ES2749717A1 - Composition and its uses (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Compositions and their uses. The present invention belongs to the field of compositions of a dietary nature, specifically, it refers to a composition comprising at least one milk protein hydrolyzate and an extract of Ginkgo biloba. Likewise, it refers to different uses of said composition, among others, to improve endothelial function. (Machine-translation by Google Translate, not legally binding)

Description

DESCRIPTION

Composition and its uses

Field of the Invention

The present invention belongs to the field of compositions of a dietary nature, specifically refers to a composition comprising at least one milk protein hydrolyzate and an extract of Ginkgo biloba, and its use, in particular to improve endothelial function.

Background of the Invention

In recent years, food supplements and functional foods have gained great interest in pharmacies and in the food sector. Due to the high incidence of diseases related to vascular dysfunction in developed countries, those products that improve endothelial health are of increasing interest.

When the vascular endothelium works normally, it responds, among others, to different signals and changes (chemical, hormonal, hemodynamic, friction force with the blood, etc.), and helps regulate blood clotting, helps the immune response of the body, controls the volume of fluid and the amount of electrolytes and other substances that pass from the blood to the tissues, causing dilation or constriction of the blood vessels. When there is endothelial dysfunction, however, the ability to perform one or more of these functions is reduced. One of the main causes suggested for endothelial dysfunction is the reduced bioavailability of nitric oxide (NO ). NO is a small molecule with a short life and a gaseous nature resulting from the combination of an oxygen atom and a nitrogen atom. From a chemical point of view it is a molecule that has an unpaired electron, making it highly reactive and unstable. NO is synthesized by enzymatic conversion of L-arginine in the presence of molecular oxygen carried out by nitric oxide synthase (NOS). NO participates in the regulation of vascular tone, in non-specific immune defense and also acts as a neurotransmitter.

There are 3 isoenzymes of NOS that present different location and regulation, endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS). The eNOS isoforms and nNOS are activated by binding calmodulin to the enzyme, a process that depends on the concentration of intracellular Ca + 2. ENOS is also regulated by its phosphorylation in response to pulsatile flow and mechanical friction stress between blood and endothelium ( shear stress).

Unlike most molecules that transmit information between cells, NO is not stored. It is synthesized in response to a stimulus and thanks to its physicochemical properties it spreads rapidly through biological membranes, after which it reacts with various intracellular targets. But the action of NO does not depend so much on the target on which it acts as on its concentration in the intracellular environment in which it acts. This process is hindered in the endothelium by the presence of ROS (reactive oxygen species), among which the superoxide anion that reacts with NO, producing toxic compounds, and reduces the intracellular concentration of Ca + 2, making it difficult to activate eNOS.

NO of endothelial origin behaves like a first messenger that, when it diffuses in the membranes of vascular smooth muscle cells, activates the soluble guanylate cyclase enzyme, with the corresponding increase in cyclic guanosine monophosphate (cGMP), which acts as a second messenger. CGMP is a smooth muscle relaxing factor of great importance in different processes of vasodilation and flexibilization of arteries and arterioles.

There are many disorders characterized by or related to endothelial dysfunction that, in addition to being an early marker of systemic vascular damage, often appear as a consequence of concomitant diseases that can precede and even predict them. Thus, for example, they are related to endothelial dysfunction, atherosclerosis, cardiovascular disease, hypertension, hypercholesterolemia, hyperglycemia, stroke, stroke, myocardial infarction, peripheral vascular disease, angina pectoris, heart failure, diastolic and / or systolic ventricular dysfunction, macro and microangiopathy in patients with diabetes, tissue injury related to ischemia and reperfusion, sexual dysfunction, etc. In addition, vascular endothelial dysfunction is a precedent for cerebral hypoperfusion related to various neurodegenerative diseases, among which is Alzheimer's disease.

A well-studied case is one that associates endothelial dysfunction as a cause of organic erectile dysfunction (ED) in men and organic female sexual dysfunction (DSF). in women. In recent decades, a multitude of studies and compositions have been described for the treatment of ED. Regarding natural remedies for sexual dysfunction, particularly ED, different compositions have been described comprising plant extracts, for example, ginseng, ginger, guarana or maca (see, for example, WO2008 / 089815, WO2012 / 067745). However, there is much controversy as to whether or not these natural products actually have an effect on improving sexual function and whether they have more effect than placebo.

On the other hand, pharmaceutical products have been developed that try to maintain for longer the effects achieved by the cGMP molecules generated by ON. For this, the enzyme phosphodiesterase 5 (PDE5) is temporarily inhibited, an enzyme that is responsible for the degradation of cGMP. Among them, sildenafil citrate was the first effective drug after oral administration for the treatment of ED, achieving its effect between 25 and 60 minutes after oral administration. However, this product has significant side effects. The main ones are headache, dyspepsia, rhinitis and visual disturbances, all of them related to the inhibition of phosphodiesterase at other levels. Furthermore, sildenafil is contraindicated in patients receiving nitrovasodilators and should be taken with caution by people with various cardiovascular disorders (coronary ischemia, hypertension, hypotension) and with active peptic ulcer. New derivatives with relative selectivity for phosphodiesterease 5 are taladafil (Cialis®) and vardenafil (Levitra®), whose action lasts for longer hours because their half-lives are longer, allowing less dependence on the moment of ingestion. However, the side effects are similar to those of sildenafil.

Thus, there remains a need to develop compositions that effectively improve endothelial function, and are therefore useful for the treatment of disorders related to endothelial dysfunction, and that have fewer side effects than known treatments or no side effects. Surprisingly, the authors of the present invention have developed a synergistic composition, derived from natural food products, capable of improving endothelial function, thus serving, among others, to improve physical performance, treat ED and DSF and act as an adjuvant to pathologies characterized by vascular endothelial dysfunction.

Object of the invention

In a first aspect, the present invention relates to a composition comprising: a) a milk casein hydrolyzate characterized in that it increases eNOS expression and / or activity in vivo and / or increases plasma arginine levels in vivo and / or has antioxidant activity;

b) an extract of Ginkgo biloba, and optionally

c) a hydrolyzate of milk whey proteins characterized in that it has inhibitory activity of the enzyme DDP-IV in vitro and / or has antioxidant activity.

In a second aspect, the present invention relates to a medicine, a food supplement, a drink or a food product comprising a composition according to the first aspect of the invention.

In a third aspect, the present invention relates to the use of a composition according to the first aspect of the invention for the preparation of a medicine, a food supplement, a drink or a food product for the treatment of a disorder related to endothelial dysfunction . It also refers to the use of the composition according to the first aspect of the invention to improve physical, sports and / or sexual performance.

In a fourth aspect, the present invention relates to a kit comprising:

a) a first container with a milk casein hydrolyzate as defined in the first aspect of the invention,

b) a second container with an extract of Ginkgo biloba, and

c) optionally a third container with a whey protein hydrolyzate as defined in the first aspect of the invention.

In a fifth aspect, the present invention relates to a method of preparing the composition of the first aspect of the invention.

Other objects, features, advantages and aspects of the present application will be apparent to the person skilled in the art from the description and the attached claims.

Brief description of the figures

Figure 1.- Chromatogram obtained after RP-HPLC-MS / MS analysis of casein hydrolyzate subjected to a selective precipitation process of caseinphosphopeptides.

Detailed description of the invention

As used in the present application, the singular forms "one / one", "one" and "the" include their corresponding plurals, unless the context clearly indicates otherwise. Unless otherwise defined, all technical terms used herein have the meaning that one skilled in the art to which this invention pertains usually understands.

The present invention relates in a first aspect to a composition (hereinafter referred to as the composition of the invention) comprising:

a) a milk casein hydrolyzate characterized in that it increases eNOS expression and / or activity in vivo, and / or increases plasma arginine levels in vivo, and / or has antioxidant activity (hereinafter referred to as HC or hydrolyzed HC);

b) an extract of Ginkgo biloba (hereinafter referred to as GB or GB extract); and optionally

c) a hydrolyzate of whey proteins of milk characterized in that it has inhibitory activity of the enzyme DDP-IV in vitro and / or has antioxidant activity (hereinafter referred to as HPS or hydrolyzed HPS).

As seen in Examples 10 and 11, the HC + GB composition is effective in the treatment of disorders related to endothelial dysfunction, such as ED and DSF. Thus, in a particular embodiment, the composition of the invention comprises a) and b). However, the HC + HPS + GB composition is even more effective in such treatment. Thus, in a preferred embodiment, the composition of the first aspect of the invention comprises a), b) and c).

Milk casein hydrolyzate increases eNOS expression in vivo, as shown in Example 5. This, in view of the effects seen in human trials (Examples 10 and 11), corroborates an increase in activity eNOS in vivo and cGMP formation. Thus, in a particular embodiment, the HC is characterized in that it increases the expression and / or the activity of eNOS in vivo.

