.03,8.015, 20]henicosa- 2( 11 ),3(8),9, 15, 17, 19-hexaene-6,9, 17,19,21 -pentol .
• propelargonidin refers to the polyphenol with IUPAC name 2-[[2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3,4-dihydro-2H-chromen-3- yl]oxy]-2-(4-hydroxyphenyl)-3,4-dihydrochromene-3,4,5,7-tetrol.
• the at least one polyphenol other than hydroxytyrosol of the group of lignans is selected from the group consisting of secoisolariciresinol diglucoside, secoisolariciresinol, matairesinoside, lariciresinol, pinoresinol, sesamin, and secoisolariciresinol.
• the terms “secoisolariciresinol diglucoside”, “matairesinoside”, “lariciresinol”, “pinoresinol”, “sesamin”, and “secoisolariciresinol” refer to the corresponding term as understood by one skilled in the art.
• the at least one polyphenol other than hydroxytyrosol of the group of stilbenoids is selected from the group consisting of resveratrol, piceatannol, pinosylvin, pterostilbene, and rhapontienin.
• resveratrol As used in the present invention, the terms “resveratrol”, “piceatannol”, “pinosylvin”, “pterostilbene”, and “rhapontienin” refer to the corresponding term as understood by one skilled in the art.
• the at least one polyphenol other than hydroxytyrosol is part of a plant extract.
• said plant extract is not an extract of a plant product of the tree O. europaea , preferably it is not an extract of the leaf or of the fruit of the tree (). europaea.
• the plant extract of which the at least one polyphenol other than hydroxytyrosol is part is an extract selected from the group consisting of an extract of a plant product of Prunus dulcis (Miller) D.A. Webb an extract of a plant product of Citrus paradisi MacFad an extract of a plant product of Linum usitatissimum L an extract of a plant product of Polygonum cuspidatum Sieb. et Zucc
• each of the plant products mentioned in the preceding embodiment is one of those indicated in the definition of “plant product” provided above.
• the plant extract of which the at least one polyphenol other than hydroxytyrosol is part is an extract selected from the group consisting of an extract of the skin of the fruit of Prunus dulcis (Miller) D.A. Webb an extract of the fruit of Citrus paradisi MacFad or an extract of the seed of the fruit of Citrus paradisi MacFad an extract of the seed of the fruit of Linum usitatissimum L an extract of the root of Polygonum cuspidatum Sieb. et Zucc.
• the plant extract of which the at least one polyphenol other than hydroxytyrosol is part specified in the preceding embodiments is obtained from any plant or vegetable belonging to the species indicated in each case, preferably of any variety belonging to the species indicated in each case.
• the tree is of the species Prunus dulcis (Miller) D. A. Webb.
• the term Prunus dulcis used in any aspect of the invention is interchangeable with the term Prunus dulcis (Miller) D.A. Webb.
• the term Citrus paradisi used in any aspect of the invention is interchangeable with the term Citrus paradisi MacFad.
• the term Linum usitatissimum used in any aspect of the invention is interchangeable with the term Linum usitatissimum L.
• the term Polygonum cuspidatum used in any aspect of the invention is interchangeable with the term Polygonum cuspidatum Sieb. et Zucc.
• the plant extract comprising the polyphenol other than hydroxytyrosol obtained from a specific species is obtained from more than one plant product of the tree, plant or vegetable of said species, wherein each of said products is the same or different.
• each of said products is obtained from different trees, plants, or vegetables, wherein each of said trees, plants, or vegetables are of the same species, preferably of the same variety.
• the plant extract of which the at least one polyphenol other than hydroxytyrosol is part is an extract selected from the group consisting of an extract of at least two plant products, each selected from the list of plant products indicated in the definition of “plant product” above and obtained from at least two different trees of Prunus dulcis, wherein each tree is preferably of a different variety an extract of at least two plant products, each selected from the list of plant products indicated in the definition of “plant product” above and obtained from at least two different trees of Citrus paradisi, wherein each tree is preferably of a different variety an extract of at least two plant products, each selected from the list of plant products indicated in the definition of “plant product” above and obtained from at least two different plants of Linum usitatissimum, wherein each plant is preferably of a different variety. an extract of at least two plant products, each selected from the list of plant products indicated in the definition of “plant product” above and obtained from at least two different plants of Polygonum cuspidatum, wherein each plant
• the at least one polyphenol other than hydroxytyrosol which is part of any aforementioned extract of one or several plant products, preferably the extract of the skin of the fruit, of one or several trees of Prunus is a flavonoid, preferably selected from the group consisting of procyanidins, propelargonidins, prodelphinidins, catechin, epicatechin, quercetin, kaempferol, isorhamnetin, rutin, and genistein.
• the at least one polyphenol other than hydroxytyrosol which is part of any aforementioned extract of one or several plant products, preferably of the seed of the fruit, of one or several trees
• Citrus paradisi is a flavonoid, preferably selected from the group consisting of naringin, naringenin, quercetin, catechin, rutin, and genistein.
• the at least one polyphenol other than hydroxytyrosol which is part of any aforementioned extract of one or several plant products, preferably of the seed of the fruit, of Linum usitatissimum, is a lignan, preferably selected from the group consisting of secoisolariciresinol diglucoside, secoisolariciresinol, matairesinoside, lariciresinol, pinoresinol, and sesamin.
• the at least one polyphenol other than hydroxytyrosol which is part of any aforementioned extract of one or several plant products, preferably of the root, of one or several plants of Polygonum cuspidatum , is a stilbene, preferably selected from the group consisting of resveratrol, piceatannol, pinosylvin, pterostilbene, and rhapontienin.
• the at least one polyphenol other than hydroxytyrosol which is part of the extract of the skin of the fruit of Prunus dulcis is a flavonoid, preferably selected from the group consisting of procyanidins, propelargonidins, prodelphinidins, catechin, epicatechin, quercetin, kaempferol, isorhamnetin, rutin, and genistein.
• the at least one polyphenol other than hydroxytyrosol which is part of the extract of the fruit of Citrus paradisi or the extract of the seed of the fruit of Citrus paradisi is a flavonoid, preferably selected from the group consisting of naringin, naringenin, quercetin, catechin, rutin, and genistein.
• the at least one polyphenol other than hydroxytyrosol which is part of the extract of the seed of the fruit of Linum usitatissimum is a lignan, preferably selected from the group consisting of secoisolariciresinol diglucoside, secoisolariciresinol, matairesinoside, lariciresinol, pinoresinol, and sesamin.
• any aforementioned extract of one or several plant products preferably the extract of the skin of the fruit, of one or several trees Prunus dulcis contains between 1-100% (w/w), 1-90% (w/w), 5-80% (w/w), 5-70% (w/w), 5-80% (w/w), 5-60% (w/w), 5-50% (w/w), 5-40% (w/w), 10-30% (w/w) of flavonoids, preferably between 5-60% (w/w) of flavonoids.
• said flavonoids are selected from the group consisting of procyanidins, propelargonidins, prodelphinidins, catechin, epicatechin, quercetin, kaempferol, isorhamnetin, rutin, genistein, and combinations thereof.
• a particular embodiment contains 1% (w/w), 2% (w/w), 3% (w/w), 4% (w/w), 5% (w/w), 6% (w/w), 7% (w/w), 8% (w/w), 9% (w/w), 10% (w/w), 12% (w/w), 15% (w/w), 20% (w/w), 22% (w/w), 25% (w/w), 27% (w/w), 28% (w/w), 29% (w/w), 30% (w/w), 31% (w/w), 32% (w/w), 35% (w/w), 40% (w/w), 45% (w/w), 50% (w/w), 60% (w/w), 70% (w/w), 80% (w/w), 90% (w/w), 95% (w/w), 97% (w/w), 98% (w/w), 99% (w/w), 99.5% (w/w), 99.8% (w/w), 99.9% (w/w), 100% (w/w
• said flavonoids are selected from the list of flavonoids indicated previously.
• the determination of the polyphenol content is carried out using the method based on the Folin-Ciocalteu reagent (Singleton VL et al. , 1999, Methods in Enzymology , vol. 299: 152-78).
• the extract of the skin of the fruit of Prunus dulcis contains between 1-100% (w/w), 1-90% (w/w), 5-80% (w/w), 5-70% (w/w), 5-80% (w/w), 5-60% (w/w), 5-50% (w/w), 5-40% (w/w), 10-30% (w/w) of flavonoids, preferably between 5-60% (w/w).
• said flavonoids are selected from the group consisting of procyanidins, propelargonidins, prodelphinidins, catechin, epicatechin, quercetin, kaempferol, isorhamnetin, rutin, genistein, and combinations thereof.
• the extract contains 1% (w/w), 2% (w/w), 3% (w/w), 4% (w/w), 5% (w/w), 6% (w/w), 7% (w/w), 8% (w/w), 9% (w/w), 10% (w/w), 12% (w/w), 15% (w/w), 20% (w/w), 22% (w/w), 25% (w/w), 27% (w/w), 28% (w/w), 29% (w/w), 30% (w/w), 31% (w/w), 32% (w/w), 35% (w/w), 40% (w/w), 45% (w/w), 50% (w/w), 60% (w/w), 70% (w/w), 80% (w/w), 90% (w/w), 95% (w/w), 97% (w/w), 98% (w/w), 99% (w/w), 99.5% (w/w), 99.8% (w/w), 99.9% (w/w), 100%
• said flavonoids are selected from the list of flavonoids indicated previously.
• the determination of the polyphenol content is carried out using the method based on the Folin-Ciocalteu reagent (Singleton VL et al. , 1999, Methods in Enzymology , vol. 299: 152-78).
