WO2021000057A1

WO2021000057A1 - Formulation that allows the protection of polyphenols, particularly those obtained from bee pollen extract

Info

Publication number: WO2021000057A1
Application number: PCT/CL2019/050055
Authority: WO
Inventors: José Vicente GONZÁLEZ ARAMUNDIZ; María Javiera ÁLVAREZ FIGUEROA; Rayen Yanara VALDIVIA OLIVARES; Raquel BRIDI; Gloria Del Carmen MONTENEGRO RIZZARDINI
Original assignee: Pontificia Universidad Católica De Chile
Priority date: 2019-07-04
Filing date: 2019-07-04
Publication date: 2021-01-07

Abstract

The invention consists of a formulation that allows the protection of polyphenols, particularly those obtained from bee pollen extract, such as to maintain their antioxidant activities and enable their use as food additives. This formulation corresponds to a triple nanoemulsion (water-oil-water, W/O/W) which comprises chitosan, polyphenols and other components that make it possible to properly maintain the W/O/W phases.

Description

FORMULATION THAT ALLOWS PROTECTING POLYPHENOLS, IN PARTICULAR THOSE OBTAINED FROM AN EXTRACTION OF APICULTURAL POLLEN

Field of the invention.

The invention consists of a triple nanoemulsion (water-oil-water, W / O / W, for its acronym in English) that allows to protect polyphenols, in particular those obtained from pollen extraction, in order to maintain and enhance their antioxidant activities , antibacterial and be able to use them as food additives. The nanoemulsion comprises chitosan, polyphenols, and other components that allow to maintain the W / O / W phases correctly.

Background.

Oral administration is usually the most common route for the delivery of bioactive agents that have preventive or therapeutic effects, due to the good acceptance and adherence of patients. However, many of the bioactive compounds cannot be delivered by this route, due to the intrinsic physiological conditions of the gastrointestinal tract (Gl) that can decrease its solubility, stability and absorption; therefore, its bioavailability will be reduced. Different nanosystems have been developed in the art, which present a series of advantages compared to a larger-scale delivery system (micrometers). The main nanosystems that have been developed include: liposomes, polymeric micelles, polymeric nanoparticles, nanoemulsions and multiple nanomulsions.

Multiple nanoemulsions have their origin in multiple emulsions, which can be classified into two types: oil-in-water-in-oil (or in English: oil-in-water-in-oil, 01 / W / 02) or water in oil in water (in English: water-in-oil-in-water, W1 / 0 / W2) being the latter one of the most used. It is then a 3-phase system; in the latter case, 2 of them are aqueous (internal and external) and one oily; being the interfaces stabilized by various surfactants. Thus, when multiple emulsions reach a size on the nanometer scale, they are called multiple nanoemulsions. In recent decades, these types of nanoemulsions have attracted the interest of researchers in the cosmetic, pharmaceutical and food industries. Among its main advantages is its ability to encapsulate hydrophilic components and allow their absorption orally, unlike conventional O / W nanoemulsions that only have the ability to encapsulate lipophilic compounds. In addition, they give the possibility of jointly administering hydrophilic and lipophilic components in a single vehicle, since it is possible to dissolve active ingredients in both the aqueous phases and the oily phase together. These nanosystems also provide other advantages for bioactive compounds, including: masking the unpleasant taste or odor of the compounds, protecting labile components during digestion, improving the absorption profile or controlling the release of assets, among others. In the process of formulating nano-sized vehicles and their future administration in vivo, the choice of biocompatible excipients is essential, one of the most used being chitosan. On the other hand, although the incorporation of high-quality excipients and compatible with the design of nanocarriers is very relevant, the development of novel nanosystems for the delivery of bioactive compounds is not only reduced to the choice of components. A great challenge in the formulation and design phase is the optimization of the manufacturing processes for nano-sized vehicles, as well as the concentrations of each component. Therefore, a great deal of experimental effort is required to achieve a specific combination of excipients for a particular bioactive compound, and the choice of such excipients is not at all obvious.

Products derived from bee hives (honey, pollen and propolis) represent a rich source of potentially bioactive compounds that have been used for various purposes over time, including the manufacture of nutraceuticals. It is because of this, that the existence of a beekeeping product that stands out for its beneficial properties for health and that has been rarely used is striking, this is the case of bee pollen. Bee pollen is produced by bees from thousands of pollen grains that it collects in various flowers, mixing them with nectar and salivary substances to form a compact granule called bee or corbicular pollen. The chemical composition of bee pollen depends on its botanical and geographical origin. In general, it includes amino acids, lipids, vitamins, macro and micronutrients, and polyphenols such as quercetin, caffeic acid, rutin, pinocembrin, apigenin, chrysin, galangin, campherol, among others. Studies indicate that these phenolic compounds, like flavonoids, are largely responsible for the biological activities of bee pollen, including its antioxidant and antibacterial potential. The antioxidant capacity of bee pollen is due to the ability of polyphenols to inactivate reactive oxygen species, such as free radicals. In various pathologies and conditions such as aging, there is an increase in the production of these reactive species that can lead to various oxidative damages. These oxidative processes are counteracted through the consumption of antioxidant compounds such as polyphenols.

Unfortunately, part of the benefits that the incorporation of polyphenols and antioxidant substances in nutraceuticals could bring are limited. Due to the poor stability of polyphenols, using complex matrices rich in these components is currently a challenge, since these compounds are affected by processing and storage conditions, in addition to having low solubility in water and poor organoleptic properties. To this is added that the bioavailability of polyphenols is reduced due to their low bioaccessibility, susceptibility to degradation in the gastrointestinal tract and poor absorption profile. There is evidence of the use of nanotechnology to preserve the antioxidant activity of molecules with characteristics similar to phenolic compounds, however, There is an unmet need in the art for the development of new nano-sized systems that are capable of encapsulating complex matrices to improve their absorption and hence their bioavailability.

To evaluate the merits of the invention described herein, a brief summary of the most relevant documents present in the art for the present invention is presented. The search was focused on documents related to composition that protect the polyphenols extracted from different matrices, which allow the controlled release of these compounds when administered orally. In general, the analysis carried out suggests that there is no triple nanoemulsion in the prior art, such as that proposed in the present invention.

In order to assess the patentability requirements, a prior art search was conducted, finding some related documents, which are summarized below.

US2015182472A1 describes nanoparticles formed by an active compound (nutraceutical) that can be a polyphenol and where the core (shell core) is formed by chitosan. The problems that polyphenols are sensitive to oxidation and not very stable in aqueous solutions are mentioned.

US2013309313A1 describes a composition as a food product additive. The composition is based on polyphenols (green tea extract), protected by 2 layers (shell) that can be both or just one of chitosan. Layers are described as primary shell and outer shell. The primary layer forms microcapsules, and the secondary layer allows agglomeration of microcapsules.

US2012064136A1 describes an anti-wrinkle cosmetic composition comprising, among other components, polyphenols as antioxidants, and chitosan as humectant.

WO2010134206A1 describes a cosmetic composition that can be in the form of a nanoemulsion, and that comprises polyphenols as antioxidants, among others, and chitosan as a moisturizer, among others.