Likewise, HC increases plasma arginine level in vivo, as shown in Example 6. Thus, in a particular embodiment according to any one of the previous embodiments, HC is characterized in that it increases plasma arginine levels. in vivo.

Furthermore, as shown in Example 9, HC and HPS have activity antioxidant. Thus, in a particular embodiment according to any one of the previous embodiments, the HC is characterized in that it has antioxidant activity, preferably antioxidant activity in vitro and / or in vivo. In another particular embodiment according to any one of the previous embodiments, the HPS is characterized in that it has antioxidant activity, preferably antioxidant activity in vitro and / or in vivo.

In a preferred embodiment according to any one of the previous embodiments, e1 HC is characterized in that it increases the expression and / or activity of eNOS in vivo, and increases plasma arginine levels in vivo, and has antioxidant activity. In this way, without wishing to be bound by any theory, the HC and the composition of the present invention can provide both the enzyme and the substrate necessary for the synthesis of NO, and correlatively the formation of cGMP facilitated by antioxidant activity. In fact, it is expected that the levels of NO and correlatively cGMP in plasma increase due to the improvement in ED shown in Examples 10 and 11. In view of the synergistic effect regarding antioxidant activity (see Example 9), in A more preferred embodiment, e1HPS is characterized in that it has DDP-IV enzyme inhibitory activity in vitro and has antioxidant activity.

In a particular embodiment according to any one of the previous embodiments, the composition of the invention does not comprise as an additional ingredient the isolated amino acid L-arginine. This is an important advantage since, even without additional arginine, it is possible to efficiently treat DE and DSF (Examples 10 and 11), avoiding the problems described for oral compositions comprising arginine (Salvatore et al, Acta Biomed 2014, vol . 85, no.3: 222-228).

In a particular embodiment of the composition of the invention according to any one of the previous embodiments, the HC comprises at least one caseinphosphopeptide (CPP). In the context of the present invention, CPPs refer to peptides that comprise between 7 and 25 amino acids with different degrees of phosphorylation.

In a particular embodiment according to any one of the previous embodiments, the HC comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20 or 21 CPPs selected from the group consisting of the sequences SEQ ID No. 1 to SEQ ID No. 21. Preferably, the composition of the invention comprises all (21) the CPPs of sequences SEQ ID No. 1a SEQ ID No. ° 21.

In a particular embodiment, the CPPs are attached to at least one mineral. The HCs comprising such CPPs are referred to herein as mineral-enriched HC, mineral-aided HC, or mineral-conjugated HC, and are denoted HC-M, with M being the mineral. Thus, for example, potassium enriched HC is denoted HC-K. In a particular embodiment according to any one of the embodiments of the first aspect of the invention, the mineral is selected from the group consisting of zinc, magnesium, iron, potassium, calcium and combinations thereof, more particularly the mineral is zinc, magnesium, iron and / or calcium. Preferably, the mineral is zinc which, as seen in Example 10, represents an improvement in the treatment of ED.

Advantageously, as shown in Examples 2 and 3, the CPPs of the present invention are resistant to simulated gastrointestinal digestion in vitro, are capable of transporting minerals during said digestion. Furthermore, these CCPs provide phosphate groups to the plasma, increasing the phosphorylation capacity and with it the activation of eNOS.

The authors of the present invention have seen that the use of the complete HC hydrolyzate (without separation of fractions) has an improved effect, among others, in terms of increasing the expression of eNOS, compared to the use of each of the fractions by separated (less and greater than 3,000 Da) or even against the effect of the sum of both fractions. In addition, thus maintaining the CPPs in the HC with the advantages that this entails. Thus, in a preferred embodiment according to any one of the previous embodiments, the HC hydrolyzate is a complete hydrolyzate, i.e. It is not an isolated / purified fraction, nor is it a combination of isolated / purified fractions.

In a particular embodiment according to any one of the previous embodiments, the HC hydrolyzate comprises one, two, three, four, five or six peptides selected from the group consisting of the sequence peptides (peptide consists of the sequence) YFY, YLG, YLGY ( SEQ ID No. 22), RYLG (SEQ ID No. 23), YFYPE (SEQ ID No. 24), YFYPEL (SEQ ID No. 25). These peptides, when HC is separated into fractions greater than and less than 3,000 Da, are found in the fraction less than 3,000 Da. These peptides, as shown in Example 7, have angiotensin converting enzyme (ACE) inhibitory activity in vitro. Furthermore, the authors of the invention have seen that they have antihypertensive activity in vivo. In a particular embodiment of the composition of the invention, the HC comprises one, two or three peptides selected from the group consisting of the sequence peptides YFY, YFYPE and YFYPEL. More particularly, it comprises one or two peptides YFYPE and YFYPEL. Advantageously, all of these peptides are resistant to gastrointestinal digestion. In a Preferred embodiment, the HC comprises the peptide of sequence YFY and / or YLG, which are smaller in size and therefore, in addition to being resistant to digestion, are more easily absorbable in the gastrointestinal tract.

In the present invention, milk casein can be any casein. In a particular embodiment according to any one of the previous embodiments, the casein comprises at least one casein selected from the group consisting of as1-casein, as2-casein, p-casein, K-casein and mixtures thereof. Preferably the casein comprises as1-casein, as2-casein, p-casein and K-casein, thus having all the CPPs in Table 1.

In another particular embodiment according to any one of the previous embodiments, the HC hydrolyzate is obtainable by simple digestion (without additional digestions) with a gastric enzyme, preferably pepsin, or by sequential double digestion (without additional digestions), first with a gastric enzyme, preferably pepsin, and then with an intestinal enzyme, preferably trypsin. More particularly, it is obtainable by the methods for preparing HC described in the fifth aspect of the invention.

The whey hydrolyzate has DDP-IV enzyme inhibitory activity in vitro, as shown in Example 8.

In a particular embodiment according to any one of the previous embodiments, the HPS hydrolyzate comprises one, two, three, four or five peptides selected from the group consisting of the peptides of sequence DAQSAPLR (SEQ ID No. 26), HTSGYDTQ (SEQ ID No. 27), EQLTQ (SEQ ID No. 28), KIPAVF (SEQ ID No. 29) and IPAVF (SEQ ID No. 30). Preferably, the HPS hydrolyzate comprises the peptides SEQ ID No. 26 and / or SEQ ID No. 27 and / or SEQ ID No. 30, which are resistant to gastrointestinal digestion, and more preferably HPS comprises one or two selected peptides from SEQ ID No. 27 and SEQ ID No. 30 having better inhibitory activity of the DDP-IV enzyme in vitro (see Example 8).

In a particular embodiment according to any one of the previous embodiments, the HPS hydrolyzate is obtainable by simple digestion with trypsin (without additional digestions). More particularly, it is obtainable by the method for preparing HPS described in the fifth aspect of the invention.

Whey protein can be any whey protein milk preparation comprising beta-lactoglobulin and alpha-lactalbumin, preferably in their proportions natural. In a preferred embodiment the whey protein is a whey protein concentrate (WPC ). More preferably it is a WPC that contains more than 90% of the native whey proteins, including both beta-lactoglobulin and alpha-lactalbumin, in their original proportions.

In a particular embodiment according to any one of the previous embodiments, the milk casein and the whey protein come from cow, buffalo, sheep, goat, mare, camel, moose, sow milk and combinations thereof, preferably come from milk. cow.

Surprisingly, the combination of HC and HPS has, compared to the hydrolyzates separately, an improved and even synergistic effect in terms of antioxidant activity (see Example 9). This is an important advantage, since, not wanting to be bound by theory, it can help protect NO, produced by HC-increased eNOS, from oxidation, thus favoring the effects produced by NO, in particular the formation of cGMP . Thus, as indicated above, in a preferred embodiment of the invention according to any one of the previous embodiments, the composition of the present invention comprises HC, GB and HPS.

As indicated above, the composition of the invention comprises an extract of Ginkgo biloba, specifically a dry extract of GB. The whole plant or some of its parts can be used, for example, root and / or leaves, preferably the leaf. In a preferred embodiment, the GB extract comprises a minimum of 18% flavonol glycosides, and a minimum of 4% terpenic lactones. Preferably the GB extract comprises 22% -27% of flavonoids expressed as flavonic glycosides and 5-7% of terpenic lactones. More preferably the GB extract is GB standardized dry extract which is the one used in the Examples.