• any aforementioned extract of one or several plant products preferably of the fruit or of the seed of the fruit, of one or several trees Citrus paradisi , contains between 1-100% (w/w), 1-90% (w/w), 5-80% (w/w), 5-70% (w/w), 5-80% (w/w), 10-80% (w/w), 10-60% (w/w), 15-50% (w/w), 20-50% (w/w), 30-50% (w/w), preferably between 10-80% (w/w) of flavonoids.
• said flavonoids are selected from the list consisting of naringin, naringenin, quercetin, catechin, rutin, genistein, and combinations thereof.
• the extract contains 1% (w/w), 2% (w/w), 3% (w/w), 4% (w/w), 5% (w/w), 6% (w/w), 7% (w/w), 8% (w/w), 9% (w/w), 10% (w/w), 12% (w/w), 15% (w/w), 20% (w/w), 22% (w/w), 25% (w/w), 27% (w/w), 28% (w/w), 29% (w/w), 30% (w/w), 31% (w/w), 32% (w/w), 35% (w/w), 40% (w/w), 45% (w/w), 50% (w/w), 60% (w/w), 70% (w/w), 80% (w/w), 90% (w/w), 95% (w/w), 97% (w/w), 98% (w/w), 99% (w/w), 99.5% (w/w), 99.8% (w/w), 99.9% (w/w), 100%
• said flavonoids are selected from the list consisting of naringin, naringenin, quercetin, catechin, rutin, genistein, and combinations thereof.
• the determination of the polyphenol content is carried out using the method based on the Folin-Ciocalteu reagent (Singleton VL et al. , 1999, Methods in enzimology , vol. 299: 152-78).
• the extract of the fruit of Citrus paradisi or the extract of the seed of the fruit of Citrus paradisi contains between 1-100% (w/w), 1-90% (w/w), 5-80% (w/w), 5-70% (w/w), 5-80% (w/w), 10-80% (w/w), 10-60% (w/w), 15-50% (w/w), 20-50% (w/w), 30-50% (w/w), preferably between 10-80% (w/w) of flavonoids.
• said flavonoids are selected from the list consisting of naringin, naringenin, quercetin, catechin, rutin, genistein, and combinations thereof.
• the extract contains 1% (w/w), 2% (w/w), 3% (w/w), 4% (w/w), 5% (w/w), 6% (w/w), 7% (w/w), 8% (w/w), 9% (w/w), 10% (w/w), 12% (w/w), 15% (w/w), 20% (w/w), 22% (w/w), 25% (w/w), 27% (w/w), 28% (w/w), 29% (w/w), 30% (w/w), 31% (w/w), 32% (w/w), 35% (w/w), 40% (w/w), 45% (w/w), 50% (w/w), 60% (w/w), 70% (w/w), 80% (w/w), 90% (w/w), 95% (w/w), 97% (w/w), 98% (w/w), 99% (w/w), 99.5% (w/w), 99.8% (w/w), 99.9% (w/w), 100%
• said flavonoids are selected from the list consisting of naringin, naringenin, quercetin, catechin, rutin, geni stein, and combinations thereof.
• the determination of the polyphenol content is carried out using the method based on the Folin-Ciocalteu reagent (Singleton VL et al, 1999, Methods in Enzymology, vol. 299: 152-78).
• any aforementioned extract of one or several plant products, preferably the extract of the seed of the fruit, of one or several plants of Linum usitatissimum contains between 1-100% (w/w), 1-90% (w/w), 5-80% (w/w), 5-70% (w/w), 5- 80% (w/w), 5-60% (w/w), 5-50% (w/w), 5-40% (w/w), 10-30% (w/w) of lignans, preferably between 5-50% (w/w) lignans.
• said lignans are selected from the group consisting of secoisolariciresinol diglucoside, secoisolariciresinol, matairesinoside, lariciresinol, pinoresinol, and sesamin, and combinations thereof.
• the extract contains 1% (w/w), 2% (w/w), 3% (w/w), 4% (w/w), 5% (w/w), 6% (w/w), 7% (w/w), 8% (w/w), 9% (w/w), 10% (w/w), 12% (w/w), 15% (w/w), 20% (w/w), 22% (w/w), 25% (w/w), 27% (w/w), 28% (w/w), 29% (w/w), 30% (w/w), 31% (w/w), 32% (w/w), 35% (w/w), 40% (w/w), 45% (w/w), 50% (w/w), 60% (w/w), 70% (w/w), 80% (w/w), 90% (w/w), 95% (w/w), 97% (w/w), 98% (w/w), 99% (w/w), 99.5% (w/w), 99.8% (w/w), 99.9% (w/w), 100%
• said lignans are selected from the aforementioned list.
• the determination of the polyphenol content is carried out using the method based on the Folin-Ciocalteu reagent (Singleton VL et al ., 1999, Methods in enzimology , vol. 299: 152-78).
• the extract of the seed of Linum usitatissimum contains between 1-100% (w/w), 1-90% (w/w), 5-80% (w/w), 5-70% (w/w), 5-80% (w/w), 5-60% (w/w), 5- 50% (w/w), 5-40% (w/w), 10-30% (w/w) of lignans, preferably between 5-50% (w/w) lignans.
• said lignans are selected from the group consisting of secoisolariciresinol diglucoside, secoisolariciresinol, matairesinoside, lariciresinol, pinoresinol, and sesamin, and combinations thereof.
• the extract contains 1% (w/w), 2% (w/w), 3% (w/w), 4% (w/w), 5% (w/w), 6% (w/w), 7% (w/w), 8% (w/w), 9% (w/w), 10% (w/w), 12% (w/w), 15% (w/w), 20% (w/w), 22% (w/w), 25% (w/w), 27% (w/w), 28% (w/w), 29% (w/w), 30% (w/w), 31% (w/w), 32% (w/w), 35% (w/w), 40% (w/w), 45% (w/w), 50% (w/w), 60% (w/w), 70% (w/w), 80% (w/w), 90% (w/w), 95% (w/w), 97% (w/w), 98% (w/w), 99% (w/w), 99.5% (w/w), 99.8% (w/w), 99.9% (w/w), 100%
• said lignans are selected from the aforementioned list.
• the determination of the polyphenol content is carried out using the method based on the Folin-Ciocalteu reagent (Singleton VL et al., 1999, Methods in enzimology , vol. 299: 152-78).
• any aforementioned extract of one or several plant products, preferably of the root, of one or several plants Polygonum cuspidatum contains between 10-100% (w/w), 20-100% (w/w), 30-100% (w/w), 40-100% (w/w), 50-100% (w/w), 60-100% (w/w), 70-100% (w/w), 80-100% (w/w), 85-100% (w/w), 90-99.9% (w/w), 90- 99.8% (w/w), 90-99.5% (w/w), 90-99.25% (w/w), 90-99% (w/w), 92-99% (w/w), 95-99% (w/w) of stilbenes, preferably between 50-100% (w/w) of stilbenes.
• said stilbenes are selected from the list consisting of resveratrol, piceatannol, pinosylvin, pterostilbene, rhapontienin, and combinations thereof, more preferably resveratrol. In a particular embodiment, it is a combination of resveratrol and at least one of the other stilbenoids indicated in the preceding list.
• the extract contains 50% (w/w), 60% (w/w), 65% (w/w), 70% (w/w), 75% (w/w), 80% (w/w), 85% (w/w), 90% (w/w), 91% (w/w), 92% (w/w), 93% (w/w), 94% (w/w), 95% (w/w), 96% (w/w), 97% (w/w), 98% (w/w), 99% (w/w), 99.5% (w/w), 99.75% (w/w), 99.8% (w/w), 99.85% (w/w), 99.9% (w/w), 99.95% (w/w), 99.99% (w/w), 100% (w/w), preferably 98% (w/w) of stilbenes.
• said stilbenes are selected from the list consisting of resveratrol, piceatannol, pinosylvin, pterostilbene, rhapontienin, and combinations thereof, more preferably resveratrol. In a particular embodiment, it is a combination of resveratrol and at least one of the other stilbenoids indicated in the preceding list.
• the determination of the polyphenol content is carried out using the method based on the Folin-Ciocalteu reagent (Singleton VL et al ., 1999, Methods in Enzymology , vol. 299: 152-78).
• the extract of the root of Polygonum cuspidatum contains between 10-100% (w/w), 20-100% (w/w), 30-100% (w/w), 40-100% (w/w), 50-100% (w/w), 60-100% (w/w), 70-100% (w/w), 80-100% (w/w), 85-100% (w/w), 90-99.9% (w/w), 90-99.8% (w/w), 90-99.5% (w/w), 90-99.25% (w/w), 90-99% (w/w), 92-99% (w/w), 95-99% (w/w) of stilbenes, preferably between 50-100% (w/w) of stilbenes.
• said stilbenes are selected from the list consisting of resveratrol, piceatannol, pinosylvin, pterostilbene, rhapontienin, and combinations thereof, more preferably resveratrol. In a particular embodiment, it is a combination of resveratrol and at least one of the other stilbenoids indicated in the preceding list.
• the extract contains 50% (w/w), 60% (w/w), 65% (w/w), 70% (w/w), 75% (w/w), 80% (w/w), 85% (w/w), 90% (w/w), 91% (w/w), 92% (w/w), 93% (w/w), 94% (w/w), 95% (w/w), 96% (w/w), 97% (w/w), 98% (w/w), 99% (w/w), 99.5% (w/w), 99.75% (w/w), 99.8% (w/w), 99.85% (w/w), 99.9% (w/w), 99.95% (w/w), 99.99% (w/w), 100% (w/w), preferably 98% (w/w) of stilbenes.
• said stilbenes are selected from the list consisting of resveratrol, piceatannol, pinosylvin, pterostilbene, rhapontienin, and combinations thereof, more preferably resveratrol. In a particular embodiment, it is a combination of resveratrol and at least one of the other stilbenoids indicated in the preceding list.
• the determination of the polyphenol content is carried out using the method based on the Folin-Ciocalteu reagent (Singleton VL et al. , 1999, Methods in Enzymology , vol. 299: 152-78).