WO20071 12366A2 describes a method to improve the absorption or bioavailability of resveratrol in animal products, where polyphenols and chitosan, among several other components, are used as additives to improve the bioavailability of resveratrol.

US2007085058A1 describes a nanoemulsion comprising, among others, polyphenols and chitosan. The stability of polyphenols is indicated to be improved. WO2006131234A1 describes cosmetic compositions consisting of nanoemulsions comprising polyphenols and chitosan, among other components.

US20061 10415A1 describes a cosmetic composition for topical application comprising, among various other components, polyphenols and chitosan. In paragraph 50 it is indicated that the composition can be a nanoemulsion.

WO2005084452A1 describes micro- or nanoemulsions comprising polyphenols and chitosan, in addition to other excipients.

The documents considered closest to the state of the art correspond to US2015182472A1, US2013309313A1, WO2010134206A1, US2007085058A1, which generally refer to emulsions, where in some cases these may correspond to simple nanoemulsions, comprising chitosan and polyphenols. The problem to be solved by the present invention can be considered in general, as providing W / O / W nanoemulsions that protect within their interior active nutraceutical compounds, the nanoemulsions being stabilized at their interface by surfactants. In no document in the art the same objective technical problem arises since most nanoemulsions are of the simple type, and do not require a rigorous selection of the excipients used, such as surfactants that stabilize the W / O interactions in the two interfaces. There are no documents in the art which indicate the use of the excipients described in the application that seek to solve the same objective technical problem.

In summary, there are no patents that describe a triple nanoemulsion containing polyphenols and chitosan that has the same composition or combination of excipients as the formulation described in the present invention.

Brief description of the invention

The invention consists of a formulation that makes it possible to protect polyphenols, in particular those obtained from bee pollen extraction, in order to maintain their antioxidant activities and to be able to use them as food additives. This formulation corresponds to a triple nanoemulsion (water-oil-water, W / O / W), which comprises chitosan, polyphenols, and other components that allow maintaining the W / O / W phases correctly.

Detailed description of the invention

The present invention consists of a formulation, which corresponds to three phases: a first internal phase which comprises a hydrophilic solution composed mainly of water; a second intermediate layer which comprises a hydrophobic solution composed mainly of lipids; and a third outer layer which comprises a solution hydrophilic in contact with the external environment, whose function is to protect the active compounds that are inserted in this formulation, delivering a controlled release of said active compounds.

In one embodiment, the inner phase can mainly comprise different amounts of ethanolic pollen extract.

In a preferred embodiment, the ethanolic extract is from bee pollen.

In another preferred embodiment, the composition of the present invention comprises between 0.1 ml_ and 2.0 ml_ of ethanolic extract of bee pollen.

In a more specific embodiment, the amount of bee pollen ethanolic extract is between 0.6 ml_ and 1.5 ml_.

In another embodiment, the hydrophobic solution comprises at least type II propylene glycol monolaurate in combination with other hydrophobic excipients.

In a more specific preferred embodiment, the amount of type II propylene glycol monolaurate is between 100 mg and 1500 mg, preferably between 600 and 1200 mg.

In another embodiment, the hydrophobic solution comprises at least polysorbate 80 surfactant, in combination with other excipients.

In another more specific embodiment, the amount of polysorbate 80 is between 100 and 500 mg, preferably 360 mg.

In another preferred embodiment, the hydrophobic solution comprises at least Lecithin, in combination with other hydrophobic excipients.

In another preferred embodiment, the amount of soy phospholipids with 70% phosphatidylcholine is between 10 and 100 mg, preferably 60 mg.

In another preferred embodiment, the hydrophobic solution comprises at least the poloxamer nonionic surfactant, in combination with other excipients.

In another preferred embodiment, the amount of poloxamer is between 100 and 800 mg, preferably between 620 and 670 mg.

In a specific embodiment, the hydrophobic solution corresponding to the intermediate phase of the formulation comprises propylene glycol monolaurate type II, polysorbate 80, soy phospholipids with 70% phosphatidylcholine, and poloxamer. In another more specific embodiment, the hydrophobic solution corresponding to the intermediate phase of the formulation comprises between 100 and 1500 mg propylene glycol monolaurate type II, between 100 and 500 mg polysorbate 80, between 10 and 100 mg soy phospholipids with 70% phosphatidylcholine, and between 100 and 800 mg poloxamer.

In a more specific embodiment, the hydrophobic solution corresponding to the intermediate phase of the formulation comprises 1.129 mg propylene glycol monolaurate type II, 360 mg polysorbate 80, 60 mg soy phospholipids with 70% phosphatidylcholine, and 699.7 mg poloxamer.

In another preferred embodiment, the hydrophilic solution of the outer layer comprises chitosan HCI.

In another more specific embodiment, the amount of chitosan HCI is between 6 and 30 mg, preferably 16.7 mg.

In another preferred embodiment, the formulation is a nanoemulsion comprising: (1) an internal solution comprising between 0.6 and 1.5 ml_ of a solution of ethanol extract of bee pollen, (2) an intermediate solution comprising between 100 and 1500 mg of propylene glycol monolaurate type II, between 100 and 500 mg polysorbate 80, between 10 and 100 mg Lipoid P75 ^® , and between 100 and 800 mg poloxamer, and (3) an external solution comprising chitosan HCI between 6 and 30 mg.

In an even more specific preferred embodiment, the formulation is a nanoemulsion that comprises: (1) an internal solution comprising 1.2 ml_ of a solution of ethanolic extract of bee pollen, (2) an intermediate solution comprising 1.129 mg Propylene glycol monolaurate type II, 360 mg polysorbate 80, 60 mg soybean phospholipids with 70% phosphatidylcholine, and between 699.7 mg poloxamer, and (3) an external solution comprising 16.7 mg of chitosan HCI.

In a second aspect, the invention also considers the method for obtaining the formulation of the present invention. This method comprises: (1) mixing the components of the intermediate solution by magnetic stirring for 15 min at room temperature, (2) mixing the intermediate solution with the internal solution by magnetic stirring between 5 min to 30 min at room temperature, (3 ) Prepare the external solution with different amounts of chitosan HCI (6, 18 or 30 mg) and poloxamer (700, 600, 360 or 180 mg) dissolved in 3.5 ml_ of ultra pure water, and (4) The multiple nanoemulsion is It is carried out on a nanoscale homogenizing at a speed of between 21,500 and 25,000 RPM using a basic homogenizer for 10 min in an ice bath to keep the temperature below 37 ° C.

In a third aspect of the invention, the formulation of the present invention is used to protect active compounds found in the inner solution. In another preferred embodiment, the composition of the present invention is used to protect active compounds from aggressive environments for their activity.

In a more specific preferred embodiment, the composition of the present invention is used to protect active compounds, where the aggressive environment is the gastrointestinal tract.

In a specific preferred embodiment, the active compound to be protected can be compounds having phenyl radicals in their structure, preferably phenolic compounds.

In an even more specific embodiment, the phenolic compound refers to polyphenols.

