ES2990173B2 - Quaternary inclusion complex of hydroxytyrosol, tryptophan, arginine and zinc gluconate, method of obtaining and using - Google Patents

Description

DESCRIPTION

QUATERNARY INCLUSION COMPLEX OF HYDROXYTYROSOL, TRYPTOPHAN,

ARGININE AND ZINC GLUCONATE, METHOD OF OBTAINING AND USE

TECHNICAL SECTOR

The present invention falls within the general field of chemistry, more specifically organic chemistry, and has applications in the pharmaceutical sector. More specifically, it falls within the field of compositions that act as immune system stimulators and also serve to control and regulate myeloid stem cell (MDSC) suppressor cells.

BACKGROUND OF THE INVENTION

Myeloid-derived suppressor cells (MDSCs) were discovered 50 years ago in cancer patients, and their role in the immune system is focused on downregulating immune responses in cancer and other infectious diseases.

The first studies on the accumulation of immunosuppressive myeloid cells associated with tumor progression were published in the early 1970s. During the 1980s and early 1990s, work from the laboratories of Diana López, Jim Talmadge, M. Rita Young, and Hans Schreiber demonstrated that several types of myeloid cells inhibited immune function during tumor cell progression.

MDSCs are present in virtually all individuals with cancer, impair adaptive and innate antitumor immunity, and promote disease progression through non-immune mechanisms. Their widespread presence, combined with a multitude of peritumoral activities, makes them a significant obstacle to immunostimulatory therapies, as well as antibody treatments.

They are a heterogeneous population of cells that expand beyond their physiological regulation during pathologies such as cancer, inflammation, and bacterial and viral infections. Their key characteristic is their remarkable ability to suppress T cell and killer cell responses. MDSCs regulate the immune system and associated responses in the context of various diseases. Although the primary function of polymorphonuclear MDSCs is to dampen T cell and killer cell responses, they also produce reactive oxygen species (ROS) and reactive nitrogen species (RNS).

MDSC cells are formed from bone marrow progenitor cells when myelopoietic processes are disrupted and proliferate under pathological conditions. They differ from other types of myeloid cells in that they exhibit immunosuppressive activity, as opposed to immunostimulatory properties, and they interact with other immune cells such as T cells, dendritic cells, macrophages, and natural killer (NK) cells, and even regulate their functions.

The main targets of MDSCs are T cells, and the main factors involved in immune suppression include arginase ARG1, iNOS, TGF-β, IL-10, COX2, tryptophan sequestration by indolamine 2,3-dioxygenase IDO, downregulation of L-selectin expression by T cells, and several others. M-MDSCs and PMN-MDSCs utilize different mechanisms of immune suppression, with the former (M-type) suppressing T cell responses both specifically and nonspecifically using mechanisms associated with nitrous oxide (NO) and cytokine production. PMN-MDSCs suppress immune responses by producing reactive oxygen species (ROS), which are essential for maintaining this ability.

MDSC activation is mediated by the expression of inflammatory cytokines and transcriptional regulators. Growing tumors produce cytokines and other substances that promote MDSC development, such as the colony-stimulating factors G-CSF and GM-CSF and the MDSC-promoting interleukin IL-6, preventing T cell activation and limiting the availability of amino acids necessary for T cell proliferation, such as arginine, or producing substances that block antigen recognition. MDSCs produce arginase 1 (ARG1), which competes for the consumption of the substrate arginine, depleting it, resulting in the loss of the T cell receptor chain essential for their activation. Recent results in tumor-bearing mice and human cancer patients indicate that increased ARG1 metabolism by MDSCs is a major factor in the inhibition of T cell responses.