Advantageously, the combination of the extract of GB with HC or with HC + HPS results in an improvement in the effective treatment of a condition or disorder related to endothelial function, such as, for example, DE and DSF (see Examples 10 and 11) . As seen in the Examples, the HC + GB composition is effective in treating such disorders, and the HC + HPS + GB composition is even more effective, thus in a preferred embodiment according to any one of the above embodiments, the Composition of the invention comprises HC, HPS and GB. Likewise, the inventors have seen that the administration of the compositions of the invention (HC + GB and HC + HPS + GB), also improve physical, sports and sexual performance in healthy subjects. These subjects do not suffer from any disorder related to endothelial dysfunction and benefit from the improvement in endothelial function that is achieved with the compositions of the present invention. Surprisingly, these improvements, both in subjects with endothelial dysfunction and in healthy subjects, are synergistic, as they are superior to the additive effect of the components of the composition separately.

In a particular embodiment of the invention according to any one of the previous embodiments, the composition comprises:

- 45% - 95% HC, preferably 65-95%;

- 1% - 9% GB, preferably 1-6%; Y

- Optionally HPS, preferably 0-30% HPS.

Preferably the composition comprises 3-30% HPS, more preferably 3-15% HPS.

More particularly, the composition according to one of the previous embodiments further comprises a mineral, in which case the mineral content is 0.2% - 20%, depending on the recommendations of the health-related organisms for daily consumption in each mineral , preferably 0.2% -10%, and more preferably 0.2-3%. The mineral is selected from the group consisting of zinc, magnesium, iron, potassium, calcium, and combinations thereof, more particularly the mineral is zinc, magnesium, iron, and / or calcium. Preferably the mineral is zinc.

In one embodiment, the composition of the invention comprises 45-95% HC and 1-9% GB, preferably it comprises 65-95% HC and 1-6% GB. Preferably any one of these compositions comprises 0-30% HPS, more preferably 3-30% HPS, and more preferably still 3-15% HPS.

In the context of the present invention, the percentages are given by weight with respect to the total weight of the composition, unless otherwise indicated.

In a preferred embodiment, the composition of the invention consists of:

- 45% - 95% HC, preferably 65-95%;

- 1% - 9% GB, preferably 1-6%,

- 0% -30% HPS, preferably 3-30% and more preferably 3-15%; Y

- optionally 0.2% -20% a mineral, preferably 0.2-10%, and more preferably 0.2-3%,

so that the total sum is 100%.

The mineral can be one or more minerals. The particular and preferred embodiments defined for HC, HPS, GB and the mineral throughout the first aspect of the invention are applicable to the preferred embodiment of the preceding paragraph.

In a particular embodiment of the composition of the invention according to any one of the previous embodiments in which the composition of the invention comprises HC and HPS, the ratio HC: HPS is from 2: 1 to 15: 1, preferably from 7: 1 at 12: 1 and more preferably 10: 1. The composition thus obtained has, for example, an improved antioxidant activity, taking into account the lower dose of HPS (see Example 9).

The first aspect of the invention also relates to the hydrolyzates comprised in the composition of the present invention according to any one of the defined embodiments given in the first aspect of the invention. Thus, it refers to a casein hydrolyzate as defined in any of the embodiments of the composition of the invention according to the first aspect of the invention. It also refers to a hydrolyzate of whey proteins as defined in any of the embodiments of the composition of the invention according to the first aspect of the invention. It also refers to a composition comprising these HC and HPS hydrolysates (without GB), preferably in an HC: HPS ratio of 2: 1 to 15: 1, more preferably 7: 1 to 12: 1, and more preferably still 10: 1. In a particular embodiment of the composition comprising HC and HPS, e1 HC increases the expression and / or activity of eNOS in vivo and increases plasma arginine levels in vivo and has antioxidant activity and HPS has DDP-enzyme inhibitory activity. IV in vitro and has antioxidant activity. This composition, as indicated above, has an improved antioxidant effect. Thus, the invention also relates to its use as an antioxidant agent.

In view of the components of the composition of the invention the composition of the present invention can be considered a composition of a food nature or character (eg by the origin of the components, by the method of preparation). Thus, in a particular embodiment according to any one of the previous embodiments, the composition of the invention is a composition of a food nature. More particularly, the composition does not comprise any synthetic molecules or pharmaceutical active ingredients additional; and preferably HC, GB and optionally HPS and the mineral are the only active ingredients in the composition, and more preferably, with HC being unfractionated and HPS being unfractionated.

In a particular embodiment, the composition of the invention does not comprise other plants or parts or seeds thereof and / or does not comprise extracts of said plants or parts or seeds thereof. Thus, for example, the composition of the invention does not comprise ginseng ( Panax ginseng), or maca ( Lepidium meyeneii), or chives, or guarana, or ginger, or combinations thereof, or parts or seeds thereof.

The fact that the composition is of a dietary nature or character has important advantages especially with regard to the toxicity and side effects associated with pharmaceutical compositions. In the present case, in fact, the composition of the present invention is non-toxic and practically devoid of side effects. In addition, it can be considered as a food supplement.

The composition of the invention according to the first aspect of the invention is preferably an oral composition. Thus, it can be in the form of a tablet, capsule or syrup, for example.

The composition of the present invention and its components can be incorporated in food products, food supplements, beverages and in the manufacture of pharmaceutical products. Thus, in a second aspect, the invention relates to a medicament, a food supplement or supplement, a drink or a food product comprising a hydrolyzate and / or a composition according to any one of the embodiments of the first aspect of the invention. Preferably, it refers to a medicine, a food supplement, a drink or a food product comprising the composition of the invention according to any one of the embodiments described in the first aspect of the invention.

Dietary supplement or supplement refers to any dietary component that provides specific nutritional or medicinal components and does not provide the full required energy value (i.e. generally less than 2,000 or 2,500 kcal / day) and includes dietary supplements in powder or tablet form , like diet products, such as diet drinks. Also included are ingredients that can be added to food before consumption or a preparation that can be consumed as such.

The composition of the invention, or the medicines, food supplements, beverages or food products that comprise it, serve to improve vascular endothelial function. Thus, they serve to treat a disorder or condition related to endothelial dysfunction (e.g. disorder or condition that occurs with and / or is caused by endothelial dysfunction).

Likewise, they serve to improve those conditions that depend on good endothelial function or even improve when endothelial function improves, such as the physical, sports and / or sexual performance of a subject, particularly a subject that does not have any disorder or condition related to endothelial dysfunction and more particularly in a healthy subject.

Thus, in a third aspect, the present invention relates to the use of a composition according to any one of the embodiments of the first aspect of the invention, for the preparation of a medicine, a food supplement, a drink or a food product for the treatment of a disorder related to endothelial dysfunction.

The third aspect of the invention also relates to a composition according to any one of the embodiments of the first aspect of the invention, for use in the treatment of a disorder related to endothelial dysfunction.

Furthermore, it refers to a method of treatment of a disorder related to endothelial dysfunction in a subject (mammal, preferably human), comprising the administration, preferably of a therapeutically effective amount, to said subject of a composition according to any one of the Embodiments of the first aspect of the invention or of a medicament, food supplement, beverage or food product according to the second aspect of the invention. In a preferred embodiment, the administration is carried out orally.

In a particular embodiment according to any one of the three preceding paragraphs of the third aspect of the present invention, the disorder related to endothelial dysfunction is selected from the group consisting of sexual dysfunction (for example, erectile dysfunction, female sexual dysfunction), macular degeneration, chronic rhinitis, Schamberg's purpura, atherosclerosis, cardiovascular disease, hypertension, hypercholesterolemia, hyperglycemia, stroke, stroke, myocardial infarction, peripheral vascular disease, angina pectoris, heart failure, diastolic and / or systolic ventricular dysfunction, macro and microangiopathy in patients with diabetes, tissue injury related to ischemia and reperfusion, neurodegenerative diseases (eg Alzheimer) and combinations thereof. Preferably, the disorder related to endothelial dysfunction is sexual dysfunction (eg, erectile dysfunction, female sexual dysfunction), macular degeneration, chronic rhinitis or Schamberg's purpura, and more preferably it is sexual dysfunction (eg, erectile dysfunction or female sexual dysfunction). ).

The term "sexual dysfunction" generally includes any sexual dysfunction in a patient, including an animal, preferably a mammal, preferably a human. The patient can be male or female. Sexual dysfunctions can include, for example, disorders of sexual desire, disorders of sexual arousal, orgasmic disorders, disorders of pain during sex, and combinations thereof. Female sexual dysfunction refers to any female sexual dysfunction, including, for example, desire disorders, sexual arousal dysfunctions, orgasmic dysfunctions, pain disorders during sex, dyspareunia, vaginismus, and combinations thereof. The woman can be premenopausal or menopausal. Male sexual dysfunction refers to any male sexual dysfunction, including, for example, erectile dysfunction and impotence.