• the extract of the skin of the fruit of Prunus dulcis contains between 5-60% (w/w), preferably 30% (w/w) of flavonoids, wherein the flavonoids are preferably selected from the group consisting of procyanidins, propelargonidins, prodelphinidins, catechin, epicatechin, quercetin, kaempferol, isorhamnetin, rutin, genistein, and combinations thereof
• the extract of the fruit of Citrus paradisi or the extract of the seed of the fruit of Citrus paradisi contains between 10-80% w/w, preferably 45% (w/w) of flavonoids, wherein the flavonoids are preferably selected from the group consisting of naringin, naringenin, quercetin, catechin, rutin, genistein, and combinations thereof.
• the extract of the seed of the fruit of Linum usitatissimum contains between 5- 50% (w/w), preferably 20% (w/w) of lignans, wherein the lignans are preferably selected from the group consisting of secoisolariciresinol diglucoside, secoisolariciresinol, matairesinoside, lariciresinol, pinoresinol, and sesamin, and combinations thereof.
• the extract of the root of Polygonum cuspidatum contains between 50-100% (w/w), preferably 98% (w/w) of stilbenoids, wherein the stilbenoids are preferably selected from the group consisting of resveratrol, piceatannol, pinosylvin, pterostilbene, rhapontienin, and combinations thereof.
• the polyphenol other than hydroxytyrosol is used at least at 1.25, 1.5, 1.75, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.4, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 10, 11, 12, 51, 18, 20, 21, 22, 23, 24, 24.2, 24.3, 24.5, 25, 26, 27, 28, 29, 30, 32, 35, 40 preferably 2.7 times in excess by weight with respect to the amount by weight of the orally administered hydroxytyrosol.
• the w/w ratio of hydroxytyrosohpolyphenol other than hydroxytyrosol used in the first, second, or third aspect is between 1:0.5 and 1:100, 1:1 and 1:50, 1:2 and 1:40.1:2 and 1:30, 1:2 and 1:28, 1:2 and 1:25, 1:2 and 1:24.5, 1:2 and 1:24.3, 1:2 and 1:24, 1:2 and 1:20, 1:2 and 1: 17, 1:2 and 1:15, 1:2 and 1:10, 1: 2 and 1:7, 1:2 and 1:5, 1:2 and 1: 4, 1:2 and 1:3, preferably between 1:1 and 1:50.
• the w/w ratio of hydroxytyrosohpolyphenol other than hydroxytyrosol used in the first, second, or third aspect is of at least 1:0.5, 1:1, 1:1.5, 1:2, 1:2.3, 1:2.5, 1:2.7, 1:2.8, 1:2.9, 1:3, 1:3.2, 1:3.5, 1:3.7, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:15, 1:17, 1:20, 1: 21, 1:22, 1:23, 1:24, 1:24.1, 1:24.2, 1:24.3, 1:24.4, 1:24.5, 1:24.6, 1:24.7, 1:24.8,.
• the w/w ratio of plant extract comprising hydroxytyrosohplant extract comprising the at least one polyphenol other than hydroxytyrosol used in the first, second, or third aspect is between 1:0.5 and 1:100, 1:1: and 1:75, 1:2 and 1:50, 1:3 and 1:40, 1:4 and 1:30, 1:5 and 1:25, 1:7 and 1:20, 1:10 and 1: 15, preferably between 1:2 and 1:50.
• the w/w ratio of plant extract comprising hydroxytyrosohplant extract comprising a polyphenol other than hydroxytyrosol is of at least 1:1, 1:2, 1:3, 1 :4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:17, 1:20, 1:22, 1:25, 1:27, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:70, 1:80; 1:90, 1:100.
• the invention in a fourth aspect, relates to a composition or kit of parts comprising hydroxytyrosol and at least one polyphenol that is not hydroxy tyro sol, at least one polyphenol that does not occur naturally in the tree O. europaea , or at least one polyphenol other than hydroxytyrosol and that does not occur naturally in the tree O. europaea.
• the hydroxytyrosol and the at least one polyphenol that is not hydroxytyrosol, the at least one polyphenol that does not occur naturally in the tree O. europaea, or at least one polyphenol other than hydroxytyrosol and that does not occur naturally in the tree O. europaea are called the compounds of the composition of the invention.
• composition refers to a combination of compounds or ingredients.
• the ingredients of the composition can be supplied separately, or in a dosage form. Therefore, if the composition is administered to a subject, the compounds of the composition could be mixed in said dosage form, mixed before administration, despite being supplied separately, being supplied and administered separately, but mixed once taken by the subject, i.e., inside the subject’s body. Furthermore, some ingredients of the composition can be administered together and others separately, but they are all mixed once taken by the subject, i.e., inside the subject’s body.
• composition according to the invention can be supplied with different formats.
• Non limiting examples include tablets, coated tablets, pills, aqueous or oily suspensions, solutions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs, pastes, gels, or the like.
• Said composition can be prepared according to any known method, and such compositions may contain in addition to the compounds indicated above in the composition of the invention, one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents, and preserving agents to provide pharmaceutically elegant and pleasing compositions.
• Any of the formats in which the composition is supplied may contain the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients that are suitable for manufacturing said supply formats.
• excipients can be, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulating and disintegrating agents, for example, corn starch or alginic acid; binding agents, for example, starch, gelatin, or acacia gum; and lubricating agents, for example, magnesium stearate, stearic acid, or talc.
• the different formats in which the composition is supplied can be uncoated or coated by known techniques to delay disintegration and absorption in the digestive system and thereby provide an action sustained over a longer period.
• a time delay material such as glyceryl monostearate or glyceryl distearate can be used.
• delayed-release pharmaceutical form releases a product or substance at a different time rapidly after the administration.
• delayed-release systems include repeat-action tablets and capsules and tablets with an enteric coating where the scheduled release is achieved through a barrier coating.
• composition according to the invention may also be formulated for oral use as hard gelatin capsules, wherein the active ingredient(s) is/are mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate, or kaolin, or soft gelatin capsules, wherein the active ingredient(s) is/are mixed with water and an oily medium, for example, peanut oil, liquid paraffin, or olive oil.
• an inert solid diluent for example, calcium carbonate, calcium phosphate, or kaolin
• an oily medium for example, peanut oil, liquid paraffin, or olive oil.
• composition according to the invention can be formulated as aqueous suspensions wherein the active ingredient(s) is/are in a mixture with excipients suitable for manufacturing aqueous suspensions.
• excipients are suspension agents, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum, and acacia gum;
• the dispersing or wetting agents can be a natural phosphatide such as lecithin, or products of condensation of an alkylene oxide with fatty acids, for example, polyoxyethylene stearate, or products of condensation of ethylene oxide with long-chain aliphatic alcohols, for example, heptadecaethylenoxyketanol, or products of condensation of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or products of condensation of ethylene oxide with partial esters derived from
• composition according to the invention can be formulated as oily suspensions by suspending the active ingredient in a vegetable oil, for example, peanut oil, olive oil, sesame seed oil, or coconut oil, or in a mineral oil such as liquid paraffin.
• the oily suspensions may contain a thickening agent, for example, beeswax, solid paraffin, or cetyl alcohol. Sweetening agents such as those shown above and flavoring agents may be added to provide an agreeable oral composition.
• These compositions can be preserved by adding an antioxidant such as ascorbic acid.
• composition according to the invention can be formulated in dispersible powder and granule form suitable for the composition of an aqueous suspension by adding water.
• the active ingredient in such powders and granules is provided in a mixture with a dispersing or wetting agent, suspension agent, and one or more preservatives.
• the dispersing or wetting agents and suspension agents are exemplified by those already mentioned above. Additional excipients, for example, sweetening agents, flavoring agents, and coloring agents, may also be present.
• the composition according to the invention can be in the form of oil in water emulsions.
• the oil phase can be a vegetable oil, for example, olive oil or peanut oil, or a mineral oil, for example, a liquid paraffin, or a mixture thereof.
• Suitable emulsifying agents can be natural gums, for example, acacia gum or tragacanth gum, natural phosphatides, for example, soy lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example, sorbitan monooleate, and products of condensation of partial esters with ethylene oxide, for example, polyoxyethylene sorbitan monooleate.
• the emulsions may also contain sweetening agents and flavoring agents.
• composition according to the invention can be formulated as syrups and elixirs.
• the syrups and elixirs can be formulated with sweetening agents, for example, glycerol, propyleneglycol, sorbitol, or sucrose.
• Such formulations may also contain a demulcent, a preservative, and flavoring agents and coloring agents.
• Demulcents are protective agents primarily used to alleviate irritation, particularly of mucous membranes or depleted tissues.
• Others include acacia gum, agar, benzoin, carbomer, gelatin, glycerin, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, propyleneglycol, sodium alginate, tragacanth, hydrogels, and the like.
• Formulations suitable for oral administration include tablets and coated tablets comprising the compounds of the composition of the invention in a flavored base, such as sucrose, acacia gum, or tragacanth; and pills comprising said compound in an inert base, such as gelatin and glycerin or sucrose and acacia gum.
• a flavored base such as sucrose, acacia gum, or tragacanth
• pills comprising said compound in an inert base, such as gelatin and glycerin or sucrose and acacia gum.
• composition of the invention which contains a suitable amount of each of the compounds of the composition of the invention, depending on the additive or support selected.
• the expression “kit of parts” refers to a product comprising different ingredients, components, or compounds, wherein said ingredients, components, or compounds are physically separated, preferably by separately packaging each ingredient, component, or compound in the kit such that it allows being transported and stored.
• the individual active ingredients, components, or compounds represent therapeutic agents and, provided that the use of those compounds, whether simultaneously, separately, or sequentially, produces the new and unexpected therapeutic effect together, as described in the present document, which is not achieved by the compounds independently with respect to one another.