Tumor immunity has revolutionized cancer therapy in recent years and represents a new avenue for improving cancer therapy by restoring the immune response, but immune system evasion is a key characteristic of tumors. To successfully establish a presence in a host and continue to grow, tumor cells use biochemical signals to conceal themselves from the host immune response and go undetected. MDSC cells remain a major obstacle to immunotherapy and contribute to treatment failure, reduced life expectancy, and poor prognosis. Checkpoint blockade immunotherapy (CBI) has revolutionized cancer treatment because the patient's adaptive immune system can eradicate malignant cells once immunosuppressive mechanisms are neutralized. Immune checkpoint inhibitors have successfully improved outcomes in several tumor types, and immune cell-based therapy is also gaining attention. However, this treatment is only effective in a small group of cancer patients, as other immunosuppressive mechanisms block T-cell-induced antitumor immunity.

Recent evidence indicates that most of these therapies are threatened by a complex network of immunosuppressive mechanisms that dominate the tumor microenvironment. These immunosuppressive strategies include the selective expansion and recruitment of a wide variety of regulatory cells, among which MDSCs play a prominent role.

The overproduction of these cells is considered an important factor in the success or failure of cancer immunotherapy. Elevated levels of MDSCs in the tumor microenvironment correlate with poorer survival in patients with solid tumors and may mediate resistance to immunotherapy. Although their primary function is to suppress T cell and killer cell responses, PMN-MDSCs can generate reactive oxygen species (ROS) and reactive nitrogen species (RNS).

Ultimately, it is now clear that successful cancer immunotherapy will only be possible with a strategy that involves eliminating suppressive factors, as they represent a critical obstacle to the success of these new strategies.

The following documents can be mentioned as background to the invention:

Pérez de la Lastra J.M. et al.: “Hydroxytyrosol and Arginine as Antioxidant, Anti-Inflammatory and Immunostimulant Dietary Supplements for COVID-19 and Long COVID’’, Foods, 2023, Vol. 12, ISSN 2304-8158, describes the joint use of arginine and hydroxytyrosol to treat problems related to COVID 19. Although this publication refers to the improvement in the immune response to certain pathological processes that occur with an increase in MDSCs and, consequently, with a weakening of the immune response, it does not disclose or suggest the possibility of creating an inclusion complex that, in addition to arginine and hydroxytyrosol, includes tryptophan and zinc gluconate, which has been shown to surprisingly improve the bioavailability of hydroxytyrosol and tryptophan, avoiding the need to use organic solvents, surfactants or emulsifiers.

ES 2731554 describes a method for creating an inclusion complex that can comprise hydroxytyrosol. This method is based on the addition and mixing with sonication of the different components of the inclusion complex. However, ES 2731554 does not disclose or suggest the possibility of developing a method capable of obtaining a quaternary inclusion complex according to the present invention.

DESCRIPTION OF THE INVENTION

A first object of the invention is a quaternary inclusion complex of hydroxytyrosol, arginine, tryptophan and zinc gluconate in aqueous solution, comprising:

a) between 5% and 10% by weight of the polyphenol hydroxytyrosol, preferably 7% by weight, b) between 6% and 12% by weight of the amino acid arginine, preferably 10% by weight, c) between 3% and 5% by weight of the amino acid tryptophan, preferably 4% by weight, d) between 1.4% and 2.3% by weight of zinc gluconate, preferably 1.8% by weight, e) water, in the percentage necessary to complete 100% by weight,

and where said inclusion complex in aqueous solution does not comprise any organic solvent, surfactant or chemical emulsifier in its composition.

In a particular embodiment of the invention, the inclusion complex may be presented in solid form, without including water in its composition. In particular, as will be described later in relation to its production process, once the inclusion complex is obtained in aqueous solution, it may then be subjected to a lyophilization process, resulting in the inclusion complex in a solid state, which is completely soluble in water. This is because the claimed inclusion complex is a salt, the components of which are primarily linked by hydrogen bonding molecular interactions, but also by certain ionic and/or covalent interactions.

One of the main advantages offered by the quaternary inclusion complex object of the invention compared to other products described in the prior art is the surprising improvement it presents in terms of the solubility of its active ingredients, hydroxytyrosol, tryptophan, and arginine, in water. In particular, it has been demonstrated that the claimed quaternary inclusion complex allows the solubilization percentage of hydroxytyrosol in water to be increased fourfold and the solubilization of tryptophan in water to be increased fiftyfold, which are completely unexpected values. Furthermore, a solubility of arginine in water of up to 200 g/l is achieved.