The third aspect of the invention also relates to a composition according to any one of the embodiments of the first aspect of the invention, for use as an adjunct in the treatment of a disorder related to vascular endothelial dysfunction. In particular, said disorder is selected from the group consisting of atherosclerosis, cardiovascular disease, hypertension, hypercholesterolemia, hyperglycemia, stroke, stroke, myocardial infarction, peripheral vascular disease, angina, heart failure, diastolic and / or systolic ventricular dysfunction, macro and microangiopathy. in patients with diabetes, ischemia and reperfusion-related tissue injury, neurodegenerative diseases (eg Alzheimer's), sexual dysfunction, macular degeneration, chronic rhinitis, Schamberg's purpura, and combinations thereof. Preferably macular degeneration, chronic rhinitis or Schamberg purpura.

Likewise, the third aspect refers to a method of treating a disorder related to endothelial dysfunction in which the composition of the invention is administered as an adjuvant to a subject. It also refers to the use of the composition of the invention to prepare an adjuvant for the treatment of a related disorder. with endothelial dysfunction, and in particular a disorder as defined in the paragraph.

The third aspect of the invention also relates to the use of a composition according to any one of the embodiments described in the first aspect of the invention, to improve any condition that improves by improving endothelial function, for example, physical, sports performance and / or sexual. It also refers to the use of said compositions for the preparation of a food supplement, drink or food product to improve physical, sports and / or sexual performance. Likewise, it refers to a method to improve physical, sports and / or sexual performance, which comprises the administration to a subject of an effective amount of a composition according to any one of the embodiments described in the first aspect of the invention and / or of a food supplement, beverage or food product according to any one of the embodiments described in the second aspect of the invention.

The terms physical, sports and sexual performance are widely known to the person skilled in the art.

In a particular embodiment, improvement in sports performance includes at least one of the following aspects: improving strength performance, changing physiological responses to resistance exercises, increasing blood volume of muscles during exercise recovery resistance, improved muscle blood perfusion, increased total work, increased resistance to fatigue.

Without being bound by theory, the improved antioxidant capacity of the composition of the invention (see Example 9) is important for any related and / or NO-dependent endothelial function, and is especially important in terms of using the composition to improve the physical, sports and / or sexual performance since NO is a molecule that reacts quickly with reactive oxygen species, the level of which increases after exercise. Thus, in a preferred embodiment according to any one of the embodiments of the third aspect of the invention, the composition comprises HC, GB and HPS. This preferred embodiment is also advantageous because the authors of the present invention have seen that there is a synergistic effect in terms of increasing expression and / or activity of eNOS in vivo when combining HC with HPS (data not shown), thus favoring the production of NO and correlatively cGMP.

The particular and preferred embodiments described in the first aspect of the invention in relation to the composition, the hydrolyzates and the GB, are applicable to the third aspect of the invention.

The use of the composition of the present invention is a great advantage over chemical treatments and supplements since enzymatic hydrolysates of dietary proteins and GB extract are naturally derived food ingredients that lack toxicity or have minimal toxicity.

For use in a medicine or food supplement, the composition of the invention, or its components, according to any one of the embodiments of the first aspect of the invention, can be combined with any carrier, diluent, adjuvant, excipient, etc. suitable, to obtain the medicine or supplement in the desired administration form. Thus, they can be presented in any form of administration, solid or liquid, and administered by any appropriate route, oral, respiratory, rectal or topical. Advantageously, said composition, medicine or food supplement is administered orally. Examples of such formulations, which can be prepared using well known methods and excipients, such as those described in "Remington's Phamaceutical Sciences Handbook" Mack Pub. Co., NYUSA, are tablets, capsules, syrups and the like for oral administration, while For parental administration the suitable forms are sterile solutions or suspensions in acceptable liquids, implants, etc. Preferred examples of excipients are microcrystalline cellulose, xanthan gum, magnesium stearate, silicon dioxide, and combinations thereof.

For use in a beverage or food product, the composition of the invention, or its components, according to the first aspect of the invention can be combined with any common food ingredient. The term "beverage" is intended to include liquids and syrups, as well as powder formulations to be dissolved in water or another liquid component for the preparation of instant beverages.

The dosage will depend on various factors such as the subject's weight, sex, type and severity of the conditions to be treated, etc. and it will be easily determined by the expert professional. Preferably, the composition of the invention is administered at a level of between 1 mg and 60 mg per day and per kg of the subject to which it is administered. Preferably at least 300 mg of composition is administered daily. The maximum dose can be determined by the maximum allowed by the legislation that applies in each country. So by For example, in Europe, for the GB (considered novel food), said maximum dose currently refers to a daily maximum of 21.6 mg of flavonol glycosides and 5.4 mg of terpene lactones.

The components of the composition of the invention, HC, GB and optionally HPS can be provided separately for combined use. Thus, the present invention in a fourth aspect refers to a kit of parts comprising:

a) a first container with a casein hydrolyzate as defined in any one of the embodiments of the first aspect of the invention;

b) a second container with an extract of Ginkgo biloba; and optionally c) a third container with whey protein hydrolyzate as defined in any one of the embodiments of the first aspect of the invention.

A preferred embodiment of the kit of the invention comprises the three containers a), b) and c), for the advantages indicated above for the use of the composition with HC, GB and HPS.

The particular and preferred embodiments described in the first aspect of the invention in relation to hydrolyzates and GB, are applicable to the kit of the fourth aspect of the invention. In a particular embodiment, the kit comprises a) and c), and optionally b).

The present kit provides the ingredients that in combination serve for the treatment of a disorder related to endothelial dysfunction, as an adjuvant to said disorder, and to improve physical, sports and / or sexual performance, as previously indicated for the composition and medically, supplement, etc. who understand it (see third aspect of the invention). Thus, in a particular embodiment, the invention relates to said kit for use in the treatment of a disorder related to endothelial dysfunction and to the use of said kit to improve any condition that improves by improving endothelial function, for example, performance physical, sports and / or sexual. By providing the hydrolyzates and the GB separately, the user can prepare the composition that interests him according to his needs. In a particular embodiment, the kit also includes instructions for use. The particular and preferred embodiments of the disorder related to endothelial dysfunction and physical, sports and sexual performance defined in the third aspect of the invention are applicable to the fourth aspect of the invention.

In a fifth aspect, the present invention relates to the methods for preparing the HC and HPS hydrolyzates, and the composition of the invention.

Hydrolyzates are obtained by dissolving or dispersing the starting material (serum or casein) at a suitable concentration in water or in a buffer solution that is at an optimal pH for the activity of gastric and intestinal enzymes (pepsin and / or trypsin, or any other similar functions). The hydrolysis conditions pH, temperature, type of substrate, substrate enzyme ratio, hydrolysis time, order of addition of the enzymes and inactivation of the enzyme / s are such that they allow to select the hydrolyzates with the therapeutic action of interest.

Thus, the fifth aspect of the present invention relates to a method for preparing a casein hydrolyzate as defined in the first aspect of the invention (hereinafter referred to as the HC-1 method), comprising the following steps:

a) Provide an appropriate dairy substrate, preferably casein;

b) Dissolve or suspend said substrate in water or in a buffer solution, at a pH suitable for the proteolytic action of the gastric enzyme, preferably pepsin, under gastric conditions, preferably at a pH between 2 and 4 and at a temperature between 37 ° C and 40 ° C;

c) Add the gastric enzyme, in an enzyme / substrate ratio of between 1/100 and 10/100 (w / w); d) Maintain the reaction for a time of between 6 and 28 hours, preferably 25-28 hours, and during this time add between 1-10% enzyme, preferably the same amount of enzyme as in step c), in increasing intervals between 4 and 8 hours;

e) inactivate the enzyme, preferably raising the pH to 7-9;

f) Optionally, add a water soluble compound containing a mineral and incubate, preferably at between 37 ° C and 40 ° C and / or for between 0.5 and 1 hours;

g) Desiccate.

The HC-1 method results in an HC that together with GB, and optionally HPS, is effective in improving endothelial function and thus serves for the treatments and improvements described in the third aspect of the invention. Advantageously, the authors of the present invention have further optimized said method so that an even more efficient treatment and improvement is achieved than with the hydrolyzate obtainable with HC-1. Said optimized method further comprises digestion under intestinal conditions after gastric digestion of HC-1. Said method (hereinafter referred to as method HC-2), comprises steps a) -e) as defined above for HC-1 and then the following steps:

f) Establish a temperature between 37 ° C and 40 ° C and a pH of intestinal conditions (if it has not been set to this pH in the previous step), preferably pH 7-9, add enzyme intestinal, preferably trypsin, in an enzyme / substrate ratio of between 1/100 and 10/100 (w / w), and incubate for a time of between 2 and 6 hours, preferably 3-5 hours;

g) Optionally, add a water-soluble compound containing a mineral and incubate, preferably at between 37 ° C and 40 ° C, more preferably at the same temperature and pH as in the previous step, and / or for between 0.5 and 1 hour;

h) Inactivate the intestinal enzyme; Y

i) drying out.