• the kit of parts typically comprises its components in suitable containers.
• the materials suitable for packaging the components of the kit include glass, plastic (polyethylene, polypropylene, polycarbonate, and the like), bottles, vials, paper, pouches, and the like.
• each container may be in the form of vials, bottles, squeeze bottles, jars, sealed sleeves, sachets or pouches, tubes or blisters or any other suitable form provided that the container is configured to prevent the premature mixing of the components.
• kits of the invention may contain instructions for the simultaneous, sequential, or separate use of the different components that are in the kit.
• Said instructions may be in the form of printed material or in the form of an electronic medium capable of storing instructions such that they can be read by a subject, such as electronic storage media (magnetic discs, tapes, and the like), optical media (CD-ROM, DVD), and the like.
• the medium may furthermore or alternatively contain Internet addresses providing said instructions.
• compositions or kits of parts of the invention may comprise, essentially consist, or consist of the aforementioned ingredients, compounds or components.
• the term “comprising” or “comprises” is used to indicate that the composition being described must contain the ingredient(s) listed, but it may optionally contain additional ingredients.
• the term “essentially consisting of’ or “essentially consists of’ is used to indicate that the composition being described must contain the ingredient(s) listed, and it may also contain a small amount (for example, up to 5 percent by weight, or up to 1 percent by weight, or 0.1 percent by weight) of other ingredients provided that no additional ingredient affects the essential properties of the extract or composition.
• the term “consisting of’ or “consists of’ is used to indicate that the composition being described must contain only the ingredient(s) listed.
• the composition or kit of parts according to the fourth aspect of the invention comprises additional ingredients in addition to hydroxytyrosol and the at least one polyphenol other than hydroxytyrosol.
• it comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100 ingredients in addition to hydroxytyrosol and the at least one polyphenol other than hydroxytyrosol.
• the hydroxytyrosol and the at least one polyphenol other than hydroxytyrosol comprise at least 0.25%, at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, at least 100% of the total amount of ingredients forming the kit.
• polyphenol as well as the expression “polyphenol that is not hydroxytyrosol wherein the at least one polyphenol other than hydroxytyrosol and/or does not occur naturally in the tree O. europaea” have been defined in the first, second, and third aspects of the invention. Said definitions, as well as the particular embodiments for describing said term and said expression apply to this aspect of the invention.
• the hydroxytyrosol is part of a plant extract.
• the compound or ingredient “hydroxytyrosol” of the composition and of the kit of parts of the fourth aspect of the invention consists of a plant extract comprising hydroxytyrosol.
• plant extract as well as “is part of a plant extract” have been defined in the first, second, and third aspects of the invention. Therefore, as one skilled in the art will understand, in a particular embodiment, the hydroxytyrosol is supplied in the composition of the fourth aspect of the invention as a plant extract.
• the plant extract comprising the hydroxytyrosol of this aspect of the invention is like the extract comprising hydroxytyrosol described in any of the embodiments and definitions of the first, second, and third aspects of the invention.
• the plant extract comprising hydroxytyrosol is obtained from any plant product of the tree O. europaea, wherein the plant product is any of those indicated in the definition of “plant product” indicated in the first, second, or third aspect of the invention.
• the plant extract of which the hydroxytyrosol is part is an extract of the leaf or of the fruit, preferably the leaf, of the tree O. europaea.
• said extract is any extract mentioned in the first, second, and third aspects of the invention, of one or several plant products, preferably of the leaf or of the fruit, more preferably of the leaf, of one or several trees of O. europaea.
• the hydroxytyrosol content in the extract comprising hydroxytyrosol of this aspect is any of those indicated in the first, second, and third aspects of the invention for the extract comprising the hydroxytyrosol of said aspects.
• the plant extract comprising hydroxytyrosol contains 10-90%, preferably 10% (w/w) of hydroxytyrosol.
• the at least one polyphenol that is not hydroxytyrosol of the fourth aspect of the invention is the at least one polyphenol that is not hydroxytyrosol defined and described in the first, second, and third aspects of the invention. Therefore, the polyphenol that is not hydroxytyrosol of the fourth aspect of the invention is any of the polyphenols indicated in the first, second, and third aspects of the invention and defined as belonging to the group of polyphenols that are not hydroxytyrosol.
• the at least one polyphenol other than hydroxytyrosol is selected from the group of polyphenols consisting of flavonoids, lignans, and stilbenes.
• the at least one polyphenol other than hydroxytyrosol of the group of flavonoids is selected from the group consisting of procyanidins, propelargonidins, prodelphinidins, catechin, epicatechin, quercetin, kaempferol, isorhamnetin, naringin, naringenin, genistein, and rutin.
• the procyanidins are type B procyanidins, preferably selected from the group consisting of type B1 procyanidin, type B2 procyanidin, type B3 procyanidin, type B4 procyanidin, type B5 procyanidin, type B6 procyanidin and type B7 procyanidin.
• it is a type Cl procyanidin.
• it is a type A procyanidin, preferably selected from the group consisting of procyanidin Al, procyanidin A2.
• the propelargonidins are type A, more preferably type B propelargonidins.
• the prodelphinidins are type A.
• the at least one polyphenol other than hydroxytyrosol of the group of lignans is selected from the group consisting of secoisolariciresinol diglucoside, secoisolariciresinol, matairesinoside, lariciresinol, pinoresinol, and sesamin
• the at least one polyphenol other than hydroxytyrosol of the group of stilbenes is selected from the group consisting of resveratrol, piceatannol, pinosylvin, pterostilbene, and rhapontienin, preferably resveratrol.
• the at least one polyphenol other than hydroxytyrosol is part of a plant extract.
• the ingredient or compound that is “polyphenol other than hydroxytyrosol” of the composition and kit of parts of the fourth aspect of the invention consists of a plant extract.
• the plant extract of which the at least one polyphenol other than hydroxytyrosol is part is not an extract of a plant product of the tree O. europaea , preferably it is not an extract of the leaf or of the fruit of the tree O. europaea.
• the plant extract of which the at least one polyphenol other than hydroxytyrosol is part is as defined and describe in the first, second, and third aspects of the invention.
• the plant extract of which the at least one polyphenol other than hydroxytyrosol is part is an extract selected from the group consisting of: an extract of the skin of the fruit of Prunus dulcis, an extract of the fruit of Citrus paradisi or an extract of the seed of the fruit of Citrus paradisi an extract of the seed of the fruit of Linum usitatissimum an extract of the root of Polygonum cuspidatum.
• the at least one polyphenol other than hydroxytyrosol comprised in each extract comprising a polyphenol other than hydroxytyrosol of this aspect of the invention is that indicated in the first, second, or third aspect of the invention for said extract.
• the at least one polyphenol other than hydroxytyrosol which is part of the extract of the skin of the fruit of Prunus dulcis is a flavonoid, preferably selected from the group consisting of procyanidins, propelargonidins, prodelphinidins, catechin, epicatechin, quercetin, kaempferol, isorhamnetin, rutin, and genistein.
• the at least one polyphenol other than hydroxytyrosol which is part of the extract of the fruit of Citrus paradisi or of the extract of the seed of the fruit of Citrus paradisi is a flavonoid, preferably selected from the group consisting of naringin, naringenin, quercetin, catechin, rutin, and genistein.
• the at least one polyphenol other than hydroxytyrosol which is part of the extract of the seed of the fruit of Linum usitatissimum is a lignan, preferably selected from the group consisting of secoisolariciresinol diglucoside, secoisolariciresinol, matairesinoside, lariciresinol, pinoresinol, and sesamin.
• the at least one polyphenol other than hydroxytyrosol which is part of the extract of the root of Polygonum cuspidatum is a stilbene, preferably selected from the group consisting of resveratrol, piceatannol, pinosylvin, pterostilbene, and rhapontienin, more preferably is resveratrol.
• the content and type of flavonoids contained in any extract mentioned in the present invention of one or several plant products, preferably the extract of the skin of the fruit, of one or several trees Prunus dulcis, is that indicated in the first, second, and third aspects of the invention for said extract.
• the content and type of flavonoids contained in any extract mentioned in the present invention of one or several plant products, preferably of the fruit or of the seed of the fruit, of one or several trees Citrus paradisi , is that indicated in the first, second, and third aspects of the invention for said extract.
• the content and type of lignans contained in any extract mentioned in the present invention of one or several plant products, preferably the extract of the seed of the fruit, of one or several plants of Linum usitatissimum, is that indicated in the first, second, and third aspects of the invention for said extract.
• the content and type of stilbenes contained in any extract mentioned in the present invention, of one or several plant products, preferably of the root, of Polygonum cuspidatum, is that indicated in the first, second, and third aspects of the invention for said extract.
• the extract of the skin of the fruit of Prunus dulcis contains between 5-60% (w/w), preferably 30% (w/w) of flavonoids, wherein the flavonoids are preferably selected from the group consisting of procyanidins, propelargonidins, prodelphinidins, catechin, epicatechin, quercetin, kaempferol, isorhamnetin, rutin, genistein, and combinations thereof.
• the extract of the fruit of Citrus paradisi or the extract of the seed of the fruit of Citrus paradisi contains between 10-80% w/w, preferably 45% (w/w) of flavonoids, wherein the flavonoids are preferably selected from the group consisting of naringin, naringenin, quercetin, catechin, rutin, genistein, and combinations thereof.
• the extract of the seed of the fruit of Linum usitatissimum containing between 5-50% (w/w), preferably 20% (w/w) of lignans, wherein the lignans are preferably selected from the group consisting of secoisolariciresinol diglucoside, secoisolariciresinol, matairesinoside, lariciresinol, pinoresinol, and sesamin, and combinations thereof.
• the extract of the root of Polygonum cuspidatum contains between 50-100% (w/w), preferably 98% (w/w) of stilbenoids, wherein the stilbenoids are preferably selected from the group consisting of resveratrol, piceatannol, pinosylvin, pterostilbene, rhapontienin, and combinations thereof.