Additionally, it offers the advantage of being a harmless complex based on natural compounds, and its formulation does not require the use of chemical compounds, such as solvents (glycerin, propylene glycol, etc.), emulsifiers (such as soy lecithin), or surfactants (such as lipids or proteins, among others), unlike other common products in the state of the art. It is important to note that the components of the claimed quaternary inclusion complex are generally recognized as safe (GRAS), thus favoring its use in nutraceutical, pharmacological, or cosmetic compounds. It is, therefore, an environmentally friendly product that does not harm ecosystems and does not contain toxic substances, based on natural, food-grade compounds. Furthermore, thanks to the use of water as a solvent, the gastrointestinal absorption process is highly effective, unlike products based on organic solvents, surfactants, or chemical emulsifiers, which have poorer water solubility, intestinal absorption, and poor bioavailability.

T lymphocytes are extremely sensitive to tryptophan deficiency, as it causes their cell cycle to arrest. By depleting tryptophan in local tissue environments, the enzyme indoleamine 2,3-dioxygenase (IDO1) (a catalyst for tryptophan) induces T cells to undergo apoptosis. Furthermore, tryptophan deprivation blocks T cell differentiation into cytolytic effector cells. Once they have differentiated into effector cells, T lymphocytes can remain in lymphatic organs to assist B lymphocytes or migrate to sites of infection or the peritumoral environment, where they can perform various functions, including macrophage activation. For this reason, it is important for lymphocytes to mature and differentiate.

Metabolic activity is closely related to T cell fate and function, and specifically, metabolism is essential for their differentiation, polarization, and survival. Elevated L-arginine levels induce a shift from glycolysis to oxidative phosphorylation in activated T cells and promote the generation of central memory-like cells, endowed with increased survival capacity and antitumor activity. Thus, intracellular L-arginine concentrations directly influence the metabolic fitness and survival capacity of T cells, which are crucial for antitumor responses. Indeed, T cells cultured in the presence of high L-arginine concentrations retain the ability to differentiate into Th1 effector cells. Additionally, L-arginine supplementation significantly increases the survival of activated CD4+ and CD8+ cells, indicating that reduced intracellular L-arginine availability negatively affects their survival.

The polyphenol hydroxytyrosol belongs to the class of organic compounds known as tyrosols, which contain an aliphatic alcohol fraction with one or two hydroxyl groups in the benzene group. It is found mainly in olives (Olea europaea), olive oil, grape skin (Brassica napus var. napus), and wine, but it has also been detected in foods such as cloves (Syzygium aromaticum) and fruits. Hydroxytyrosol is an ortho-diphenol with significant biological activity, either in free form or as an ester (oleuropein), and provides olive oil with high stability due to its high antioxidant activity. It also has anti-inflammatory properties, typical of polyphenols in general.

Hydroxytyrosol is considered safe (as a novel food) for human consumption up to a level of 50 mg per kg of body weight per day, according to the European Food Safety Authority (EFSA). In the United States, hydroxytyrosol is also considered a safe ingredient (GRAS).

In the context of the present invention, an immunomodulator is understood to mean a substance or complex that favors and improves the performance of the immune system, both innate and adaptive, in particular by downregulating the expression of MDSC cells. In particular, the claimed quaternary inclusion complex (as well as any composition comprising it) has the ability to modulate and reduce the appearance of MDSC cells in the environment of a tumor, an infection or chronic inflammation, and thus reduce the suppression of T and NK cells, which are fundamental cells in the defense mechanisms of the human immune system.

One of the main advantages of the invention is that it is a quaternary inclusion complex based on natural, safe and biodegradable substances, while improving the bioavailability of hydroxytyrosol, and very significantly that of tryptophan, allowing us to eliminate the presence of organic solvents, surfactants or emulsifiers.