In a preferred embodiment of this embodiment the polyphenolic compounds are derived from pollen.

In another preferred embodiment, the phenolic compounds derived from pollen correspond to flavonoids.

In a more specific preferred embodiment, the phenolic compounds and pollen flavonoids are selected from: chlorogenic acid, syringic acid, coumaric acid, synapic acid, ferulic acid, cinnamic acid, epicatechin, myricetin, quercetin, apigenin, kaempferol, chrysin, narigenin, rhamnetin, pinocembrine, galangine, pinobanksin, hesperetin, CAPE (caffeic phenyl ester acid), luteolin, 3-methylquercetin, isorramnetin, tectocrisin, ellagic acid, and rutin. In a particular preferred embodiment, the flavonoids present in pollen correspond to myricetin and quercetin.

Brief description of Figures

Figure 1. Graphic representation of the multiple nanoemulsion designed and the location of the components in each phase.

Figure 2. Evaluation of the amounts of the different components of the multiple nanoemulsions in the particle size function T (F). Evaluation of the variation of components of the external aqueous phase (A). Evaluation of the components of the internal aqueous phase (bee pollen) and the oily phase (Lauroglycol®90) (B).

Figure 3. Evaluation of the amounts of the different components of multiple nanoemulsions in the potential function ZP (F). Evaluation of the variation of components of the external aqueous phase (A). Evaluation of the components of the internal aqueous phase (bee pollen) and the oily phase (Lauroglycol®90) (B). Figure 4. Stability of multiple bee pollen nanoemulsions (NEMP). Under simulated gastrointestinal tract conditions. (A): Simulated gastric fluid without USP pH 1.2 enzyme. (B): Simulated intestinal fluid without USP enzyme pH 6.8. Temperature: 37 ^e C. (Average ± SD, n = 3)

Figure 5. Stability of multiple bee pollen nanoemulsions (NEMP) in phosphate buffered saline at pH 7.4. Assay carried out for 48 h. (Average ± S.D., n = 3)

Figure 6. Stability of multiple nanoemulsions under storage conditions. Trial carried out for 4 months. (A): NEMP: multiple nanoemulsions of bee pollen. (B): NEMB: multiple white nanoemulsions. (Average ± S.D., n = 3).

Figure 7. Stability of polyphenolic compounds during 4 months of storage. Stability of non-encapsulated bee pollen extract (A). Stability of multiple optimized nanoemulsions (Average ± SD, n = 3) (B). Markers used myricetin, quercetin, and cinnamic acid.

Examples

The following are examples of embodiments for the present invention as described above:

Example 1: Preparation of prototype multiple nanoemulsions

The design of multiple water-oil-water (WOW) nanoemulsions was started using the 2-phase emulsification method described by A. Silva-Cunha et al with some modifications. A prototype was developed using a rational design, different excipients for the oil phase, interface surfactants and polymeric coating were tested. Regarding the optimization of the formulation, a set of experiments was developed. Briefly, a primary W / O emulsion was formed using as organic phase Lauroglycol ^® 90 in different amounts (600; 900 or 1200 mg), and the surfactants Span 80 ^® (360 mg), and Lipoid P75 ^® (60 mg). The 3 components of this organic phase were mixed under magnetic stirring for 15 min at room temperature. Then the internal aqueous phase was added, consisting of different amounts of ethanolic extract of bee pollen (1, 5, 1, 0 or 0.6 mL), stirring for an additional 15 min. Subsequently, the second emulsification phase was prepared by adding the external aqueous phase, consisting of different amounts of chitosan HCI (6, 18 or 30 mg) and Pluronic F-68 ^® (700, 600, 360 or 180 mg) dissolved in 3, 5 mL of ultra pure water. The multiple nanoemulsion was carried out on a nanoscale homogenizing at 21,500 RPM using the basic UltraTurrax homogenizer, IKA ^® T25 (IKA, Brazil) for 10 min in an ice bath to keep the temperature below 37 ° C. A series of formulations were made with the objective of incorporating the excipients of interest in the various phases of the multiple nanosystem and developing a prototype formulation. The internal aqueous phase at all times corresponded to the concentrated bee pollen extract in 100% ethanolic solution. The ethanolic extract of bee pollen was incorporated into the formulation in as much quantity as possible, this is due to the fact that nanosystems allow the incorporation of a large quantity of active ingredient versus conventional or simple nanoemulsions.

In order to stabilize interface 1 (internal aqueous phase-oil), the surfactants Lipoid P-75 ^® (lecithin) and Span ^® 80 (sorbitan monooleate) were used. Lipoid P-75 ^® is a naturally occurring amphoteric surfactant that comprises a high percentage of phosphatidylcholine. This surfactant has proven to be safe and has been widely used by industry, primarily because it is a GRAS excipient. Span ^® 80 is a non-ionic surfactant with FDA approval for use in food, so it can be used to stabilize nanocarriers for oral administration of bioactives. The oil phase was optimized by testing various continuous phases or oils of low hydrophilic-lipophilic index (HBL). It was initially tested using Maisine ^® CC as the oil core due to its low HBL (around 1), but it was not possible to stabilize a nanosystem, these formulations did not form emulsions, therefore, this excipient is discarded to be incorporated into the prototype . Later, Labrafac CC ^® was used, this oil corresponds to a saturated fatty acid composed of a mixture of triglycerides (capric and caprylic), an excipient widely used in pharmaceutical formulations and nanomedicine. Favorable results were obtained, however, this oil did not allow to obtain multiple nanosystems with particle sizes lower than 300 nm. Subsequently, an attempt was made to reduce the particle size through the incorporation of another oil, and that would also provide the vehicle with additional characteristics, this is how Gelucire ^® 44/14 and Lauroglycol ^® 90 were used, which have a history of being promoters of the oral bioavailability. The tests carried out with Gelucire ^® 44/14 did not constitute smaller emulsions, probably due to the intermediate HBL that this oil possesses (around 1 1), so it could not constitute the oily phase of the nanosystem. It is important to highlight these results since they demonstrate that the choice of stabilizers described in the art is not obvious, but rather that the inventors demonstrate that only some can be used in the formulation of the invention. The tests with Lauroglycol ^® 90 were successful allowing to obtain nanosystems with smaller particle size compared to those containing Labrafac CC ^® . Lauroglycol ^® 90 is an oil made up of propylene glycol mono- and diesters of lauric acid (C12) [1 16]. This excipient has been shown to increase the bioavailability of low-absorption bioactive compounds and is also suitable for oral administration.

For interface 2 (oil-external aqueous phase) an anionic surfactant (Lipocol HCO-40 ^® ) was initially tested, however, this did not allow to obtain a small particle size and It contributed a negative charge to the system, which contributed to reducing the total positive charge sought for the Z potential. It was then tested, using Pluronic F-68 ^® as surfactant, initially in concentrations of 1 and 3% but it was not possible to stabilize the nanosystem, for this reason it was increased to 20% with respect to the volume of the external aqueous phase (3.5 ml_). Pluronic F68 ^® corresponds to a set of polyoxyethylene-polyoxypropylene-polyoxyethylene (PEO-PPO-PEO) block polymers, widely used as wetting, solubilizing or binding agents; having an adequate profile regarding its safety and compatibility with the oral route. Additionally, Pluronic F68 ^® is known to be an inhibitor of P-glycoprotein, a protein that participates in the transport of active ingredients with substrate specificity, which is widely distributed in the gastrointestinal epithelium and is the main cause of drug efflux.