That is, the HC-2 method comprises the same steps as the HC-1 method, with steps g) and i) of HC-2 corresponding to steps f) and g) of HC-1, respectively, and additionally comprising steps f ) and h) in the order indicated above.

When step e) is carried out on HC-1 or g) on HC-2, the HC is a mineral-enriched HC (HC-M), that is, the HC comprises at least one CPP bound to a mineral. In a particular embodiment, the mineral is selected from magnesium, iron, potassium, zinc, calcium and combinations thereof, preferably it is magnesium, iron, calcium and / or zinc, and more preferably the mineral is zinc.

The fifth aspect of the present invention also relates to a method for preparing the whey protein hydrolyzate according to any one of the embodiments defined in the first aspect of the invention (hereinafter referred to as the HPS method), comprising the following stages:

a) Providing an appropriate dairy substrate, preferably whey protein; b) Dissolve or suspend said substrate in water or in a buffer solution, at a pH suitable for the proteolytic action of an intestinal enzyme, preferably trypsin, in intestinal conditions, preferably at a pH between 7 and 9, and at a temperature between 37 ° C and 40 ° C;

c) Add intestinal enzyme, preferably trypsin, in an enzyme / substrate ratio of between 1/100 and 10/100 (w / w);

d) Maintain the reaction for a time of between 6 and 24 hours, preferably between 8 and 14 hours;

e) Inactivate the intestinal enzyme; Y

f) Desiccate.

In a particular embodiment of any of the above methods, pH adjustments are made with HCl or KOH, as appropriate. In another embodiment, the inactivation of trypsin it is carried out by heat or by adjusting the pH, preferably to a pH comprised between 3 and 5. In another particular embodiment, drying is carried out by lyophilization or by heat in an oven, oven or spray.

Likewise, the fifth aspect of the invention refers to a method for preparing the composition of the invention according to any one of the embodiments of the first aspect of the invention, comprising the following steps:

i) carrying out the HC-1 or HC-2 method, and optionally the HPS method; Y

ii) mix in the desired quantity of HC, and optionally HPS, obtained (s) in step i) and the extract of Ginkgo biloba.

In a particular embodiment, in step i) the method is the HC-M method described above.

The particular and preferred embodiments defined in the first aspect of the invention, in particular as regards the amounts, proportions and components of the composition of the invention are applicable to the methods of the fifth aspect of the invention.

The fifth aspect of the present invention also refers to the HC, HPS hydrolyzates, and compositions obtainable by any of the methods described in the preceding paragraphs of the fifth aspect of the invention in any of its particular embodiments. Furthermore, it refers to compositions comprising HC and HPS obtainable by the HC methods (HC-1 or HC-2) and the HPS method, respectively. Furthermore, it refers to medicines, food supplements, beverages, food products or kits that comprise them and their use, as defined in the second, third and fourth aspects of the invention.

Examples

Specific examples of embodiments of the invention are detailed below to illustrate the invention.

EXAMPLE 1.- Casein hydrolyzate

1.1.- Obtaining the casein hydrolyzate

Casein hydrolyzate (HC) was obtained using bovine casein as substrate. dispersed in 2% (w / v) water and then the pH was adjusted to between 2 and 4 with HCl and the temperature was maintained between 37 and 40 ° C throughout the process. The gastric enzyme, in this particular case, porcine pepsin was then added in an enzyme / substrate ratio of between 1/100 and 10/100 (w / w). To compensate for the degradation of pepsin, the same amount of enzyme was added again as before at 4, 10 and 17 hours after the start. After 25 hours, the reaction was stopped, inactivating the enzyme by raising the pH to pH 8, adding KOH. Subsequently, a second hydrolysis was carried out using as a substrate the product of the first hydrolysis to which the intestinal enzyme was added, in this particular case trypsin, in an enzyme / substrate ratio of between 1/100 and 10/100 (w / w ) and incubating at the same temperature of 40 ° C for 2 h. The enzyme was inactivated, lowering the pH to between 3-5. The hydrolyzate was subsequently dried in an oven at 90 ° C.

1.2.- Obtaining the HC enriched with mineral molecules conjugated to CPPs

The previous process was followed but adding the mineral of interest at the end of the 2 h hydrolysis with trypsin and kept at 40 ° C and pH 8 for one hour before lowering the pH to 4.5. Each of the minerals of interest was added as follows:

Potassium: KCl in ratio (dry weight) HC: KCl of 3: 1;

Magnesium: MgO in proportion (dry weight) HC: MgO of 5: 1;

Iron: C4H8FeN2O4 in ratio (dry weight) HC: C4H8FeN2O4 of 5: 1;

Zinc: ZnSO4 in ratio (dry weight) HC: ZnSO4 of 90: 1;

Calcium: Ca3 (PO4) 2 in proportion (dry weight) HC: Ca3 (PO4) 2 of 5: 1.

The HC, HC-K, HC-Zn, HC-Ca, HC-Mg, HC-Fe cited in the following examples were produced following the methods described in this section, as appropriate. No separation of fractions from the hydrolyzate, i.e. a complete hydrolyzate was used.

EXAMPLE 2.- Determination of the digestion resistance capacity of the CPPs generated in the casein hydrolyzate

After the trypsin hydrolysis of Example 1.1, it was observed that the hydrolyzate obtained retained 78% of the phosphorus content of the starting casein, indicating the presence of the phosphorylated regions of the caseins. The identification of these phosphorylated peptides was performed by HPLC-Ms / MS and is described in figure 1 and table 1. Of the 55 peptide sequences that were identified (figure 1), 21 fragments corresponded to CPPs, six of them from the as1-casein, eight of as2-casein, six derivatives of p-casein and one of K-casein. It should be noted that four CPPs contained the sequence SpSpSpEE, specifically the peptides like-casein (61-74) 4P, as2-casein (51-64) 3P, p-casein (1-25) 4P and p-casein (2-25 ) 4P. Of the 34 non-phosphorylated peptides found, nine were derived from as-casein, seven from as2-casein, 16 from pcasein, and two from K-casein. Most of these non-phosphorylated peptides contained negatively charged residues. Some peptides do not correspond to those that would be formed taking into account the specificity of trypsin, so they may have been formed by the action of the pepsin used during the first part of the hydrolysis.

Table 1.- Peptides identified in the hydrolyzate.

Sp, phosphoserine.

a Numbering as indicated in the chromatogram of Figure 1.

Obs .: observed

Cal .: calculated

A comparison of the resulting CPPs was made after digestion with pepsin and trypsin, with simulated gastrointestinal digestion in vitro using the protocol of Martos et al., 2010 ( Egg white ovoalbumin digestion mimicking physiological conditions. J. Agric. Food Chem. , 2010, 58, 5640-5648). It should be noted that after digestion, the phosphorylated peptides belong to the same protein domains as those generated by hydrolysis. Furthermore, the patterns obtained are similar, revealing the resistance of these phosphorylated regions to gastrointestinal digestion. Only small differences were found between the sequences and, in addition, the present known CPPs, such as p-casein f (1-25) 4P and as1-casein f (43-58) 2P and as2-casein f (56 -68) 3P and fragments thereof were found after simulated digestive digestion. The results showed a high homology (73%) between the sequences identified by both hydrolysis procedures, thus demonstrating that the set of peptides formed are capable of resisting digestion and therefore of transporting to the intestine the minerals that could carry conjugates.

EXAMPLE 3.- Determination of the mineral transport capacity during gastric and intestinal digestion

To verify the efficiency of the conjugation of the CPPs formed with the minerals (in the present case K, Zn, Ca, Mg, Fe), the concentration of these minerals in the soluble phase of a simulated digestion in vitro was compared using the protocol of Martos et al., (2010) with that obtained with the same amount of the unconjugated compound at three moments: 0 and 60 minutes in gastric conditions and 60 min in intestinal conditions. The results in% of soluble mineral are shown in Table 2.A and 2.B.

Table 2.A.- Percentage of soluble mineral

Table 2.B.- Percentage of soluble mineral

The determination of the mineral quantity was made by atomic absorption spectroscopy.

As can be seen in Tables 2.A and 2.B, the percentage of soluble mineral in the intestinal phase, where absorption is carried out, is clearly higher when the mineral has been conjugated with the CPPs, favoring its joint use with hydrolyzates of the present invention, for example, to balance testosterone levels and antioxidant activity (Zn), or decrease tiredness and fatigue (Mg, Fe). In the case of Fe, the gastrointestinal side effects normally associated with the consumption of the preparations that seek the absorption of Fe, of special interest to women, would also be avoided.