The invention also relates to a composition characterized in that it comprises the claimed quaternary inclusion complex, either in aqueous solution or in solid state, once the inclusion complex has been subjected to a lyophilization process in aqueous solution which, being a salt, retains its solubility in water.

Additionally, the object of the invention is a method for obtaining a quaternary inclusion complex of hydroxytyrosol, tryptophan, arginine and zinc gluconate in aqueous solution comprising the following steps:

a) add between 5% and 10% by weight of the polyphenol hydroxytyrosol, preferably 7% by weight, in preferably deionized or distilled water and stir preferably at a stirring speed of between 300 and 1000 rpm until obtaining a dispersed mixture, partially solubilized in the medium;

b) adding, under stirring (preferably at a stirring speed of between 300 and 1000 rpm), between 6% and 12% by weight of the amino acid arginine, preferably 10% by weight, to the mixture obtained in step (a);

c) adding, under stirring (preferably at a stirring speed of between 300 and 1000 rpm), between 3% and 5% by weight of the amino acid tryptophan, preferably 4% by weight, to the mixture obtained in step (b);

d) adding, under stirring (preferably at a stirring speed of between 300 and 1000 rpm), between 1.4% and 2.3% by weight of zinc gluconate, preferably 1.8% by weight, to the dispersed mixture obtained in step (c);

e) heating to a temperature between 40°C and 60°C, preferably between 45°C and 55°C, maintaining stirring preferably at a speed of between 300 and 1,000 rpm, stopping stirring once said temperature is reached and applying ultrasound with a frequency preferably between 15 and 25 kHz, and more preferably between 20 and 25 kHz, until the total dissolution of hydroxytyrosol and the formation of the quaternary inclusion complex is complete. Preferably, the application of ultrasound (sonication) can be carried out for periods of 2 minutes each, for a total time of 10 minutes; and

f) optionally, allowing the inclusion complex in aqueous solution to cool to a temperature between 20°C and 25°C,

and where said method is carried out without adding organic solvents, surfactants or chemical emulsifiers.

In a particular embodiment of the invention, the claimed method may comprise an additional final drying or lyophilization step (preferably by vacuum evaporation at a temperature of between 20 and 25°C), resulting in a lyophilized inclusion complex in a solid state. As described above, since the inclusion complex itself is a salt, once lyophilized it retains the property of being completely soluble in water.

Additionally, the object of the invention is a quaternary complex as claimed, either in aqueous solution or in solid state, as well as any composition comprising it, for use as a medicine, preferably for oral administration, although it can also be supplied parenterally.

Additionally, the object of the invention is a quaternary complex as claimed, either in aqueous solution or in solid state, as well as any composition comprising it, for use as an immunomodulator (i.e., capable of stimulating the immune system), as a regulator of the vascular system and/or to down-regulate the concentration of MDSC cells.

Finally, the object of the invention is the use of the claimed quaternary complex (as well as any composition comprising it) as a nutraceutical (understood as an alternative product to a medicine with physiological benefits) or as a cosmetic, particularly for topical use in creams.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1.- FIG. 1A. Image of the structure of the resonance forms of the guanidinium group; and FIG. 1B. Spatial average structure of guanidinium.

Figure 2.- FIG. 2A. Molecular structure of hydroxytyrosol and FIG. 2B. Spatial structure of hydroxytyrosol. Energy content of -5.91 kcal/mol.

Figure 3.- FIG. 3A. Molecular structure of arginine and FIG. 3B. Spatial structure of arginine. Energy content of 7.2 kcal/mol.

Figure 4.- FIG. 4A. Molecular structure of tryptophan and FIG. 4B. Spatial structure of tryptophan. Energy content of 3.76 kcal/mol.

DETAILED DESCRIPTION OF THE INVENTION

As previously described, the present invention offers a novel development for increasing the solubilization of hydroxytyrosol, tryptophan, and arginine in water, which has not been described to date in the technical literature. The novel quaternary inclusion complex claimed increases the solubility of hydroxytyrosol in water by up to four times and the solubility of tryptophan by 50 times. Additionally, it increases the solubility of arginine in water by up to 200 g/l and is especially indicated for use in the development of immunomodulatory and vascular system regulating compositions.