Finally, we worked on the external aqueous phase incorporating a polymeric chitosan HCI coating. Among the reasons for incorporating chitosan into the formulation are its excellent properties that it can deliver to the multiple nanoemulsion. Among them that it is not toxic, that it does not induce inflammation or provoke the response of the immune system, in addition to being biodegradable. Another advantage of this active is its mucoadhesiveness, since its positive charge allows interaction with the sialic acid of the mucus, which translates into an increase in the residence times of the active ingredients in the absorption site, which leads to increase. Figure 1 graphically shows the multiple nanosystem designed and how its components would be oriented in each layer and interface. Finally, a prototype formulation was obtained that incorporates all the excipients of interest for the rational design of a nanosystem. Table 1 shows the composition of the prototype formulation, which has a particle size of 184 nm, with an IPD of 0.084, which is indicative of a homogeneous population of particles, and a Z potential of +21 mV, highlighting the positive character by the presence of chitosan on the surface of the emulsion.

Table 1. Composition of prototype W / O / W multiple nanoemulsion.

Example 2: Development of improved formulations.

The desired characteristics for the multiple nanoemulsion were the smallest possible size (hopefully less than 100 nm), and a positive Z potential, ideally close to +30. Based on these desired effects, different formulations were produced varying the concentrations of the excipients according to what is indicated in Table 2.

Table 2. Composition and physicochemical characterization of the 6 multiple nanoemulsions made in triplicate.

(Average ± S.D., n = 3).

Figure 2 shows the graphs obtained for the size function T (F) setting in A, the amount of bee pollen extract at 1.5 ml and of Lauroglycol ^® 90 at 1200 mg and in B, the amount of chitosan HCI in 18 and Pluronic F-68 ^® in 700 mg. In Figure 2A it is observed that by varying the amount of Pluronic F-68 ^® , the particle size decreases as the amount of this excipient is increased. On the other hand, it is observed that as the amount of chitosan HCI increases, the particle size also increases, this situation could be due to a greater steric hindrance granted by the hydrophobic tails of the biopolymer. There are studies where the same phenomenon is seen in other types of nanocarriers. On the contrary, in Figure 2B, it can be seen that no difference was found when varying the amount of Lauroglycol ^® 90 or the amount of bee pollen extract. In the case of the Lauroglycol ^® 90 variation, the applicants are the first to report a multiple W / O / W nanoemulsion with these characteristics that incorporates Lauroglycol ^® 90 in its oily phase. There are reports in the literature for primary W / O nanoemulsions where a different phenomenon is observed from that observed in the multiple system, where as the concentration of Lauroglycol ^® 90 increases, the droplet size also increases. increases; which confirms the different behavior between a simple nanoemulsion and a multiple one like the one developed in this study.

In addition, to see if the physicochemical properties of the nanoemulsions varied by varying the batch of bee pollen extract; A study was carried out replicating the same formulation, but with pollen from a different batch and multiple white nanoemulsions. The results for the size did not change drastically, therefore, from what was obtained experimentally, we can have a formulation that encapsulates any batch of bee pollen, since it will not affect its physicochemical properties (data not shown).

For the potential function Z, the three-dimensional graphs obtained are shown in Figure 3. In this case the same variables mentioned above were fixed, but this time the variation of the potential Z was evaluated. In Figure 3A, it is observed that the quantity of Lauroglycol ^® 90 is influential and that there is a limited region in which the Z potential is greater. In the case of bee pollen extract, there are no significant differences in the variation of potential Z when its concentration increases or decreases. In the case of bee pollen, the same behavior was observed as was seen in the case of size, there was no variation in the Z potential when changing the concentration or batch of extract. This reaffirms that the extract constitutes the innermost phase of the multiple nanosystem and that it is protected by the continuous oil phase. In Figure 3B, a situation similar to that described above is observed, that is, there is a very limited region where Z potentials of around +30 mV are obtained. Thus, it is observed that the surfactant Pluronic F68 ^® does not significantly influence the increase in Z potential, however, it does vary significantly as the concentration of chitosan HCI is increased, the highest values for Z potential being found in the range from 10 to 20 mg of this polysaccharide. In the case of Pluronic F68 ^® there is no history of its influence on the potential, however, as it is a non-ionic surfactant it is known that it does not provide electrostatic charge to the system. In the case of chitosan HCI, the variation in Z potential depends on the way in which this biopolymer is incorporated into the formulation. In this case, it is found in solution in the external aqueous phase, so as its concentration increases, the Z potential increases. However, at a given point, as the concentration of this polymer continues to increase, the hydrophobic sites of the biopolymer probably they begin to block the load, reflecting this in a decrease in the surface load.

Based on all the experiments performed, the optimal formulation composition was obtained for the smallest particle size and the highest positive Z potential. This formulation corresponds to the so-called F. To facilitate the understanding of the rest of the work carried out, from now on, this formulation will be called NEMP (multiple nanoemulsion that encapsulates bee pollen extract). And that equivalent formulation that used ethanol as the internal phase aqueous, it was called NEMB (white multiple nanoemulsion). The compositions of both nanosystems are shown in Table 3.

Table 3. Composition of NEMP and NEMB.

NEMP: multiple nanoemulsions of bee pollen. NEMB: multiple white nanoemulsions.

Example 3: Physicochemical characterization of the developed systems.

The characterization of multiple nanosystems was determined by measuring their physicochemical properties such as particle size and polydispersity index using dynamic light scattering (DLS) technique. Furthermore, its Z potential was studied using the Doppler laser electrophoresis technique (Malvern Instruments, UK). To measure size and polydispersity index, 1 pL of formulation (nanosystems) was diluted in 999 pL of Milli Q water. To measure potential Z, the same dilution was used, but this time dispersing the nanosystems in 999 pL of potassium chloride solution 1 mM. Additionally, the optimized nanosystem was characterized using the Nanosight equipment (Malvern Instruments, UK). For this, a dilution of the sample of 1: 45000 was carried out by dispersing the nanosystem in ultra-pure water. a) Study of Morphology.

The morphology of the formulations was observed through scanning transmission electron microscopy (STEM) (FEI Company, United States). Briefly, a drop of the formulation was placed on the surface of a carbon-coated copper grid, subsequently stained for two minutes with 2% w / v phosphotungstic acid and finally the grid was washed with ultra-pure water. The grids were left in a desiccator (Normax, Portugal) for 48 hours to be subsequently analyzed through STEM.