EXAMPLE 4.- Hydrolyzate of whey protein

The whey protein hydrolyzate (HPS) was obtained using as a substrate a whey protein concentrate (WPC) of bovine milk rich in a-lactoalbumine and plactoglobulin that was dispersed in water at a concentration of 5% (w / v). The hydrolysis was carried out with trypsin at pH between 7 and 9, and with an enzyme / substrate ratio of 1% -10% (w / w) at a temperature of between 37 ° C and 40 ° C for 10 h. The inactivation of the enzyme was carried out by reducing the pH to 4.5. It was subsequently dried in an oven at 90 ° C.

The HPS cited in the following examples were produced following this method, unless otherwise indicated. No separation of fractions from the hydrolyzate, i.e. a complete hydrolyzate was used.

EXAMPLE 5.- Determination of the expression of the enzyme Nitric Oxide Synthase Epithelial (eNOS) in the aorta of experimental animals.

Worked with 10 WKY rats, kept under controlled temperature conditions (23 ± 2 ° C) and humidity (40-70%) with light-dark cycles of 12 hours. The standard diet and water were available ad libitum. Two groups of studies detailed in Table 3 were performed, with 5 individuals in each group. In the control group, the standard diet was supplemented with cellulose and in the HC group, the standard diet was supplemented with HC.

Frozen aortic samples obtained from said rats were homogenized and centrifuged at 2,000 rpm for 1 min at 4 ° C in RIPA lysis buffer, the lysates were collected and centrifuged at 10,000 rpm for 5 min at 4 ° C. Supernatants were collected and protein concentration was determined using a standard assay (Bio-Rad Laboratories Inc., USA). Proteins were eluted in Laemmli buffer, and separated by electrophoresis using 10% SDS-PAGE gel, and transferred to a nitrocellulose membrane (Whatman, Germany). The membranes were blocked with 5% skimmed milk in Tris buffer with 0.1% Tween for 1h at room temperature and then incubated with the rabbit polyclonal antibody eNOS (1: 5000, Bio-Rad, Laboratories Inc., USA) for overnight at 4 ° C with stirring. The membrane was then washed several times with TBS-Tween buffer and incubated with a secondary anti-rabbit antibody bound to the HRP enzyme (1: 1000, Bio-Rad, Laboratories Inc., USA) for 1h at room temperature. The membrane was developed chemiluminescent using the Western blotting ECL system (Amersham-Pharmacia-Biotech) and X-ray exposure (Fuji, India). Densitometry was performed using the Flurochem program, (Alpha Innotech Corp., USA). The eNOS protein corresponds to the 145-kDa band and was visualized with reference to molecular weight markers. The results were expressed as the ratio between the immunoblot signal between the eNOS band and actin (load control) (Table 3).

The duration of treatment was 6 weeks. Weight was monitored twice a week. The statistical analysis was carried out using the analysis of variance (ANOVA) with the SPSS statistical program.

Table 3.- Determination of the expression of the eNOS enzyme. The increase in% is calculated between week 6 and week 0 of each group.

As can be seen in this table, the measurements stabilized from the fourth week. The administration of the HC causes a significant increase in the expression of eNOS in vivo, specifically an increase that becomes greater than 62% with respect to the control.

Interestingly, the inventors have seen that when HC is separated into two fractions, one smaller and one greater than 3,000 Da, the sum of the eNOS induction of each of these two fractions is much less than when using an HC complete (not subjected to fraction separation, as in this example) (data not shown).

EXAMPLE 6.- Determination of plasma availability of Arginine

It was analyzed whether HC has any effect on plasma arginine levels. Arginine was quantified by ion exchange chromatography. Samples from 6 adult pigs (35-45 Kg) fed the usual diet once a day, without access to water, were analyzed. 7 hours after the food intake, they were allowed access to water including 10 g of HC (group HC, 3 pigs) or 10 g of undigested casein (control, 3 pigs) and taking blood samples, every 30 minutes for 3 hours. The results are shown in Table 4.

Table 4.- Arginine concentration (^ mol / L) over time.

As shown in this table, the arginine concentration is significantly higher than the control, especially during the three hours following HC intake.

Arginine is necessary for the production of NO. Thus, without being bound by any theory, the increase in plasma arginine levels produced by HC allows obtaining NO as long as eNOS is active.

EXAMPLE 7.- Determination of ACEI activity in vitro

ACEI activity was determined spectrophotometrically. The compound used as substrate of the ACE was Hipuril-Histidil-Leucina (HHL) (Sigma). HHL was dissolved in 0.1M sodium borate buffer with 0.3M NaCl to obtain a final concentration of 5mM HHL at a pH of 8.3. To 100 ^ L of substrate was added 40 ^ L of each of the samples whose ACE inhibitory activity was wanted to determine. 2 mUs of the enzyme ACE (EC 3.4.15.1, 4.7 U / mg protein, Sigma), dissolved in 50% glycerol, and diluted 1/10 in Milli-Q water were added at the time of the assay. The reaction was carried out at 37 ° C for 30 minutes. The enzyme was inactivated by lowering the pH with 150 ^ L of 1N HCl. The hippuric acid formed was extracted with 1000 ^ L of ethyl acetate. After vortexing for 20 seconds, it was centrifuged at 4,000 * g for 10 minutes at room temperature. 750 ^ L of the organic phase were extracted and evaporated by heating at 95 ° C for 15 minutes. The hippuric acid residue was re-dissolved in 800 ^ L of Milli-Q water and, after stirring, the absorbance at 228 nm was measured on a DU-800 spectrophotometer (Beckman Coulter, Fullerton, CA, USA). The blank and positive control received the same treatment as the rest of the samples, however, water was added to the blank instead of enzyme and water was added to the control.

ACEI activity was expressed as the peptide concentration (^ M) necessary to inhibit 50% of the enzyme activity (Table 5). To carry out its calculation, the percentage of ACE inhibitory activity was plotted against the concentration of the sample (minimum 5 points) and a non-linear adjustment of the data was performed with the PRISM version 4.02 software for Windows (GraphPad Software , San Diego, CA, USA). The activity of each sample was determined in triplicate, and the following formula was used to calculate the percentage of ACE inhibitory activity:

ACE inhibitory activity (%) = (AC-AM) / (AC-AB) * 100

AC: Absorbance of hippuric acid formed after ACE action without inhibitor (control). Ab: Absorbance of the Hipuril-Histidil-Leucine compound that has not reacted and that has been extracted with ethyl acetate (white).

Am: Absorbance of hippuric acid formed after ACE action in the presence of inhibitory substances (sample).

Table 5.- ACE inhibitory activity expressed in ^ M of peptides.

This table shows the sequences of the identified peptides and their location in the protein of origin, together with the inhibitory activity of ACE determined in vitro. Furthermore, these peptides show antihypertensive activity in vivo in hypertensive rats (data not shown).

Interestingly, the inventors have seen that when HC is separated into two fractions, one less and one greater than 3,000 Da, the ACE activity of the entire HC is greater than that which would be assigned to the fraction less than 3,000 Da (data not shown)

EXAMPLE 8.- Determination of the inhibitory activity of the DPP-IV enzyme in vitro

The whey protein hydrolyzate produced according to Example 4 was used. The inhibitory activity of the DPP-IV enzyme was determined by an in vitro enzymatic method . This assay was carried out in 96-well plates using diprotin A (Enzo Life Sciences Inc., Farmingdale, NY, USA) as a positive control. 15 ^ L of the recombinant human enzyme DPP-IV (0.01 mg / mL) (Sigma-Aldrich, St. Louis, MO, USA) and 35 ^ L of the sample to be analyzed (at different concentrations) were mixed and incubated at room temperature for 10 minutes. After this time, 50 ^ L of the assay buffer containing the H-Gly-Pro-p-nitroaniline chromogenic substrate (Enzo Life Sciences Inc.) at a concentration of 200 ^ M was added and the absorbance was measured at 405 nm in a plate reader (BMG LABTECH Inc., Champigny surMarne, France) for 30 minutes at 2 minute intervals. Absorbance data were plotted against time and expressed as the percentage of residual enzyme activity in the presence of the sample compared to the control (no sample added). For each sample, three different tests were performed to represent the dose-response curve and calculate the IC50 value (protein concentration necessary to inhibit 50% of the activity of the DPP-IV enzyme).

In order to analyze the inhibitory activity of the DPP-IV enzyme, the hydrolyzate was fractionated and subsequently, in those fractions with the highest activity, the most abundant peptide sequences, potentially responsible for the activity of the fraction, were studied using HPLC-MS.