In the context of the present invention, the formation of the complex occurs as a consequence of the loss of the proton by a phenolic hydroxyl from the aromatic ring of hydroxytyrosol, which is transferred from this molecule to the resonant structure of the guanidinium group, which becomes positively charged. The phenolic hydroxyl is more acidic than that of the aliphatic group and is therefore more reactive, with a greater tendency to donate the proton and become charged with a free radical that is delocalized by resonance along the benzene aromatic ring.

The efficacy of the product of the present invention, combined with its high bioavailability, makes it a truly advantageous product compared to the nutraceutical, pharmacological, and cosmetic compounds known on the market, which barely dissolve 0.012% of tryptophan and 3% of hydroxytyrosol in water. Furthermore, the claimed quaternary inclusion complex is composed of effective natural components, thus avoiding the use of chemicals responsible for toxic effects on the environment or with undesirable secondary consequences for the human body.

As described above, it is worth noting that, thanks to the chemical composition of the claimed quaternary inclusion complex, the solubility of tryptophan has been surprisingly and unexpectedly increased, as has that of hydroxytyrosol, albeit to a lesser extent. In fact, it has been shown that the solubility of hydroxytyrosol increases with the concentration of arginine, present between 6% and 12% by weight, thus facilitating the formation of the quaternary inclusion complex between the three compounds through weak or short-range molecular interactions between the molecules of said compounds in water. In particular, these interactions consist of hydrogen bonds between the compounds described, that is, the water-soluble ones (arginine and gluconate), the insoluble ones (tryptophan), and the partially soluble ones (hydroxytyrosol). Hydrogen bonding is an electrostatic force that provides great stability, but less than covalent or ionic bonds. The action of ultrasound, which is applied during the method of obtaining the quaternary inclusion complex, is essential for the appearance of these bonds.

In particular embodiments of the present solution, the quaternary inclusion complex is formed by the described compounds, giving rise to a composition in aqueous solution with a pH between 8 and 11, of cationic character due to the presence of the guanidinium group of arginine, as shown in Figure 1, with a great tendency to capture the proton of the phenolic hydroxyl group of hydroxytyrosol and the carboxyl group of tryptophan, and become positively charged, giving the cationic character to the complex formed. In preferred embodiments, the pH of the aqueous solution will be between 9 and 10.

A major advantage of the claimed quaternary inclusion complex is that it does not require the addition of organic solvents, emulsifiers or surfactants to improve its solubility and bioavailability in aqueous solution, unlike other alternative products in the state of the art, which carry a certain risk of toxicity.

Therefore, this is a highly advantageous solution compared to the compounds currently on the market, since a simple preparation of the quaternary inclusion complex yields a highly stable product, which is transported in a more elemental manner than other alternatives based on oils and emulsifiers, polyelectrolytes, as well as other more complicated encapsulation products, with the resulting economic savings. Although it is known that the stability of solutions can be improved by adding surfactants, for reasons of environmental sustainability, the product object of the invention does not contain them in its composition.

In preferred embodiments of the present invention, the immunostimulatory and regulatory quaternary inclusion complex of MDSC cells allows dilutions in successive quantities of water, maintaining the solubility of the compounds of the quaternary inclusion complex in a surprising way, without precipitating in the water or without forming an isolated powder on the free surface of the liquid.

Because arginine forms a quaternary inclusion complex that increases the solubility of hydroxytyrosol at least 4-fold and the solubility of tryptophan 50-fold, in particular embodiments of the invention the quaternary inclusion complex will maintain its solubility in water even in quantities of 1 liter of quaternary inclusion complex diluted in water in an amount between 30 liters and 100 liters. Surprisingly, it has been verified how the claimed complex does not end up precipitating in the mixture as the amount of water in the solution increases. This is due to the phenomenon described above, regarding the bioavailability of the quaternary inclusion complex so that it can be applied to humans and can have a greater immunostimulatory and regulatory effect on MDSC cells, as is the case with the present invention. This is of significant importance, since when this composition enters the human body, it is progressively metabolized and diluted in plasma and internal body fluids, maintaining its solubility and bioavailability.