Table 4 shows the physicochemical properties of NEMP, the size and potential Z values predicted by the model being 94 nm and +33 mV respectively, very similar to what was obtained experimentally. In the case of NEMB it is possible to observe that the characterization is very similar, a fact that has been previously explained in the optimization of the formulation, where it is known that the bee pollen extract does not intervene significantly in the particle size of the nanosystems or in their surface charge.

Table 4. Physicochemical characterization of multiple nanoemulsions optimized by supervised learning mathematical model, experimental data.

(Average ± S.D., n = 3). NEMP: multiple nanoemulsions of bee pollen. NEMB: multiple white nanoemulsions. b) Quantification of polyphenol content in encapsulated bee pollen.

To quantify the polyphenol content, high performance liquid chromatography (HPLC) coupled to a diode array detector was used using a Hitachi Chromaster VWR equipment (Hitachi, Japan) with an endcapped Purospher STAR RP-18 column and pre-column (5um, 250 x 4.6 mm), using a stepped gradient method, with a flow of 0.8 mL / min, at a temperature of 37 ° C, injecting 10 mI_. A multistandard polyphenol calibration curve was performed, the running time being 80 min. For this, two flavonoid markers (corresponding to myricetin and quercetin) and a phenolic acid marker (corresponding to cinnamic acid) were chosen. The reason for this decision was the high concentration of these polyphenols in the pollen lot used. Table 5 shows the percentage of pollen encapsulation of the formulations that were made in triplicate during the optimization of the mathematical model.

Table 5. Encapsulation percentage for the markers quercetin, myricetin and phenolic acid present in the encapsulated bee pollen extract. Encapsulation percentage based on non-isolated NEMP.

From the analysis of the encapsulation percentage, it was found that all the formulations efficiently encapsulate the three components or polyphenols of the extract used as markers, finding percentages greater than 80% in all the analyzed nanosystems. Formulation F, which, as noted above, corresponds to the optimized NEMP formulation, curiously presents the same encapsulation percentage for the three markers studied. In turn, formulation C was the one that presented a higher degree of encapsulation, this may be due to the fact that this formulation had the greatest amount of oil and in turn the least amount of bee pollen extract, which could contribute to encapsulate more Efficient all polyphenols, however, it was not considered an optimal formulation due to its larger size (221 nm).

These results allow to demonstrate that not only was it possible to obtain an optimized nanosystem in terms of its physicochemical characterization, but also that the bioactive compounds are homogeneously incorporated into the formulation and in a high percentage. This shows that the composition of the multiple nanoemulsions designed is robust and that in addition to being efficient when incorporating the actives, they are capable of incorporating in a homogeneous and reproducible way bioactive compounds from highly complex natural matrices, in which a high variability.

Example 4: Stability studies of nanoemulsions

The stability of nanometric vehicles was studied under various conditions, this in order to obtain data that would allow to support that the nanosystems will maintain their nanometric size when facing the various pH conditions throughout the gastrointestinal tract in the absorption process, and in storage conditions. Furthermore, in this experimental phase it was not only wanted to evaluate the stability of the nanosystem as a vehicle, but the stability of the polyphenols encapsulated in said nanosystems was also evaluated.

To evaluate the integrity of the nanosystems, stability studies were carried out in different buffers that simulated physiological conditions by monitoring the physicochemical characterization of size and IPD, all these tests were carried out using the optimized multiple nanoemulsion NEMP. The stability of the nanosystems was evaluated in the first instance in 2 pH conditions of interest 1, 2 and 6.8 respectively. These conditions were used because the nanosystems that encapsulate bioactive compounds in their passage to be absorbed orally, will have to face various conditions, among them: a complex gastrointestinal environment due to the presence of metabolic enzymes, the solubility limitations of the active ingredients and extreme pH conditions that contribute to the degradation of bioactive compounds. I know has the antecedent for this work of susceptibility of polyphenolic compounds to extreme pH conditions, so the stability of the nanosystem plays an important role.

In Figure 4 the studies for NEMP at pH 1, 2 and 6.8 are shown; at 37 ^e C. With respect to simulated gastric fluid conditions (pH 1, 2), the nanosystem maintains its structure in the range of 100 nm throughout the test period and a low polydispersity around 0.1, this The latter is indicative that not only was the average size maintained, but also that the particle population remained homogeneous. In the case of the simulated intestinal fluid (pH 6.8), there was a small increase in the size of the nanosystems of approximately 20 nm, however, the vehicle continues to maintain an optimal size range to see its absorption favored and a low polydispersity (<0.2). Due to these results, it is demonstrated that under these pH conditions the multiple nanoemulsion that encapsulates bee pollen is stable for at least 4 hours, sufficient time for future in vivo administration to be absorbed. Finally, the stability of NEMP in phosphate buffered saline (pH 7.4) was evaluated in order to verify its stability under physiological conditions. Figure 5 shows the characterization for size and IPD of the nanosystem during the test, it is shown that the nanosystem is stable for at least 48 hours, maintaining its initial physicochemical characteristics.

Then, stability was evaluated under storage conditions at 4 ^e C, evaluated both NEMP to NEMB order to observe the evolution of the particle size over time. Figure 6 shows the variation in particle size during the 4 months since this test began. For NEMP, it was observed that in the course of 4 months, even when the nanometric size is maintained, it has doubled, however, a low IPD <0.3 was maintained, so the family of particles modified their particle size in a homogeneous way . On the other hand, NEMB tripled in size during the 4 months of the trial, but also maintained its IPD <0.3, demonstrating only one particle population. Additionally, no phase separation was observed in any of the studied nanosystems, the Z potential was maintained at all times in the approximate range of +30 mV. Considering that both nanosystems started the study with similar particle sizes, there is a phenomenon that has allowed the nanosystem that comprises bee pollen to maintain its initial size more stable over time, however, from the reduced number of studies that have multiple nanosystems in the literature , stability studies have not been performed under storage conditions. Nor is there a history of multiple nanoemulsions that contain this distribution of excipients, so it is not possible to attribute said instability to any particular excipient.

It should be noted that although there was an increase in particle size, there is a history that nanosystems below 300 nm are capable of absorbing themselves favored orally. On the other hand, the low IPD of both nanosystems is indicative that they are still stable as non-aggregated nanocarriers, despite having increased their size, this could be due to the fact that they have a favorable Z potential for their stability that has not been affected. in time (> +30).

Example 5: Stability of polyphenols from bee pollen encapsulated in NEMP

In order to verify the stability of the polyphenolic extract encapsulated in the nanosystem (NEMP), two tests were carried out. The first corresponded to the quantification of the marker compounds of said pollen (exposed in 3.2.2.3) after storing the nanosystems for 4 months at 4 ° C; using HPLC for this. The second corresponded to the determination of total phenols carried out in order to quantify the total concentration in the nanosystems of this type of compounds, using the Folin Ciocalteu test.