• the polyphenol other than hydroxytyrosol is at least at 1.25, 1.5, 1.75, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.4, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 10, 11, 12, 51, 18, 20, 21, 22, 23, 24, 24.2, 24.3, 24.4, 24.5, 25, 26, 27, 28, 29, 30, 32, 35, 40, 54, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100, preferably 2.7 times in excess by weight, more preferably 24.3 times in excess by weight, even more preferably at least 28 times in excess by weight with respect to the amount by weight of hydroxytyrosol comprised in the composition or kit of parts.
• the w/w ratio of hydroxytyrosokpolyphenol other than hydroxytyrosol is between 1:0.5 and 1:100, 1:1 and 1:50, 1:2 and 1:40, 1:2 and 1:30, 1:2 and 1:28, 1:2 and 1:25, 1:2 and 1:24.5, 1:2 and 1:24.3, 1:2 and 1:24, 1:2 and 1:20, 1:2 and 1: 17, 1:2 and 1:15, 1:2 and 1:10, 1:2 and 1:7, 1:2 and 1:5, 1:2 and 1: 4, 1:2 and 1 :3, preferably between 1:1 and 1:30 in the composition or kit of parts.
• the w/w ratio of hydroxytyrosokpolyphenol other than hydroxytyrosol is 1:0.5, 1:1, 1:1.5, 1:2, 1:2.3, 1:2.5, 1:2.7, 1:2.8, 1:2.9, 1:3, 1:3.2, 1:3.5, 1:3.7, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:15, 1:17, 1:20, 1: 21, 1:22, 1:23, 1:24, 1:24.1, 1:24.2, 1:24.3, 1:24.4, 1:24.5, 1:24.6, 1:24.7, 1:24.8,.
• the w/w ratio of hydroxytyrosokpolyphenol other than hydroxytyrosol is between 1:1 and 1:50, preferably 1:24.3, more preferably 1:28.
• the w/w ratio plant extract comprising hydroxytyrosol: plant extract comprising a polyphenol other than hydroxytyrosol is between 1:0.5 and 1:100, 1:1: and 1:75, 1:2 and 1:50, 1:3 and 1:40, 1:4 and 1:30, 1:5 and 1:25, 1:7 and 1:20, 1:10 and 1: 15, preferably between 1:5 and 1:25,
• the w/w ratio plant extract comprising hydroxytyrosol: plant extract comprising a polyphenol other than hydroxytyrosol is of at least 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:17, 1:20, 1:22, 1:25, 1:27, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:70, 1:80
• the hydroxytyrosol content in the composition of the fourth aspect of the invention is of at least 0.02-20% (w/w), at least 0.025-15% (w/w), at least 0.5- 10% (w/w), at least 0.5-7.5% (w/w), at least 0.6-5% (w/w), at least 0.7-2.5% (w/w), at least 0.8-2% (w/w), at least 0.9-1.5% (w/w), more preferably at least 0.05-10% (w/w), with respect to the total of the composition.
• the polyphenol composition of almond skin in the composition of the fourth aspect of the invention is of at least 5-100% (w/w), at least 7-75% (w/w), at least 10-50% (w/w), at least 15-40% (w/w), at least 20-30% (w/w), preferably at least 10-50% (w/w) with respect to the total of the composition.
• the polyphenol composition of almond skin in the composition of the fourth aspect of the invention is of at least 5% (w/w), at least 7% (w/w), at least 10% (w/w), at least 12% (w/w), at least 15% (w/w), at least 17% (w/w), at least 20% (w/w), at least, 21% (w/w), at least 22% (w/w), at least 23% (w/w), at least 24% (w/w), at least 25% (w/w), at least 26% (w/w), at least 27% (w/w), at least 28% (w/w), at least 29% (w/w), at least 30% (w/w), at least 32% (w/w), at least 35% (w/w), at least 40% (w/w), at least 45% (w/w), at least 50% (w/w), at least 55% (w/w), at least 60% (w/w), preferably at least 26% (w/w) with respect to the total of the composition.
• the content of hydroxytyrosol in the composition of the fourth aspect of the invention is of at least 0.05-10% w/w, preferably at least 0.9% w/w with respect to the total of the composition, wherein at least one polyphenol other than hydroxytyrosol is a polyphenol composition of almond skin, wherein the content of the polyphenol composition of almond skin is of about at least 10-50% w/w, preferably at least 26% w/w with respect to the total of the composition.
• the definitions and particular embodiments of the first, second, and third aspects of the invention are applied to the fourth aspect of the invention.
• the invention in a fifth aspect, relates to a method for obtaining the composition of the fourth aspect of the invention, which method comprises contacting a composition comprising hydroxytyrosol with a composition comprising at least one polyphenol that is not hydroxytyrosol and that does not occur naturally in the tree O. europaea.
• composition has been defined in the fourth aspect of the invention.
• hydroxytyrosol and “polyphenol that is not hydroxytyrosol” have been defined in the first, second, and third aspects of the invention.
• the polyphenol other than hydroxytyrosol is as defined in any definition and particular embodiment of the first, second, third, and fourth aspects of the invention.
• the expression “contacting” refers to the mixture or combination of the composition comprising hydroxytyrosol with the composition comprising a polyphenol other than hydroxytyrosol, such that the ingredients of one composition are surrounded by the ingredients of the other composition.
• a composition in turn comprising the composition comprising hydroxytyrosol and the composition comprising the polyphenol other than hydroxytyrosol is thus obtained.
• the product obtained by said contacting is the composition of the first, second, third, or fourth aspect of the invention.
• Said contacting can be performed in vitro as described below, or after the composition comprising hydroxytyrosol and the composition comprising a polyphenol other than hydroxytyrosol is ingested by a subject, at the same time or separately. In this case, the contacting between both compositions occurs once they are inside the subject’s body.
• the mixture or combination of the composition comprising hydroxytyrosol and the composition comprising a polyphenol other than hydroxytyrosol can be performed by placing in one and the same vessel both compositions and by moving the content of the vessel such that the ingredients of one composition are among the ingredients of the other composition with a certain homogeneity.
• at least one non-toxic pharmaceutically acceptable excipient suitable for manufacturing the composition of the first, second, third, or fourth aspect of the invention is included in the mixture.
• Said excipients can be any of the inert diluents, excipients suitable for manufacturing aqueous suspensions, vegetable oils, thickening agents, antioxidants, dispersing or wetting agents, suspension agents, emulsifying agents, sweetening agents, or flavoring agents indicated in the fourth aspect, which the composition of said aspects may comprise.
• One skilled in the art will be capable of suitably formulating the mixture, which contains a suitable amount of composition comprising hydroxytyrosol and of composition comprising a polyphenol other than hydroxytyrosol, depending on the excipient or additive selected.
• the composition comprising hydroxytyrosol is a plant extract.
• the composition comprising the at least one polyphenol other than hydroxytyrosol is a plant extract.
• the composition comprising hydroxytyrosol is a plant extract and/or the composition comprising the at least one polyphenol that is not hydroxytyrosol is a plant extract.
• the plant extract comprising hydroxytyrosol is like the plant extract comprising hydroxytyrosol defined in any of the preceding aspects of the invention.
• the plant extract comprising hydroxytyrosol is an extract of the leaf or of the fruit, preferably of the leaf, of the tree O. europaea.
• the hydroxytyrosol content in the extract comprising hydroxytyrosol is any of the hydroxytyrosol content in said extract indicated in any of the preceding aspects of the invention.
• the plant extract comprising hydroxytyrosol contains 1- 90% w/w, preferably 10% (w/w) of hydroxytyrosol.
• plant extract comprising the at least one polyphenol other than hydroxytyrosol is like any extract comprising the at least one polyphenol other than hydroxytyrosol defined in any of the preceding aspects of the invention.
• the plant extract comprising at least one polyphenol that is not hydroxytyrosol is selected from the group consisting of: an extract of the skin of the fruit of Prunus dulcis, an extract of the fruit of Citrus paradisi or an extract of the seed of the fruit of Citrus paradisi an extract of the seed of the fruit of Linum usitatissimum and an extract of the root of Polygonum cuspidatum.
• the content and type of flavonoids contained in any extract mentioned in the present invention of one or several plant products preferably the extract of the skin of the fruit, of one or several trees Prunus dulcis is that indicated in the first, second, and third aspects of the invention for said extract.
• the content and type of flavonoids contained in any extract mentioned in the present invention of one or several plant products, preferably of the fruit or of the seed of the fruit, of one or several trees Citrus paradisi c is that indicated in the first, second, and third aspects of the invention for said extract.
• the content and type of lignans contained in any extract mentioned in the present invention of one or several plant products, preferably the extract of the seed of the fruit, of one or several plants of Linum usitatissimum, is that indicated in the first, second, and third aspects of the invention for said extract.
• the content and type of stilbenes contained in any extract mentioned in the present invention, of one or several plant products, preferably of the root, of Polygonum cuspidatum, is that indicated in the first, second, and third aspects of the invention for said extract.
• the extract of the skin of the fruit of Prunus dulcis contains between 5-60% w/w, preferably 30% w/w of flavonoids, wherein the flavonoids are preferably selected from the group consisting of procyanidins, propelargonidins, prodelphinidins, catechin, epicatechin, quercetin, kaempferol, isorhamnetin, rutin, genistein, and combinations thereof.
• the extract of the fruit of Citrus paradisi or the extract of the seed of the fruit of Citrus paradisi contains 10-80% w/w, preferably 45% (w/w) of flavonoids, wherein the flavonoids are preferably selected from the group consisting of naringin, naringenin, quercetin, catechin, rutin, genistein, and combinations thereof.