EXAMPLES

1. Procedure for obtaining and characterizing a quaternary inclusion complex as claimed.

It starts with the production of the complex between the three compounds (hydroxytyrosol, arginine and zinc gluconate), according to the following composition and procedure:

To 772 g of deionized or distilled water are added 70 g of powdered hydroxytyrosol and, after 5 minutes of stirring, so that the powder is partially solubilized in water and the remainder is evenly distributed, 100 g of the amino acid arginine, 40 grams of the amino acid tryptophan and 18 g of zinc gluconate are added under stirring (500 rpm). The mixture is heated to a temperature of 40°C to 45°C and ultrasound is applied, these conditions being necessary and sufficient for the formation of the quaternary inclusion complex in water. The different components (hydroxytyrosol, arginine and zinc gluconate) are mixed by stirring and the resulting complex is stabilized and homogenized by directly introducing the sonicator head of the ultrasonic equipment into the solution (at a frequency of 20 kHz). Sonication is carried out for periods of 2 minutes each, for a total of 10 minutes, without allowing the solution to heat above 50°C. The resulting solution, protected from light, changes color to an ochre-yellow. The formed quaternary inclusion complex can then be packaged. The pH of the resulting solution must not be altered to prevent the formation of undesirable precipitates. The resulting aqueous solution can be lyophilized to obtain a powder that can be compacted into tablets, according to standard pharmacopoeial techniques.

2. Analysis and computer determination of the formation of the inclusion complex between hydroxythymsol and arginine.

Computational verification of the complex formation is based on the study of its energy content, which was calculated using ChemBio3D Ultra 16.0 software (Perkin-Elmer Inc., Waltham, MA, USA) and the Molecular Mechanics (MM2) calculation method. Calculations were performed to obtain the energy of the 3D molecular structure by analyzing the ground-state energy. The lower the final energy, the greater the stability of the molecule. MM2 is a method used to determine the geometry, molecular energies, vibrational spectra, and enthalpies of formation of molecules in their ground state. It is commonly used to determine the behavior of large molecules of biological and pharmaceutical importance. It is often used to determine the geometries of large molecules, such as those of biological and pharmaceutical importance, which are beyond the reach of more intensive molecular orbital-based methods. For this reason, MM2 is not useful for modeling transition states in chemical processes with a large spectrum of experimental steps.

Several calculations were performed to obtain the energy of different conformations of the same 3D complex, analyzing and displaying those with the lowest formation energies. Different three-dimensional conformations were estimated for the formed complex. The total energy content, expressed in kcal/mol, of each molecule is described by the sum of the following interactions:

E<Total>= E<stretch(“stretch>m<g”)>+E<Bending(“Bend>m<g”>’<)>+E<Torsion(“Torsion”)>+ E<| non-bonded interaction(“Non-bonded inte”)>

To verify the energy content of the complex formed by the two, its conformational structure and its interrelation through hydrogen bonding were studied. Different spatial possibilities for the formation of the tryptophan-arginine complex were then presented, as it is the most important, given tryptophan's zero solubility in water.

Table 1

From the energetic study of the different possibilities for salt formation between tryptophan and arginine through hydrogen bond formation, it is concluded that the sixth study is the most energetically favorable and is therefore the one that will occur, since the protonation of the guanidinium group of arginine and the free amino group of tryptophan occurs through the transfer of protons from the carboxylic groups present in both amino acids.