For the separation of the peptides, a Mediterranean Sea 18 reverse phase column (150 * 2.1 mm ID, 5 ^ m particle size) was used (Teknokroma, Barcelona). Solvent A was a mixture of water and trifluoroacetic acid (1000: 0.37) and solvent B a mixture of acetonitrile and trifluoroacetic acid (1000: 0.27). A linear gradient was used 0 to 45% B in 60 minutes, at a flow of 0.2 mL / min. The samples were injected at a concentration of 0.884 mg protein / mL with an injection volume of 50 ^ L and their elution was monitored at 214 nm. The HPLC equipment was coupled to an Esquire-3000 mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany), directing the entire flow at the detector outlet towards the mass spectrometer nebulizer. The team used nitrogen as the mist and drying gas (60 psi, 8 L / min, 350 ° C), and helium at an estimated pressure of 5 bar. The mass spectra were acquired in a range between 100 and 1500 m / z. The capillary was maintained at a voltage of 4 kV. The signal from the mass spectrometry analyzes was obtained from the average of 15 spectra and the mean value of 5 spectra was used for tandem mass spectrometry analyzes. The intensity limit for carrying out the tandem mass spectrometry analyzes was 50,000 arbitrary units. The precursor ions were isolated with an interval of 4 m / z and fragmented with a voltage ramp optimized for each fraction. The spectral data was processed and transformed to mass values using the Data Analysis program (version 4.0, Bruker Daltonik). The BioTools program (version 3.1, Bruker Daltonik) was used to process the tandem mass spectrometry (MS / MS) spectra and to carry out the sequencing of the peptides.

Table 6- Sequence of the identified peptides, location in the protein of origin, and DPP-IV inhibitory activity (expressed as percentage of inhibition at the tested concentration of 100 ^ M).

The five selected peptides contribute an interesting percentage of inhibition and the five together contribute to more than 80% of the total activity tested.

EXAMPLE 9.- Evaluation of the antioxidant activity (Scanvenger) against the superoxide radical of the hydrolyzed HC, HPS and their combination

80 ^ L of intact casein, intact serum proteins, HC, and HPS (at 5 mg / mL) were mixed with 80 ^ L of 50 mM Tris-HCl (Merk) buffer, at a pH of 8.3 in a absorbance in 96-well plates. Then 40 ^ L of Pyrogallol (Merk) 1.5 mM in HCl, 10mM were added. The rate of superoxide anion-induced pyrogallol polymerization (AAs / min) was measured by increasing absorbance at 320 nm for 5 min at 23 ° C. Glutathione was used as a positive control. The Tris-HCl buffer was used as blank (AAc / min).

The Scavenger activity index (IAS) against superoxide was measured with the following formula IAS = [(AAc / min) - (AAs / min)] / (AAc / min) x 100

The results (%) were as follows shown in Table 7. The tests are made with the same amount of protein or hydrolyzate.

Table 7.- IAS of different samples.

As seen in the previous table, the combined effect of all intact proteins against casein alone improves the result by 20% (36 vs. 30). However, when hydrolyzates are combined, the antioxidant capacity increases by 27.3% (70 vs. 55). Furthermore, when the 10: 1 ratio is tested, the improvement is 21.8%, which proves its synergistic effect. This improvement is of great interest when formulating a food supplement where the total dose must, in addition to being effective, not exceed a total weight of daily dose that is rejected by the consumer.

As Glutathione is the main antioxidant in cells, the high percentage achieved by HC + HPS (10: 1) is observed, 68% (67 vs. 98) compared to what intact proteins achieve.

The inventors have seen that the composition comprising HC and HPS has unexpectedly high antioxidant activity against the ingredients of the composition separately, which, without wishing to be bound by theory, suggests that the composition of the invention comprising HC and HPS can help to avoid oxidation of NO, allowing its activity, especially the formation of cGMP.

EXAMPLE 10.- Erectile dysfunction

A trial was carried out with 140 men who had gone to the doctor, the pharmacy, the dietitian or the doctor to resolve their ED without declaring other related pathologies.

The study was carried out for 6 months and followed the “double-blind” methodology. Furthermore, since one of the populations at risk of drugs for sexual dysfunction is hypertensive individuals, it was also analyzed in this study if the The composition of the present invention had some effect on blood pressure (AT), allowing the participation of people with mild hypertension.

Participants had:

Age: 35-79 years (mean: 59).

Weight: 72-120 Kg (average: 87 Kg).

Blood pressure:

- 92 normotensive patients: systolic 100-145 mmHg (mean 115), diastolic 58-80 mmHg (mean 70) - 48 with mild hypertension: systolic 130-170 mmHg (mean 135), diastolic 80-110 mmHg (mean 89).

The groups detailed in Table 8 were formed. The compositions were taken included in 2 capsules with water, outside of meals. The GB extract used was standardized GB dry extract.

Participants were asked to note the time when they noticed a reaction. Three checks were performed including the measurement of BP, at baseline, at 3 months and at 6 months.

Participants were asked if they had noticed: increased penile stiffness, morning erection, spontaneous erection, increased sexual desire, when they noticed improvement, and collectively how they rated the improvement (mild or significant). The results are detailed in Table 8:

Table 8.- DE test results

As can be seen in this table, the combinations HC + GB and HC + HPS + GB achieve results in the total positive reaction (70% and 85% respectively) that are much higher than HC (35%), HC + HPS (40%) and GB (10%), thus verifying the synergistic action of the combination of the hydrolyzate (s) with GB. Furthermore, significant reactions only appear when hydrolyzates with GB are used: HC + GB (60%), HC + HPS + GB (75%). And if Zn is also added (HC-Zn + HPS + GB) the results improve both in significant reaction (80%) and in total (90%)

The onset of effects was detected between the third and fourth week (mean: 21 days) and interestingly, these effects were maintained throughout the duration of the trial. Thus, a long-term treatment of ED is achieved by administering a food grade composition that shows no relevant side effects and allows its continued use. This is a great advantage since many of the existing treatments at present are for punctual use since their continued use presents health risks.

Regarding the side effects, only 9 participants noticed any side effect that consisted of a slight headache in the first week that subsided within 4 or 5 days of continuing treatment. Thus, the present invention provides a composition effective in the treatment of ED and practically free of side effects.

Regarding hypertensive individuals, it should be noted that surprisingly, more than 75% of the participants who consumed the composition with HC or HC + HPS with or without GB, showed a moderate drop in blood pressure of the order of 7 mm Hg (systolic) and 4 mm Hg (diastolic), but all normotensive participants maintained their BP levels. These results are very favorable in that they allow the use of the composition of the invention for both normotensive and hypertensive patients, seeing the latter as the product can also help them normalize their TA values.

It is also important to note that 7 of these participants, who suffered from mild chronic pathologies, reported significant improvements in their symptoms (3 in macular degeneration, 3 in chronic rhinitis and 1 in Schamberg purpura), which supports that the composition of the The present invention can serve to treat or contribute to the treatment (adjuvant) of numerous pathologies related to vascular endothelial dysfunction, such as macular degeneration, chronic rhinitis or Schamberg purpura.

Lastly, it should be emphasized that the effects are achieved with very low quantities of final product, of little more than one gram, which can be included in one or two small tablets or capsules, which gives this product a special facility for the consumer to maintain the treatment and the administration guideline, since the consumer rejects presentations in high quantities.

EXAMPLE 11.- Female sexual dysfunction

This study was carried out on 96 women, who had gone to the specialist doctor (urologists or gynecologists) to improve their sexual function, because they suffered from sexual dysfunction. The women were between 40 and 62 years old (mean age 49 years). The groups shown in Table 9 were made (16 women per group) and the dose used in the previous example was administered. The "double blind" methodology was followed.

The study was carried out for 12 weeks. During the duration of the study, a series of questionnaires were carried out in order to determine if they had experienced any improvement in their sexual function and, if so, of what type and to what extent. Thus, the different individuals were classified as "null reaction" if they had not experienced any improvement and as "positive reaction" if they had experienced any improvement. The latter were also asked about certain details of the improvement. Specifically, they were asked if they had noticed: increased clitoral sensitivity, decreased vaginal dryness, increased sexual desire, when you noticed improvement, and collectively how you rated the improvement (mild or significant).

The compositions were taken included in capsules with water, outside of meals. Three checks were performed, at baseline, at 8 weeks, and at 12 weeks. The results obtained are detailed in Table 9.

Table 9.- DSF

As can be seen in this table, the combinations HC + GB and HC + HPS + GB achieve results in the total positive reaction (56% and 69% respectively) that are much superior to HC (19%), to HC + HPS (25%) and GB (13%), thus verifying the synergistic action of the hydrolyzate (s) with GB. Furthermore, significant reactions only appear when the hydrolyzates with GB are used: HC + GB (31%) and HC + HPS + GB (44%).

The effects (mild and / or significant) were noted in all cases between the fourth and fifth week of the trial (mean 32 days) and the effects were maintained for the remaining time until the end of the study (12 weeks).