• the extract of the seed of the fruit of Linum usitatissimum containing between 5-50% w/w, preferably 20% (w/w) of lignans, wherein the lignans are preferably selected from the group consisting of secoisolariciresinol diglucoside, secoisolariciresinol, matairesinoside, lariciresinol, pinoresinol, and sesamin.
• the extract of the root of Polygonum cuspidatum contains between 50-100% w/w, preferably 98% (w/w) of stilbenes, wherein the stilbenes are preferably selected from the group consisting of resveratrol, piceatannol, pinosylvin, pterostilbene, rhapontienin, and combinations thereof, preferably is resveratrol, more preferably resveratrol in combination with at least one of the stilbenes indicated previously.
• the definitions and particular embodiments of the first, second, third, and fourth aspects of the invention are applied to the fifth aspect of the invention.
• the invention in a sixth aspect, relates to a pharmaceutical composition
• a pharmaceutical composition comprising the composition of the fourth aspect of the invention, or a composition obtained by the method of the fifth aspect of the invention and a pharmaceutically acceptable excipient.
• pharmaceutical composition refers to any physiologically tolerable composition that typically does not produce an allergic reaction or a similar unfavorable reaction such as gastric disorders, dizziness, or the like, when administered to a human or animal.
• composition comprises at least one pharmaceutically active ingredient and one or more pharmaceutically acceptable supports.
• pharmaceutically acceptable support refers to a non toxic filler, diluent, auxiliary or encapsulating material of a solid, semisolid, or liquid formulation of any conventional type.
• a pharmaceutically acceptable support is essentially non-toxic for receptors at the doses and concentrations used and is compatible with other ingredients of the formulation. Suitable supports include, but are not limited to, water, dextrose, glycerol, saline solution, ethanol, and combinations thereof.
• the support may contain additional agents such as wettings or emulsifying agents, pH regulating agents, or adjuvants which increase the efficacy of the formulation.
• the adjuvants could be selected from the group consisting of sterile liquids, such as water and oils, including those from petroleum, animal origin, plant origin, or synthetic origin, such as peanut oil, soybean oil, vaseline oil, sesame seed oil, and the like.
• Water or aqueous saline solutions and aqueous dextrose and glycerol solutions, particularly for injectable solutions, are preferably used as vehicles. Suitable pharmaceutical vehicles are described in “Remington’s Pharmaceutical Sciences” by E.W. Martin, 21 st Edition, 2005.
• compositions of said aspects may comprise.
• the pharmaceutical composition comprises a plant extract of any aspect of the invention in a therapeutically effective amount.
• the term “effective amount” is synonymous to “therapeutically effective amount”, “effective dose”, or “therapeutically effective dose”, and when used in the present invention it refers to the minimum dose of the plant extract of any aspect of the invention needed to achieve the desired therapeutic effect and includes a sufficient dose to reduce at least one cardiovascular disease symptom, preferably atherosclerosis.
• the efficacy in treating the diseases or conditions described herein can be determined observing a drop in serum oxLDL levels and oxLDL/LDL ratio.
• the efficacy in treating the diseases or conditions described herein consists in the description provided in any of the particular embodiments indicated in the first, second, or third aspect of the invention for the expression “increasing the antiatherosclerotic effect of hydroxytyrosol”.
• the corresponding reference value can be the indicated in said embodiments or the value of the corresponding parameter in a serum sample from a subject not receiving any type of treatment, or from the same subject to whom the pharmaceutical composition is administered at least 2 weeks before said administration or at least 4 weeks before administration. Therefore, methods for measuring the efficacy in treating the diseases or conditions described herein are also the described in said embodiments of the first, second, or third aspect of the invention.
• a “unit dose” refers to the amount of inventive composition or kit of parts required to produce a response of the 50 percent of the maximum effect (i.e., ED50).
• the unit dose can be evaluated by the extrapolation of dosage and response curves derived from system for testing in vitro or in animal models.
• the amount of compounds in the compositions of the present invention that will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition and may be determined by standard clinical techniques (see, for example, Goodman and Gilman’s The Pharmacological Basis of Therapeutics, Joel G. Harman, Lee E.
• an effective amount of said compounds will further depend on factors including, without limitation, the administration frequency, the half-life of the compound, or any combination thereof.
• compositions of the present invention are large enough to produce the desired therapeutic effect.
• the compositions according to the present invention are administered once or twice a day on a regular basis.
• a typical dose administered to a human is between about 20 mg and about 5 g of the composition, preferably between 200 mg and 1 g of the composition.
• compositions provided herein can be administered to a subject by a number of suitable methods known in the art.
• suitable methods include: (1) intramuscular, intradermal, intraepidermal, or subcutaneous administration, (2) oral administration, and (3) topical application (such as ocular, intranasal, and intravaginal application).
• topical application such as ocular, intranasal, and intravaginal application.
• the compositions are formulated for oral administration.
• the preferred administration route of the compositions provided herein is oral.
• the composition for oral use has the format, form, or formulation, described for the composition of the invention in the fourth aspect of the invention.
• the definitions and particular embodiments of the first, second, third, fourth, and fifth aspects of the invention are applied to the sixth aspect of the invention.
• the invention relates to a food or nutritional supplement comprising the composition of the fourth aspect of the invention, or a composition obtained by the method of the fifth aspect of the invention, and a nutritionally acceptable excipient.
• the term “food supplement” or “nutritional supplement” refers to concentrated sources of nutrients or other substances obtained from edible products, the purpose of which fin is to supplement the normal diet.
• the food supplement or nutritional supplement according to the present invention includes compositions of functional foods, i.e., food, beverage, pet chow or feed, or a food supplement, beverage, pet chow or feed, nutritional supplements, fragrances or flavoring agents, enological or cosmetic formulations.
• the terms “food supplement” or “nutritional supplement” may also mean:
• a product intended to supplement the diet which has or contains one or more of the following nutritional ingredients: [A] a vitamin, [B] a mineral, [C] a herb or other botanical element, [D] an amino acid, [E] a nutritional substance for use by man to supplement the diet by increasing the total nutritional intake; or (F) a concentrate, metabolite, constituent, extract, or combination of any ingredient described in clause (A), (B), (C), (D), or (E); or
• the “food supplement” or “nutritional supplement” according to the present invention is usually administered orally and is provided together with the diet of a subject. It may be in very different forms, including tablets, capsules, liquid suspensions, dry powder, wet composition, a dry tube feeding or wet tube feeding.
• a nutritional formulation for example, medical food, for example, in the form of tube feeding, or in oral nutritional form as a complete meal, as part of a meal, as a food additive, as a powder for dissolution, for example, health drinks, as a solution, as a prepared beverage, including juices, shakes, yogurt drink, smoothie, or soy beverage, in a bar, or dispersed in foods of any type, such as baked goods, cereal bars, dairy bars, fast foods, soups, breakfast cereals, muesli, candies, cookies, cakes.
• medical food for example, in the form of tube feeding, or in oral nutritional form as a complete meal, as part of a meal, as a food additive, as a powder for dissolution, for example, health drinks, as a solution, as a prepared beverage, including juices, shakes, yogurt drink, smoothie, or soy beverage, in a bar, or dispersed in foods of any type, such as baked goods, cereal bars, dairy bars, fast foods, soups, breakfast cereals, mues
• the term “nutritionally acceptable excipient” or “nutritionally acceptable support” refers to a support, as it has been defined above, that is edible and relates to preparing solutions to be administered orally.
• the typical nutritionally acceptable supports, diluents, and excipients will be known to one skilled in the art. Non limiting examples of such supports are provided in US patents 6,258,846, 6,576,666, and 7,112,609.
• nutraceutical compositions for humans or animals
• compositions of functional foods i.e., food, beverage, pet chow or feed, or a food supplement, beverage, pet chow or feed
• nutritional supplements i.e., nutritional, beverage, pet chow or feed
• fragrances i.e., a food supplement, beverage, pet chow or feed
• drugs pharmaceutical compositions or formulations
• veterinary compositions enological or cosmetic formulations
• the amount of the composition present in the food supplement or nutritional supplement will be from about 0.001 to about 50 percent by weight of the nutraceutical compositions, nutritional or food product, nutritional supplement, fragrance or flavoring agent, enological formulation, such as from about 0.01 to about 10 percent, or from about 0.1 to 1 percent.
• the definitions and particular embodiments of the first, second, third, fourth, fifth, and sixth aspects of the invention are applied to the seventh aspect of the invention.
• the invention relates to the composition or kit of parts of the fourth aspect of the invention, the composition obtained by the method of the fifth aspect of the invention, the pharmaceutical composition of the sixth aspect of the invention, or the food or nutritional supplement of the seventh aspect of the invention, for use in medicine.
• the invention relates to the composition or kit of parts of the fourth aspect of the invention, the composition obtained by the method of the fifth aspect of the invention, the pharmaceutical composition of the sixth aspect of the invention, or the food or nutritional supplement of the seventh aspect of the invention, for use in the treatment of a cardiovascular disease.
• CVD cardiovascular disease
• CVD includes coronary artery diseases (CAD) such as angina and myocardial infarction.
• CAD coronary artery diseases
• Other CVDs include atherosclerosis, apoplexy, heart failure, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, abnormal heart rhythms, congenital heart disease, valvular heart disease, carditis, aortic aneurysms, peripheral artery disease, thromboembolic disease, and venous thrombosis.
• Peripheral vascular disease, cerebrovascular accident, or coronary artery disease are related to atherosclerosis and commonly entail the existence of atherosclerosis.
• the cardiovascular disease of the ninth aspect of the invention is selected the group consisting of atherosclerosis, cerebrovascular accident, peripheral vascular disease, and coronary disease.
• anterior atherosclerosis has been defined in the first, second, or third aspect of the invention.