3. Treatment of cancer patients for the stimulation and improvement of the immune system and regulation of MDSC cells with the compound prepared in Example 1

A small-scale, two-year trial was conducted with five cancer patients aged between 50 and 75 years, receiving chemotherapy, radiotherapy, or immunotherapy. Treatment with the described compound was administered during the intervals between conventional treatments. The test consisted of ingesting the inclusion complex three times daily as described in Example 1. Analyzing their general condition and using blood samples, the results verified the maintenance of PMN-MDSC and M-MDSC cells at concentrations that doubled the usual values in healthy individuals, whereas in some cancers they can multiply by up to 10 times. Clinical reports indicated that these patients had fewer circulating MDSCs than the expected average, lower expression of iNOS and arginase 1 in these cells, and a higher number of spontaneously generated tumor-specific T cells. A lower production of ROS generated by MDSCs is observed through the upregulation of the transcription factor Nrf2, which regulates the expression of antioxidant proteins and enzymes that protect against oxidative damage present in the tumor microenvironment.

Claims (12)

1. Quaternary inclusion complex of hydroxytyrosol, arginine, tryptophan and zinc gluconate in aqueous solution, characterized in that it comprises: a) between 5% and 10% by weight of hydroxytyrosol, b) between 6% and 12% by weight of arginine, c) between 3% and 5% by weight of tryptophan, d) between 1.4% and 2.3% by weight of zinc gluconate, e) water, in the percentage necessary to complete 100% by weight, and where said quaternary inclusion complex in aqueous solution does not comprise any organic solvent, surfactant or chemical emulsifier in its composition. 2. Inclusion complex according to claim 1, wherein said inclusion complex comprises: a) 7% by weight of hydroxytyrosol, b) 10% by weight of arginine, c) 4% by weight of tryptophan, d) 1.8% by weight of zinc gluconate, e) water, up to 100% by weight, and where said quaternary inclusion complex in aqueous solution does not comprise any organic solvent, surfactant or chemical emulsifier in its composition. 3. Inclusion complex according to claim 1 or 2, wherein said inclusion complex is lyophilized, in a solid state, without containing water in its composition. 4. Composition characterized in that it comprises an inclusion complex according to any one of claims 1 to 3. 5. Method for obtaining a quaternary inclusion complex of hydroxytyrosol, tryptophan, arginine and zinc gluconate in aqueous solution, characterized in that it comprises the following steps: a) add between 5% and 10% by weight of the polyphenol hydroxytyrosol in water and stir until a dispersed mixture is obtained, partially solubilized in water; b) adding, under stirring, between 6% and 12% by weight of arginine to the dispersed mixture obtained in step (a); c) adding, under stirring, between 3% and 5% by weight of tryptophan to the dispersed mixture obtained in step (b); d) adding, under stirring, between 1.4% and 2.3% by weight of zinc gluconate to the dispersed mixture obtained in step (c); e) heat to a temperature between 40°C and 60°C, maintaining stirring, stop stirring once said temperature is reached and apply ultrasound until the total dissolution of the hydroxytyrosol and the formation of the quaternary inclusion complex is complete; and where said method is carried out without adding any organic solvent, surfactant or chemical emulsifier. 6. Method according to claim 5, wherein the stirring speed in steps (a) to (d) of the process is between 300 and 1000 rpm and where the application of ultrasound in step (e) of the process is carried out with a frequency between 15 and 25 kHz. 7. Method according to claim 5 or 6, wherein said method comprises a final step of lyophilizing the quaternary inclusion complex in aqueous solution until obtaining a quaternary inclusion complex in solid state. 8. Use of a quaternary inclusion complex according to any one of claims 1 to 3 as a nutraceutical. 9. Quaternary inclusion complex of hydroxytyrosol, arginine, tryptophan and zinc gluconate according to any one of claims 1 to 3 for use as a medicament. 10. Quaternary inclusion complex of hydroxytyrosol, arginine, tryptophan and zinc gluconate according to any one of claims 1 to 3 for use as a medicament for oral administration. 11. Quaternary inclusion complex of hydroxytyrosol, arginine, tryptophan and zinc gluconate according to any one of claims 1 to 3 for use in stimulating the immune system. 12. Quaternary inclusion complex of hydroxytyrosol, arginine, tryptophan and zinc gluconate according to any one of claims 1 to 3 for use in down-regulating the concentration of MDSC cells.