Claims (38)

1. - Composition characterized in that it comprises: a) a milk casein (HC) hydrolyzate characterized in that it increases the expression and / or activity of eNOS in vivo and / or increases plasma arginine levels in vivo and / or has antioxidant activity; b) an extract of Ginkgo biloba (GB); and optionally c) a hydrolyzate of milk whey protein (HPS) characterized in that it has inhibitory activity of the enzyme DDP-IV in vitro and / or has antioxidant activity. 2. - Composition according to claim 1, where the HC increases the expression and / or activity of eNOS in vivo, increases plasma arginine levels in vivo and has antioxidant activity. 3. - Composition according to claim 1 or 2, wherein the HC comprises at least one caseinphosphopeptide. 4. - Composition according to claim 3, wherein the sequence of the caseinphosphopeptide is selected from the group consisting of SEQ ID No. 1 - SEQ ID No. 21. 5. - Composition according to claim 3 or 4, wherein the HC comprises 21 caseinphosphopeptides whose sequences are SEQ ID No. 1 - SEQ ID No. 21. 6. - Composition according to any one of claims 3-5, where the CCP (s) is (are) bound to a mineral. 7. - Composition according to the preceding claim, where the mineral is selected from magnesium, iron, potassium, calcium, zinc and combinations thereof. 8. - Composition according to the preceding claim, where the mineral is zinc. 9. - Composition according to any one of the preceding claims, wherein the HC comprises one, two, three, four, five or six peptides selected from the group consisting of the peptides of sequence YFY, YLG, YLGY (SEQ ID No. 22), RYLG (SEQ ID No. 23), YFYPE (SEQ ID No. 24), YFYPEL (SEQ ID No. 25). 10. - Composition according to the preceding claim, wherein the HC comprises one, two or three peptides selected from the group consisting of the peptides of sequence YFY, YFYPE (SEQ ID No. 22) and YFYPEL (SEQ ID No. 23). 11. - Composition according to the preceding claim, wherein the HC comprises one or two peptides of sequence YFYPE (SEQ ID No. 22) and YFYPEL (SEQ ID No. 23). 12. - Composition according to any one of the preceding claims, wherein the HPS has inhibitory activity of the DDP-IV enzyme in vitro and has antioxidant activity. 13. - Composition according to any one of the preceding claims, wherein the HPS comprises one, two, three, four or five peptides selected from the group consisting of the peptides of the sequence DAQSAPLR (SEQ ID No. 26), HTSGYDTQ (SEQ ID No. 27), EQLTQ (SEQ ID No. 28), KIPAVF (SEQ ID No. 29) and IPAVF (SEQ ID No. 30). 14. - Composition according to the preceding claim, wherein the HPS comprises one or two peptides selected from SEQ ID No. 27 and SEQ ID No. 30. 15. - Composition according to any one of the preceding claims, comprising 45% -95% HC, 1% -9% GB and, optionally HPS. 16. - Composition according to the preceding claim, comprising 45% -95% of HC, 1% -9% of GB and 0% -30% of HPS. 17. - Composition according to the preceding claim, comprising 45% -95% HC, 1% -9% GB and 3% -30% HPS. 18. - Composition according to the preceding claim, comprising 65% -95% HC, 1% -6% GB and 3% -15% HPS. 19. - Composition according to any one of claims 15 to 18, which further comprises 0.2% -20% mineral, preferably 0.2-10%. 20. - Composition according to any one of the preceding claims, comprising HPS and wherein the HC: HPS ratio is from 2: 1 to 15: 1, preferably from 7: 1 to 12: 1. 21. - Medicine, food supplement, drink or food product characterized in that it comprises a composition according to any one of claims 1-20. 22. - Composition according to any one of claims 1-20 for use in medicine. 23. - Composition according to any one of claims 1 to 20, for use in the treatment of a disorder related to endothelial dysfunction. 24. - Composition according to any one of Claims 1 to 20, for use as an adjuvant in the treatment of a disorder related to endothelial dysfunction. 25. - Composition according to claim 23 or 24, wherein the disorder related to endothelial dysfunction is selected from the group consisting of atherosclerosis, cardiovascular disease, hypertension, hypercholesterolemia, hyperglycemia, stroke, stroke, myocardial infarction, peripheral vascular disease, angina pectoris , heart failure, diastolic and / or systolic ventricular dysfunction, macro and microangiopathy in patients with diabetes, ischemia and reperfusion-related tissue injury, sexual dysfunction, macular degeneration, chronic rhinitis, Schamberg purpura and combinations thereof. 26. - Composition according to claim 23 or 24 where the disorder related to endothelial dysfunction is sexual dysfunction, particularly erectile dysfunction or female sexual dysfunction. 27. - A kit of parts comprising: a) a first container with a casein hydrolyzate as defined in any one of claims 1-11; b) a second container with an extract of Ginkgo biloba; and optionally c) a third container with whey protein hydrolyzate as defined in any one of claims 1, 12, 13 and 14. 28. - Kit according to claim 27 for use in the treatment of a disorder related to endothelial dysfunction. 29.- Use of a composition according to any one of claims 1 to 20, or of a food supplement, drink or food product according to claim 21, or the kit according to claim 27 to improve physical, sports and / or sexual performance. 30. - Method for preparing an HC as defined in any one of claims 1 to 11 characterized in that it comprises the following steps: a) Provide an appropriate dairy substrate, preferably casein; b) Dissolve or suspend said substrate in water or in a buffer solution, at a pH suitable for the proteolytic action of the gastric enzyme, preferably pepsin, under gastric conditions, preferably at a pH between 2 and 4 and at a temperature between 37 ° C and 40 ° C; c) Add the gastric enzyme, in an enzyme / substrate ratio of between 1/100 and 10/100 (w / w); d) Maintain the reaction for a time of between 6 and 28 hours, preferably 25-28 hours, and during this time add between 1-10% enzyme, preferably the same amount of enzyme as in step c), in increasing intervals between 4 and 8 hours; e) inactivate the enzyme, preferably raising the pH to 7-9; f) Optionally, add a water soluble compound containing a mineral and incubate, preferably at between 37 ° C and 40 ° C and / or for between 0.5 and 1 hours; g) Desiccate. 31. - Method according to the preceding claim, comprising steps a) -e) as defined in the preceding claim and then the following steps: f) Establish a temperature between 37 ° C and 40 ° C and a pH of intestinal conditions, preferably pH 7-9, add an intestinal enzyme, preferably trypsin, in an enzyme / substrate ratio of between 1/100 and 10/100 (w / w), and incubate for a time of between 2 and 6 hours, preferably 3-5 hours; g) Optionally, add a water-soluble compound containing a mineral and incubate, preferably at between 37 ° C and 40 ° C, more preferably at the same temperature and pH as in the previous step, and / or for between 0.5 and 1 hour; h) Inactivate the intestinal enzyme; Y i) drying out. 32. - Method for preparing an HPS as defined in any one of claims 1, 12, 13 and 14 characterized in that it comprises the following steps: a) Providing an appropriate dairy substrate, preferably whey protein; b) Dissolve or suspend said substrate in water or in a buffer solution, at a pH suitable for the proteolytic action of an intestinal enzyme, preferably trypsin, in intestinal conditions, preferably at a pH between 7 and 9, and at a temperature between 37 ° C and 40 ° C; c) Add intestinal enzyme, preferably trypsin, in an enzyme / substrate ratio of between 1/100 and 10/100 (w / w); d) Maintain the reaction for a time of between 6 and 24 hours, preferably between 8 and 14 hours; e) Inactivate the intestinal enzyme; Y f) Desiccate. 33. - Method for preparing the composition according to any one of claims 1 to 20, characterized in that it comprises the following steps: i) carrying out the method according to claim 30 or 31, and optionally the method according to claim 32; Y ii) mix in the desired quantity the hydrolyzate (s) obtained in step i) and an extract of Ginkgo biloba. 34. - Composition comprising: a) a milk casein (HC) hydrolyzate characterized in that it increases eNOS expression and / or activity in vivo, and / or increases plasma arginine levels in vivo, and / or has antioxidant activity; Y b) a hydrolyzate of milk whey protein (HPS) characterized in that it has inhibitory activity of the enzyme DDP-IV in vitro and / or has antioxidant activity. 35. - Composition according to the preceding claim, where the HC increases the expression and / or activity of eNOS in vivo and increases the levels of arginine in plasma in vivo and has antioxidant activity and the HPS has inhibitory activity of the enzyme DDP-IV in vitro and it has antioxidant activity. 36. - Composition according to the preceding claim, in which the HC: HPS ratio is from 2: 1 to 15: 1, preferably from 7: 1 to 12: 1. 37. - Use of a composition according to claim 35 or 36 as an antioxidant agent. 38. - Method for preparing the composition according to claim 34, 35 or 36 characterized in that it comprises the following steps: i) carrying out the method according to claim 30 or 31; ii) carrying out the method according to claim 32; Y ii) mix the hydrolysates obtained in steps i) and ii) in the desired amount.