• peripheral vascular disease refers to the obstruction of large arteries that are not part of the coronary, aortic arch, or cerebral vasculature. It causes an acute or chronic ischemia (lack of blood supply). EVP can be a consequence of atherosclerosis, inflammatory processes which result in stenosis, an embolism, or the formation of thrombi. Generally, the term EVP is used to refer to atherosclerotic blockages in a lower limb.
• the peripheral vascular disease is due to the existence of atherosclerosis.
• the expression “cerebrovascular accident”, “cerebral attack”, “stroke”, or “cerebral infarction” refers to the situation in which the blood flow to a part of the brain is stopped, causing cell death.
• the expression “ischemic stroke” or “ischemic cardiovascular accident” refers to the situation in which the cerebral structure loses its blood supply due to the sudden and immediate interruption of blood flow due to the occlusion of any of the arteries which supply the encephalic mass.
• Said occlusion may be due to a buildup of fibrin or calcium or to an erythrocyte abnormality, but it is generally due to atherosclerosis, or due to an embolus (cerebral embolism) that comes from another location, fundamentally the heart or other arteries (such as the carotid or aortic arch bifurcation).
• embolus Cerebral embolism
• the expression “hemorrhagic stroke” or “hemorrhagic cerebral accident” refers to the situation in which a brain blood vessel ruptures, thereby depriving the area of the brain dependent of this blood vessel, generally an artery, of blood supply. The extravasated blood applies compression on cerebral structures, including other blood vessels, thereby amplifying the area of the brain affected.
• the cerebrovascular accident is ischemic, preferably due to the existence of atherosclerosis.
• coronary disease or “coronary artery disease” refers to the condition in which there is an imbalance between the blood flow of the coronary arteries or coronary flow and the oxygen requirement of the heart muscle or myocardium.
• This imbalance causes ischemia the effects of which are metabolic (increase in lactic acid, acidosis, decrease in ATP, decrease in phosphocreatines), mechanical (decrease in cardiac contractility, decrease in distensibility of the ischemic area, and others) and electrical (modification of rest and action potentials, electric instability and the subsequent rhythm disorders).
• the main cause of coronary disease is the narrowing of the coronary arteries supplying blood to the heart due to atherosclerosis.
• the coronary disease is due to the existence of atherosclerosis.
• compositions, components of the kit of parts, pharmaceutical compositions, food or nutritional supplements of the invention can be administered at variable doses (i.e., therapeutically effective doses, administered to a patient in need of same).
• dose administered to a mammal, particularly a human in the context of the present invention must be sufficient so as to affect a therapeutic response in the mammal during a reasonable space of time.
• said therapeutic effect corresponds with the efficacy in treating the diseases or conditions described herein specified in the sixth aspect of the present invention.
• the selection of the dose and exact composition and the most suitable dosage regimen will also be influenced by, among others, the pharmacological properties of the formulation, the nature and severity of the condition being treated, and the physical condition of the receptor, as well as the age, condition, body weight, sex, and response of the patient to be treated, and the stage/severity of the disease.
• the term “subject” refers to a mammal, preferably a human.
• the subject to be treated is a mammal, preferably a human.
• the subject to be treated according to the invention can be selected based on several criteria associated with cardiovascular diseases, such as serum oxLDL and/or LDL levels, or the corresponding oxLDL/LDL- cholesterol ratio.
• the subject presents hypercholesterolemia, preferably moderate hypercholesterolemia. In another particular embodiment, the subject presents acute hypercholesterolemia.
• the composition, the kit of parts, the pharmaceutical composition, or the food or nutritional supplement for use of the eighth or ninth aspect of the invention is administered to a patient with hypercholesterolemia, preferably moderate hypercholesterolemia.
• the term “hypercholesterolemia” refers to the condition characterized by high blood cholesterol levels. Given that cholesterol is insoluble in water, it is transported in blood plasma together with proteins, forming lipoproteins. Lipoproteins are classified by their density: very low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL); low-density lipoproteins (LDL), and high-density lipoproteins (HDL).
• VLDL very low-density lipoproteins
• IDL intermediate-density lipoproteins
• LDL low-density lipoproteins
• HDL high-density lipoproteins
• LDL-cholesterol LDL transporting cholesterol
• cholesterol levels are considered to be high when the concentration of total blood cholesterol is greater than 200 mg/dl, and/or the concentration of LDL-cholesterol is greater than 100 mg/dl.
• the expression “moderate hypercholesterolemia” refers to hypercholesterolemia characterized by a concentration of total blood cholesterol greater than 200 mg/dl and less than 250 mg/dl, and a concentration of LDL-cholesterol greater than 100 mg/dl and less than 175 mg/dl.
• the expression “acute hypercholesterolemia” refers to hypercholesterolemia characterized by a concentration of total blood cholesterol greater than 250 mg/dl, and a concentration of LDL-cholesterol greater than 175 mg/dl.
• compositions, components of the kit of parts, the pharmaceutical composition, or the food or nutritional supplement of the invention can be administered as such, the invention also contemplates the possibility of at least one of the components of the composition, kit of parts, pharmaceutical composition, or food or nutritional supplement to be administered separately from the rest of the components of the composition, of the kit of parts, of the pharmaceutical product, or of the food or nutritional supplement.
• the hydroxytyrosol and the polyphenols other than hydroxytyrosol are administered separately.
• the hydroxytyrosol is administered separately from the at least one polyphenol other than hydroxytyrosol or from the extract comprising the at least one polyphenol other than hydroxytyrosol.
• the hydroxytyrosol is administered separately from the at least one polyphenol other than hydroxytyrosol or from the extract comprising the at least one polyphenol other than hydroxytyrosol and from the rest of the components of the composition, kit of parts, pharmaceutical composition, or food or nutritional supplement of the invention.
• the plant extract comprising hydroxytyrosol is administered separately from the at least one polyphenol other than hydroxytyrosol or from the extract comprising the at least one polyphenol other than hydroxytyrosol.
• the plant extract comprising hydroxytyrosol is administered separately from the at least one polyphenol other than hydroxytyrosol or from the extract comprising the at least one polyphenol other than hydroxytyrosol and from the rest of the components of the composition, kit of parts, pharmaceutical composition, or food or nutritional supplement of the invention.
• the at least one polyphenol other than hydroxytyrosol is administered separately from the hydroxytyrosol or from the extract comprising the hydroxytyrosol. In another particular embodiment, the at least one polyphenol other than hydroxytyrosol is administered separately from the hydroxytyrosol or from the extract comprising the hydroxytyrosol and from the rest of the components of the composition, kit of parts, pharmaceutical composition, or food or nutritional supplement of the invention.
• the plant extract comprising the at least one polyphenol other than hydroxytyrosol is administered separately from the hydroxytyrosol or from the extract comprising the hydroxytyrosol. In another particular embodiment, the plant extract comprising the at least one polyphenol other than hydroxytyrosol is administered separately from the hydroxytyrosol or from the extract comprising the hydroxytyrosol and from the rest of the components of the composition, kit of parts, pharmaceutical composition, or food or nutritional supplement of the invention.
• the different parts of the kit for use of the eighth and ninth aspects of the invention can be administered separately or can alternatively be combined before the administration.
• the components of the kit of parts are combined before the administration.
• the administration of the composition, the components of the kit of parts, the pharmaceutical product, or the food or nutritional supplement comprises the administration of multiple doses of the composition, of the components of the kit of parts, of the pharmaceutical product, or of the food or nutritional supplement of the fourth aspect of the invention, for at least 1 day, at least 2, days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 1.5 weeks, at least 2 weeks, at least 2.5 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, preferably for at least 1 week, more preferably at least 2 weeks, even more preferably for at least 4 weeks or for at least 8 weeks.
• the administration of the composition, the components of the kit of parts, the pharmaceutical product, or the food or nutritional supplement comprises the administration of one, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, preferably 2 daily doses of the composition, of the components of the kit of parts, of the pharmaceutical product, or of the food or nutritional supplement of the invention.
• the initial indications of the suitable therapeutic dose of the compositions of the invention can be determined in vitro and in animal model systems in vivo , and in clinical trials in humans.
• One skilled in the art would know how to use animal studies and human experience to identify a dose that can be administered safely without generating toxicity or other side effects.
• the therapeutic dose is preferably close to the maximum tolerated dose.
• lower doses may be desirable due to concerns for long-term effects.
• the administration of the composition, of the components comprised in the kit of parts, of the pharmaceutical composition, or of the food or nutritional supplement comprises administering 1 mg/day, 2 mg/day, 3 mg/day, 4 mg/day, 5 mg/day, 6 mg/day, 7 mg/day, 7.5 mg/day, 8 mg/day, 8.5 mg/day, 9 mg/day, 9.5 mg/day, 10 mg/day, 10.5 mg/day, 11 mg/day, 11.5 mg/day, 12 mg/day, 13 mg/day, 14 mg/day, 15 mg/day, 17 mg/day, 20 mg/day, 22 mg/day, 25 mg/day, 30 mg/day, 35 mg/day, 40 mg/day, 45 mg/day, 50 mg/day, 60 mg/day, 70 mg/day, 80 mg/day, 90 mg/Day, 100 mg/day, preferably 7.5 mg/day of hydroxytyrosol.
• the administration of the composition, of the components comprised in the kit of parts, of the pharmaceutical composition, or of the food or nutritional supplement comprises administering 20 mg/day, 30 mg/day, 40 mg/day, 50 mg/day, 60 mg/day, 70 mg/day, 80 mg/day, 90 mg/day, 100 mg/day, 125 mg/day, 150 mg/day, 175 mg/day, 190 mg/day, 200 mg/day, 205 mg/day, 210 mg/day, 215 mg/day, 220 mg/day, 230 mg/day, 240 mg/day, 250 mg/day, 275 mg/day, 300 mg/day, 325 mg/day, 350 mg/day, 375 mg/day, 400 mg/day, preferably 210 mg/day of polyphenols other than hydroxytyrosol.