The polyphenol content present in the stored samples was quantified using the HPLC method. The samples used for this experiment were the same ones used for the particle size stability studies under storage conditions. Figure 7 shows the concentrations of the markers chosen for each of the samples. In Figure 7A, it is possible to analyze that the concentration of all the polyphenols present in the sample of non-encapsulated bee pollen extract has decreased over time, with quercetin concentration being the most affected with almost 33% degradation, followed by the Myricetin with 23% and cinnamic acid with a 19.8 loss of the active compared to the initial concentration. In Figure 7B the stability of the bee extract that has been encapsulated in the nanosystems can be observed. From the analysis it is obtained that none of the concentrations of the flavonoid markers decreased by more than 20%, in the case of the cinnamic acid marker its concentration was not affected over time, it should be noted that the concentration of quercetin, which is our most stable marker which was quite affected in the non-encapsulated pollen sample, had a much lower decrease (8%).

These results were obtained by comparing the initial concentration of each polyphenol in the extract sample with its concentration after 4 months of storage, it should be noted that these concentrations are not directly comparable with the NEMP samples, because in the encapsulation process of extract, there is a loss of assets due to the fact that this process does not have a 100% yield and the polyphenols, as mentioned in this work, are unstable. However, this does not harm the analysis, since what was sought to observe is how the concentration of the chosen markers evolved over time, and if it was affected by the storage conditions at 4 ° C or the nanosystem. With these tests it is demonstrated that NEMP is not only capable of maintaining its physicochemical stability under physiological conditions, but also that in aqueous solution, encapsulated actives are preserved in better conditions than non-encapsulated polyphenols in storage. Example 6: Antioxidant capacity of the developed nanoemulsions

As an additional evaluation strategy of the designed nanosystems, it was decided to use the ORAC-FL assay to evaluate the antioxidant potential of NEMP. This method reflects the ability of compounds containing phenolic groups to neutralize free radicals thermally induced by the activity of the compound AAPH (2,2' -AZO-bis (2-Amidine-propane) dihydrochloride). In the presence of oxygen, these radicals are interconverted to peroxyl radicals that are capable of oxidizing a fluorescent marker, in this case fluorescein (FL). Thus, when interacting with the marker, it loses its fluorescence over time, making it possible to record the decay curves of the marker in the presence or absence of antioxidant substances. To perform this analysis, the ORAC value of NEMP and NEMB was quantified on the day of their formulation and after one month of storage at 4 ° C (see Table 6). This in order to evaluate if there was a variation in the antioxidant potential of the formulation over time. The test was also carried out using bee pollen extract diluted in the same proportion as multiple nanoemulsions (NEMP) as a control with the aim of observing if there was any improvement in its antioxidant potential or deterioration when encapsulated polyphenols were found. Additionally, both nanosystems studied (NEMP and NEMB) were used in their entirety and destroyed, this in order to evaluate if there is any difference between the samples that comprise the encapsulated polyphenols and the samples in which the polyphenols from the nanosystems are released. The results obtained for this test are shown in Table 6, in it it is possible to see the ORAC values for the 5 samples analyzed at the different times. It is important, before interpreting the results, to consider that the ORAC values should be taken as reference values of the antioxidant potential that a sample has and not as absolute values, since being complex matrices originating in the vast majority of cases from natural sources. For this reason, the antioxidant potential of a species of plant origin varies in each sample, even when it is the same variety, due to the mere fact that they have experienced different environmental conditions. On the other hand, there are factors that generate variability between one experiment and another (times of exposure to light of the samples, interpretation of areas under the curve, etc.), which are specific to the experiment and are considered in the standard deviation of each point. Despite all this, this test allows us to evaluate and compare the antioxidant potential of highly complex matrices.

Table 6. Antioxidant capacity of multiple nanoemulsions in the time obtained for samples stored at 4 ° C.

(Average ± S.D., n = 3). NEMP: multiple nanoemulsions of bee pollen. NEMB: multiple white nanoemulsions.

From the analysis of the results obtained in Table 6, it can be seen that at a general level that the ORAC values obtained at time 0 and at 1 month of storage are quite different for each of the samples. Regarding the control of bee pollen extract that was stored under the same conditions as the nanoemulsions, its ORAC value decreased considerably in 1 month, this supports the results obtained in point 4.3.2.1, where it is appreciated that the non-encapsulated pollen sample It is less stable over time, showing that the conservation of polyphenols is affected. With respect to the NEMP samples, it can be seen that although its ORAC value has decreased over time, it has been a decrease in a percentage well below 60% for the non-encapsulated pollen sample. Furthermore, bee pollen encapsulated in NEMP shows to have an antioxidant potential greater than double that of the non-encapsulated sample at time zero. The foregoing can be explained by analyzing what happened by the NEMB control that corresponds to the same formulation, but without the encapsulated pollen extract, where it is observed that the formulation provides antioxidant potential and that this effect would be synergistically enhanced, since initially its ORAC value is lower than the ORAC value shown by the bee pollen sample. Finally, analyzing the difference in the antioxidant capacity of the destroyed and non-destroyed samples, it is observed that by destroying NEMP the nanosystem increases the antioxidant potential. This phenomenon could be explained by the release of polyphenols that remain encapsulated, being more available to exert their antioxidant effect. In the white NEMB formulation the same phenomenon is observed, but with a lower ORAC value increase compared to NEMP. This result is consistent with what was observed, since this formulation does not have polyphenols, so that when it is destroyed, more excipients are available that could have an antioxidant effect by this mechanism for evaluating antioxidant capacity. By way of comparison, it is possible to express the antioxidant potential of the NEMP formulation as a function of its ORAC value per 1 g of fresh pollen generated by the extract that is encapsulated. Thus, we can express your ORAC value at time zero at 451 pmol ET // 1 g of pollen. Reviewing the ORAC values provided by the Institute of Nutrition and Food Technology (INTA) and expressing them as a function of pmol ET // 1 g of fresh fruit, we can see that the values reported for each 1 g of fresh fruit, for example for blueberries they are 54.81; in the case of the Hass avocado produced in our country it is 48.53 and for a fruit like fresh maqui recognized for its antioxidant properties it is 371.74 pmol ET // 1 g of fresh fruit. This previous analysis allows us to understand that although it is true that bee pollen is an excellent source of antioxidants, encapsulated bee pollen further improves its property, being even superior and comparable to a sample of fresh maqui.

Example 7: Antibacterial capacity of optimized formulations

In the first instance, a screening of different samples and their effect against the microorganisms of interest was carried out using the agar diffusion technique. Among the samples used are multiple NEMP nanoemulsions, using white nanoemulsions (NEMB) as a control in order to evaluate if there is any antibacterial effect inherent to the nanosystem only as a vehicle. Additionally, the impact on the antibacterial potential of the non-encapsulated bee pollen extract preserved at 4 ° C and -80 ° C was explored, with the aim of evaluating whether the storage conditions harm in any way the antibacterial potential of the ethanolic extract and pollen. On the other hand, the antibacterial effect delivered by the excipient chitosan HCI in solution at the same concentration that was found in the formulations was evaluated.