• the administration of the composition, of the components comprised in the kit of parts, of the pharmaceutical composition, or of the food or nutritional supplement comprises administering 1 mg/day, 2 mg/day, 3 mg/day, 4 mg/day, 5 mg/day, 6 mg/day, 7 mg/day, 7.5 mg/day, 8 mg/day, 8.5 mg/day, 9 mg/day, 9.5 mg/day, 10 mg/day, 10.5 mg/day, 11 mg/day, 11.5 mg/day, 12 mg/day, 13 mg/day, 14 mg/day, 15 mg/day, 17 mg/day, 20 mg/day, 22 mg/day, 25 mg/day, 30 mg/day, 35 mg/day, 40 mg/day, 45 mg/day, 50 mg/day, 60 mg/day, 70 mg/day, 80 mg/day, 90 mg/day, 100 mg/day, preferably 7.5 mg/day of hydroxytyrosol and any of the doses in mg/day of polyphenols other than hydroxyt
• the administration of the composition, of the components comprised in the kit of parts, of the pharmaceutical product, or of the food or nutritional supplement of the invention comprises administering 7.5 mg/day of hydroxytyrosol and 210 mg/day of polyphenols other than hydroxytyrosol.
• the administration of the composition, of the components comprised in the kit of parts, of the pharmaceutical composition, or of the food or nutritional supplement comprises administering 10 mg/day, 15 mg/day, 20 mg/day, 25 mg/day, 30 mg/day, 35 mg/day, 40 mg/day, 45 mg/day, 50 mg/day, 55 mg/day, 57 mg/day, 60 mg/day, 61 mg/day, 62 mg/day, 63 mg/day, 64 mg/day, 65 mg/day, 66 mg/day, 67 mg/day, 68 mg/day, 69 mg/day, 70 mg/day, 72 mg/day, 75 mg/day, 77 mg/day, 80 mg/day, 82 mg/day, 85 mg/day, 87 mg/day, 90 mg/day, 95 mg/day, 100 mg/day, 110 mg/day, 120 mg/day, 150 mg/day, 170 mg/day, 200 mg/day, 225 mg/day, 250 mg/day,
• the administration of the composition, of the components comprised in the kit of parts, of the pharmaceutical composition, or of the food or nutritional supplement comprises administering 50 mg/day, 70 mg/day, 100 mg/day, 125 mg/day, 150 mg/day, 175 mg/day, 200 mg/day, 225 mg/day, 250 mg/day, 275 mg/day, 300 mg/day, 325 mg/day, 350 mg/day, 375 mg/day, 400 mg/day, 425 mg/day, 450 mg/day, 475 mg/day, 500 mg/day, 525 mg/day, 550 mg/day, 575 mg/day, 600 mg/day, 625 mg/day, 650 mg/day, 675 mg/day, 680 mg/day, 690 mg/day, 700 mg/day, 710 mg/day, 720 mg/day, 730 mg/day, 740 mg/day, 750 mg/day, 775 mg/day, 800 mg/day, 825 mg/day,
• the administration of the composition, of the components comprised in the kit of parts, of the pharmaceutical composition, or of the food or nutritional supplement comprises administering 10 mg/day, 15 mg/day, 20 mg/day, 25 mg/day, 30 mg/day, 35 mg/day, 40 mg/day, 45 mg/day, 50 mg/day, 55 mg/day, 57 mg/day, 60 mg/day, 61 mg/day, 62 mg/day, 63 mg/day, 64 mg/day, 65 mg/day, 66 mg/day, 67 mg/day, 68 mg/day, 69 mg/day, 70 mg/day, 72 mg/day, 75 mg/day, 77 mg/day, 80 mg/day, 82 mg/day, 85 mg/day, 87 mg/day, 90 mg/day, 95 mg/day, 100 mg/day, 110 mg/day, 120 mg/day, 150 mg/day, 170 mg/day, 200 mg/day, 225 mg/day, 250 mg/day,
• the administration of the composition, of the components comprised in the kit of parts, of the pharmaceutical composition, or of the food or nutritional supplement comprises administering 67 mg/day of plant extract providing hydroxytyrosol as defined in the first to fourth aspects of the invention and 700 mg/day of plant extract providing polyphenols in the first to fourth aspects of the invention.
• the definitions and particular embodiments of the first, second, third, fourth, fifth, sixth, and seventh aspect of the invention are applied to the eighth and ninth aspects of the invention.
• the term consists of, essentially consists of, and comprises are interchangeable in any embodiment of any aspect of the present invention.
• Each capsule with extract contained 3.75 mg of HT in 33.5 mg of an extract of olive tree ( Olea europaea L.), and 105 mg of ASP in 350 mg of an extract of almond skin ( Prunus dulcis (Mill.)) and 16.5 mg of maltodextrin.
• Each capsule with placebo contained 400 mg of maltodextrin.
• Both extracts were obtained in the Biosearch, S.A. production plant in Talayuela, Caceres (Spain) and the combination of the extracts was performed at the Biosearch, S.A. R&D facilities in Granada (Spain).
• the capsules were prepared in the Pharmaceutical Technology Department of the Faculty of Pharmacy of the University of Granada (Spain).
• the extracts and placebo were supplied in identical gelatin capsules, packaged in identical plastic containers, with a code number that referred to the code of the volunteer according to randomization.
• the olive tree extract can be obtained by means of methods based on the extraction of leaves of O. europaea L. with an aqueous solvent, such as water for example.
• the method includes the following steps:
• the extract of Prunus dulcis Mill can be obtained following the same method as that used for obtaining the extract of O. europaea L., wherein the starting material is the skin of the fruit instead of leaves of O. europaea.
• step (i) may include several extraction phases for extracting the plant product with the initial solvent or a different solvent, combining the extracts obtained to continue the process.
• the extract may also be concentrated by means of chromatography using polymeric adsorbent resins.
• the method can also include a final drying step for drying the extract obtained in the preceding steps of the method.
• the primary endpoint of the study was the determination of oxLDL levels.
• the secondary parameters included the oxLDL/LDL-c ratio, total cholesterol (TC), LDL cholesterol (LDL- c), HDL cholesterol (HDL-c) levels and serum triglyceride (TG) levels.
• the subjects went to the Biosearch, S.A. facilities in Granada at the start of the study and at 4 and 8 weeks. Blood samples were collected after an overnight fast that lasted at least 10 hours, before the start of the intervention and in each follow-up visit, using the Vacutainer® SSTTM II Advance Tubes (BD,NJ, USA) system containing a thixotropic gel. The serum was obtained after centrifugation of the blood samples at 1000 g for 15 min and stored at -80°C.
• the weight was determined using the Tanita BC-418 body composition analyzer (Tanita, Tokyo, Japan).
• the height was determined using a height measuring apparatus with a precision of 1 mm (range, 80-200 cm).
• the body mass index (BMI) was calculated as weight/height squared (kg/m2). All the measurements were performed by qualified personnel.
• the food intake of each subject was evaluated at the start and at the end of the study to control possible changes in their habits.
• the subjects completed a report detailing 72 hours of their dietary intake, specifying the types of foods consumed and the weights of the portions.
• the daily food, the intake of energy, the intake of nutrients, and the energy provided by the macronutrients were calculated using the Nutriber computer application (Funiber, Barcelona, Spain). Subjects were also asked about the physical activity habits (type of activity and time they practice it per week) at the start and at the end of the study.
• the levels of the lipid parameters were analyzed by an external laboratory (Reference Laboratory S.A, Barcelona, Spain) by means of standard methods.
• the determination of serum oxLDL concentration was performed by means of ELISA specific for human oxLDL (Elabscience Biotechnology, USA); and the oxLDL/LDL-c ratio was calculated.
• the grapefruit, flax, and polygonum extracts can be obtained by applying the same method for obtaining the olive tree extract indicated in section A- on materials and methods, wherein:
• the starting material for the grapefruit extract is the seed of the fruit of the tree Citrus paradisi MacFad
• the starting material for the flax extract is the seed of the fruit of Linum usitatissimum L.
• the starting material for the polygonum extract is the root of Polygonum cuspidatum Sieb. etZucc.
• the solvent used in the methods for obtaining these extracts is a hydroalcohol, wherein the alcohol can be ethanol or methanol, for example.
• Example 1 Effect of the combination of extracts of standard olive tree ( Olea europaea L.) and skin of a standard almond ( Prunus dulcis Mill.) on serum oxLDL levels
• Table 6 shows the intake of energy and nutrients at the baseline and at week 8. There were no significant differences at the baseline between intervention groups, or between the baseline and the end of the intervention in each (extract or control) group. Nor were there any changes during the study in the degree of physical activity (data not shown) and the subjects in both groups did not significantly change their BMI between the start and week 8 (Table 6).
• Lipid and oxidized LDL parameters of the subjects who took the placebo or the extract, at the start, at 4 weeks, and at 8 weeks of treatment Mean ⁇ SD adjusted for sex, age, obesity/overweight, smoker, and physical activity. P indicates differences between groups at each time. * P ⁇ 0.05 between start, 4 weeks and 8 weeks
• Example 2 Effect of mixtures of olive tree, almond, grapefruit, flax, and polygonum extract on the bioavailability of hydroxytyrosol of the olive tree extract.
• digestion assays were performed on a small scale on the extracts separately and in different combinations.
• 50 mg/mL olive tree extract and 1:1, 1:4, and 1:9 mixtures with almond skin extract were used. Based on the ratio of polyphenols of both extracts in each of the mixtures, the mixtures with the 3 other assayed extracts were defined (Table 9).
• HPLC high-performance liquid chromatography
• the polygonum extract is characterized by a high concentration of the stilbene resveratrol, with an approximate RT of 43 minutes, without the presence of compounds with an RT of 10-11 min (characteristic of hydroxytyrosol) and without this type of compounds being detected significantly in the present chromatograms in the olive tree extract ( Figure 6).

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