To complement the study, the same samples were used, but this time the nanosystems were destroyed, with the aim of releasing the encapsulated polyphenols and evaluating whether there was an improvement or a worsening of the antibacterial potential. These samples were twice diluted with respect to the aforementioned samples. Finally, a 50% ethanol control was used, in order to know the antimicrobial effect produced by the solution with which the formulations are destroyed. Experiments carried out with ethanol as a control by the botanical laboratory show that there is an antibacterial effect over 50%. Table 7 shows the results obtained for the screening experiment in 4 bacteria of interest. When analyzing the results, it is observed that only NEMP and NEMB had an important inhibition halo that could be measured for the microorganism S. pyyogenes. When this result was confirmed, an agar diffusion test was performed only for this microorganism using the same samples detailed above. It is also possible to corroborate that, at the concentrations of ethanol used, this excipient does not have any effect on the bacteria used.

Table 7. Exploratory screening tests in 4 bacteria of interest. Destroyed formulations do not exceed 50% ethanol in the sample.

Table 8 shows the results obtained for samples by diffusion on agar and serial microdilutions carried out in the sensitive microorganism S. pyogenes. When analyzing the agar diffusion test, it is possible to observe that the samples of bee pollen encapsulated in NEMP and its NEMB control show an important inhibition of bacterial growth represented as inhibition halo in mm. As the difference between them is only 1 mm, it does not allow conclusions to be drawn regarding the contribution of the encapsulated pollen extract in the antibacterial effect, however, this shows that there is an important contribution of the formulation as a vehicle.

Table 8. Diffusion assays in agar and serial microdilutions in microorganism S. pyogenes.

Regarding the analysis of the samples of non-encapsulated bee pollen extract preserved at 4 ° C and -80 ° C, there is no difference in the inhibition halo, which shows that the antibacterial effect provided is not related to the storage temperature of the extract in the short term (1 month). Additionally, this result is consistent with that previously reported for this pollen batch (FIAV1827). In the case of the formulations destroyed, unlike what was expected, there was also no significant difference between NEMP and NEMB, which only allows us to demonstrate that although the developed nanoemulsions have an important antibacterial effect; It is not possible to analyze the contribution of the encapsulated extract with this test, which It should be noted that it was only exploratory in nature. Regarding the HCI chitosan solution, this had an inhibition halo of zero in both cases, which indicates that the antibacterial effect found in literature is not only due to the incorporation of this excipient, but rather could be indicative that it is its incorporation into a nanometric vehicle which contributes to this effect. Finally, it is possible to highlight that the bee pollen extract encapsulated in NEMP has an antibacterial effect close to double with respect to its non-encapsulated simile, however, with the scope of this exploratory trial it is not possible to evaluate to what extent this nanosystem is superior to vehicle not incorporating extract (NEMB). On the other hand, the destroyed samples did not have significantly higher inhibition halos compared to the non-destroyed ones (only 1 mm difference). It should be noted that in these exploratory tests an arbitrarily defined sample concentration was used, in the case of multiple non-destroyed nanoemulsions these were not diluted, but the destroyed samples by their nature were 2 times diluted. The foregoing suggests that a lower concentration sample amount is required to obtain the same antibacterial effect. In order to analyze this last antecedent, serial microdilution tests were carried out in order to explore where the minimum inhibitory concentration (MIC) could be located in said samples. When analyzing the non-destroyed samples of NEMP and NEMB, we can conclude that there is no difference in the antibacterial contribution up to the tested dilution (8), which would be indicative that the MIC is above that dilution. If it was possible to demonstrate with this experiment and the subsequent performance of the agar diffusion test with the 8 dilutions, that there was no bacterial growth in the wells, and also bacterial death occurred, which is why it is shown that multiple nanoemulsions have a bactericidal effect on the sensitive microorganism tested. Regarding the samples of bee pollen extract preserved under different conditions, a slight improvement in the antibacterial effect provided by the extract preserved at 4 ° C is striking, however, with the scope of this test it is not possible to evaluate the reason that originated this change.

Regarding the destroyed samples, exactly the same behavior was observed as the non-destroyed ones, however, it is this antecedent that makes us suspect that the MIC is well above the 256 dilution, since it must be remembered that these destroyed samples had a a dilution factor of two prior to the test given by the incorporation of solvent to destroy the nanosystems. Regarding the chitosan sample analyzed in two different cases, a slight improvement was observed in the sample dissolved in destruction solvent. There is a history of antibacterial activity of this polymer at concentrations of 5 mg / mL, considering that the nanosystem incorporates it at 2.78 mg / mL, this could be the reason that in solution it is not active or its effect is mild. There is a history that chitosan activity is found against Gram positive strains, which is consistent with the sensitivity presented by S. pyogenes. Finally, when comparing the NEMP samples both destroyed As not destroyed with the non-encapsulated pollen extract, up to the scope of this test, the concentration of pollen extract necessary to inhibit bacterial growth is considerably lower compared to the non-encapsulated pollen, however, only subsequent MIC search tests They will be able to clarify these doubts, and explain what is the contribution of the extract to the antibacterial potential NEMP compared to NEMB.

Example 8: Multiple nanoemulsion permeability studies (NEMP) using PAMPA

In order to study the permeability of the nanosystems that encapsulate ethanolic extract of bee pollen, an in vitro PAMPA permeability study was carried out. It is necessary to remember that the polyphenols present in the bee pollen extract are not only unstable, but also that their bioavailability is greatly reduced as a result of their low permeability, which ultimately limits the possibilities of obtaining a benefit when ingesting these compounds, as already was exposed in this work. It is due to the above, that the performance of this permeability test is supported, since it seeks to evaluate if this parameter is favored when the polyphenols present in the bee pollen extract are incorporated into the optimized nanosystem (NEMP). The precedent for this test is the excellent correlation between the flux through PAMPA and the degree of absorption for drugs when they are absorbed mainly by passive diffusion. There is no absolute rule of what conditions should be used in this assay, however, Avdeef et al. Provide some suggestions, for example, regarding working concentrations that may be in the range of 50 mM to 500 mM. Said concentration will depend on the detection limit of the quantification method with which one is working. Regarding the incubation times of the assay, ranges between 4 and 15 hours are suggested, finally the suggested pH conditions vary between 5 and 7.4, however, all these recommendations refer to pure molecules and not to complex matrices with a high variability such as bee pollen extract. Thus, after an extensive search for works that incorporated PAMPA methodologies, it was decided to follow the PAMPA protocol reported for quercetin-encapsulating nanosystems, with some modifications.

Table 9 shows the permeability results for each of the samples, in order to contrast the performance of unencapsulated pollen versus that found incorporated in the NEMP. For this, bee pollen extract was used in the same dilution of NEMP. On the other hand, verapamil hydrochloride was used as a positive control at a concentration of 100 mM, as this drug is a marker of high permeability.

Table 9. Permeability of multiple optimized nanoemulsion obtained by PAMPA.

The permeability values determined after 5 h of incubation at 25 ° C in pH 6.8 saline buffer.

(Average ± S.D., n = 3).

Before interpreting the data, it is necessary to remember that compounds with Pe values <0.1 x 10 ^-6 cm / s are poorly absorbed (<30% absorption in vivo), values between 0.1 and 0.9 x10 ⁶ cm / s are moderately absorbed (30-70% absorption in vivo) and compounds with Pe values> 0.9 x 10 ⁶ cm / s have high oral absorption (<70% in vivo) [67, 68]. It was found that the permeability of the markers cinnamic acid, quercetin and myricetin in the bee pollen extract is 0; in the case of nanosystems with encapsulated bee pollen, it is possible to observe that both quercetin and cinnamic acid had permeability values greater than 0.9 x10 ⁶ cm / s. The flavonoid myricetin had quantification problems due in part to its instability. Finally, the verapamil hydrochloride control had a permeability value within the expected, which indicates that the methodology used in the trial was applied correctly. With these results, it could be estimated that the polyphenols studied in pollen that are encapsulated in NEMP will have an absorption greater than 70% in vivo [67, 68]. This situation contrasts with that obtained for the polyphenols present in the pollen extract in solution, where they were not detected in the receiving solution.

In summary, it was possible to design and develop a multiple W / O / W nanoemulsion formulation that incorporated appropriate excipients for the incorporation of a complex matrix such as bee pollen. The process was optimized by mathematical modeling of supervised learning, of which there is no history of its use applied to active molecule delivery systems (or from English: drug delivery system). There are studies that show that the vehicle developed is stable at physiologically relevant pH conditions, and in storage it favors the conservation of the complex matrix of interest. On the other hand, the innovative multiple nanoemulsion is capable of promoting the antioxidant effect already provided by synergistically encapsulated polyphenols, which has an antibacterial effect on the sensitive microorganism S. pyogenes. Finally, the new nanosystem demonstrated to be capable of improving the permeability of encapsulated polyphenolic compounds in in vitro tests under simulated biological conditions according to the step of oral absorption of compounds in a PAMPA test.

Claims

1 . Formulation for the protection of polyphenols, in order to maintain and enhance their antioxidant and antibacterial activities and to be able to use them as food additives, CHARACTERIZED because it comprises three phases: a. a first interior phase which comprises a hydrophilic solution composed mainly of water; b. a second intermediate layer which comprises a hydrophobic solution composed mainly of lipids; and c. a third external layer which comprises a hydrophilic solution in contact with the external environment, whose function is to protect the active compounds that are inserted in this formulation, delivering a controlled release of said active compounds.

2. Formulation according to claim 1, CHARACTERIZED in that the inner phase comprises ethanolic pollen extract.

3. Formulation according to claim 2, CHARACTERIZED in that the ethanolic extract is bee pollen.

4. Formulation according to claim 2 or 3, CHARACTERIZED in that it comprises between 0.1 ml_ and 2.0 ml_ of ethanolic extract of bee pollen.

5. Formulation according to claim 1, CHARACTERIZED in that the hydrophobic solution comprises at least type II propylene glycol monolaurate in combination with other hydrophobic excipients.

6. Formulation according to claim 5, CHARACTERIZED in that the amount of propylene glycol monolaurate type II is between 100 mg and 1500 mg.

7. Formulation according to claim 1 or 5, CHARACTERIZED in that the hydrophobic solution comprises at least the surfactant polysorbate 80, in combination with other excipients.

8. Formulation according to claim 7, CHARACTERIZED in that the amount of polysorbate 80 is between 100 and 500 mg.

9. Formulation according to claim 1 or 5 or 7, CHARACTERIZED in that the hydrophobic solution comprises at least Lecithin, in combination with other hydrophobic excipients.

10. Formulation according to claim 9, CHARACTERIZED in that the lecithin corresponds to soy phospholipids with 70% phosphatidylcholine.

eleven . Formulation according to claim 10, CHARACTERIZED in that it comprises between 10 and 100 mg of phosphatidylcholine.

12. Formulation according to claim 1, CHARACTERIZED in that the hydrophobic solution comprises at least one poloxamer non-ionic surfactant, in combination with other excipients.

13. Formulation according to claim 12, CHARACTERIZED in that the amount of poloxamer is between 100 and 800 mg.

14. Formulation according to claim 1, CHARACTERIZED the hydrophobic solution corresponding to the intermediate phase of the formulation comprises propylene glycol monolaurate type II, polysorbate 80, soy phospholipids with 70% phosphatidylcholine, and poloxamer.

15. Formulation according to claim 14, CHARACTERIZED in that the hydrophobic solution comprises between 100 and 1500 mg propylene glycol monolaurate type II, between 100 and 500 mg polysorbate 80, between 10 and 100 mg soy phospholipids with 70% phosphatidylcholine, and between 100 and 800 mg poloxamer.

16. Formulation according to claim 1, CHARACTERIZED in that the hydrophilic solution of the outer layer comprises chitosan HCI.

17. Formulation according to claim 16, CHARACTERIZED in that the amount of chitosan HCI is between 6 and 30 mg.

18. Formulation according to claim 1, CHARACTERIZED in that the formulation is a nanoemulsion comprising: a. an internal solution comprising between 0.6 and 1.5 ml_ of a solution of ethanolic extract of bee pollen; b. an intermediate solution comprising between 100 and 1500 mg of propylene glycol monolaurate type II, between 100 and 500 mg polysorbate 80, between 10 and 100 mg Lipoid P75 ^® , and between 100 and 800 mg poloxamer; and c. an external solution comprising chitosan HCI between 6 and 30 mg.

19. Method to obtain the formulation of claims 1 to 18, CHARACTERIZED because it comprises the following steps: a. mixing the components of the intermediate solution by magnetic stirring at room temperature; b. mix the intermediate solution with the internal solution at room temperature; c. prepare the external solution with different amounts of chitosan HCI and poloxamer dissolved in ultra pure water; and d. Prepare a multiple nanoemulsion by homogenizing at a speed of between 21,500 and 25,000 RPM in an ice bath.

20. Use of the formulation according to claims 1 to 18, CHARACTERIZED in that it serves to protect active compounds found in the internal solution of the formulation according to claims 1 to 18.

twenty-one . Use according to claim 20, CHARACTERIZED because it serves to protect active compounds from aggressive environments for their activity.

22. Use according to claim 21, CHARACTERIZED because it serves to protect active compounds, where the aggressive environment is the gastrointestinal tract.

23. Use according to claim 20, 21 or 22, CHARACTERIZED in that the active compounds to be protected are phenolic compounds.

24. Use according to claim 23, CHARACTERIZED in that the phenolic compounds are polyphenols.

25. Use according to claim 24, CHARACTERIZED in that the polyphenolic compounds are derived from pollen.

26. Use according to claim 25, CHARACTERIZED because the phenolic compounds derived from pollen correspond to flavonoids.

27. Use according to claim 24, 25 or 26, CHARACTERIZED in that the phenolic compounds and flavonoids of pollen are selected from: chlorogenic acid, syringic acid, coumaric acid, synapic acid, ferulic acid, cinnamic acid, epicatechin, myricetin, quercetin , apigenin, kaempferol, chrysin, narigenin, rhamnetin, pinocembrine, galangin, pinobanksin, hesperetin, CAPE (caffeic phenyl ester acid), luteolin, 3-methylquercetin, isorramnetin, tectocrisine, ellagic acid, and rutin.