Nanostructured aqueous formulations of propolis extracts and uses thereof

Abstract

The present invention relates to nanostructured aqueous formulations containing a high content of propolis extract, in particular green propolis extract or Brazilian red propolis extract, for use in pharmaceutical, food, cosmetic and veterinary formulations. The biological activities present in the propolis extract contribute to its antioxidant, anti-inflammatory, antitumor and antiviral activities, which are enhanced and protected by encapsulating the extract in lipid nanostructures.

Description

Detailed Description of the Invention

[0001] [Field of the Invention] The present invention belongs to the field of nanotechnology, more precisely to the fields of pharmaceuticals, food, cosmetics and veterinary medicine, and refers to nanostructured aqueous formulations of propolis extracts with a high solids content (Nano-AP), which can be used in liquid form or added to semi-solid or liquid bases to obtain products with antioxidant, antibacterial, anti-inflammatory, antitumor and antiviral activity in human or veterinary medicine.

[0002] [Background of the invention] Propolis has been used as a therapeutic agent since ancient times and is known to have antioxidant, anti-inflammatory, antitumor, antiallergic, antifungal, and antibacterial properties (Patel, 2015; Endo et al., 2018; Daugsch et al., 2008). Furthermore, it has been shown to be effective against cancers of the brain, head and neck, skin, breast, liver, pancreas, kidney, bladder, prostate, colon, and blood (Patel, 2015).

[0003] Propolis is a resin formed by honeybees, consisting of substances collected from various parts of plants, mainly from the European honeybee (Apis mellifera) species, mixed with their secretions, such as beeswax and saliva, which are used by honeybees to seal hive openings and protect the hive from insects and microorganisms (Devequi-Nunes et al., 2018; De Mendonca et al., 2015; Reis et al., 2019).

[0004] The composition of propolis varies depending on the region and the plant, but it generally consists of 50% resin rich in secondary metabolites, 30% wax, 10% essential oil, 5% pollen, and 5% other substances. Over 300 chemical compounds have already been identified, especially phenolic compounds and flavonoids (Endo et al., 2018; De Mendonca et al., 2015; Reis et al., 2019; Devequi-Nunes et al., 2018).

[0005] Propolis exhibits a diverse and complex chemical composition that varies depending on the flora of the geographical area where bees collect the material and the time of collection. Its geographical botanical origin is of great importance for quality control, standardization, and therapeutic efficiency (Lustosa et al., 2008) and is highly related to its chemical and biological composition (De Mendonca et al., 2015).

[0006] Propolis is known worldwide for its antioxidant, anti-inflammatory, antifungal, antiviral, and antibacterial properties ( Sforcin et al., 2011 ; Dantas et al., 2017 ).

[0007] Green propolis (PV) is the most widely used propolis worldwide. Baccharis dracunculifolia DC (Asteraceae), known as "alecrim-do-campo" or "vassoura," is the main botanical source of green propolis, named for its color (Bufalo et al., 2010). As discussed below, the main components of green propolis extract are phenolic compounds, particularly those derived from cinnamic acids such as artepelin C, baccharin, and drupanin, in addition to other phenolic acids such as caffeic and coumaric acids (Barretta et al., 2017; Sousa et al., 2007; Endo et al., 2018). [ka]

[0008] Brazilian red propolis (PVB) also stands out in the global market for its distinct composition from other types of propolis (Freires et al., 2016). Its main botanical source is Dalbergia ecasthophyllum, a species found in mangrove forests and on the coasts of the northeastern region of the country (Freires et al., 2016; Reis et al., 2019). The presence of polyprenylated derivatives of benzophenone in extracts of this propolis suggests the cooperative involvement of another botanical source, Symphonia globulifera (Aldana-Mejia et al., 2021).

[0009] The compounds most relevant to its biological activity are secondary metabolites of the isoflavonoid family, such as formononetin, biochanin A, and vestitol, although benzophenones and other classes of compounds have also been found ( Berretta et al., 2017 ; Reis et al., 2019 ). [ka]

[0010] Despite its many advantages, the use of propolis extract is limited by its low bioavailability, the low water solubility of its main components, the strong odor characteristic of propolis, and the presence of alcohol in the final formulation (Daudt et al., 2013; Watkins et al., 2015). Therefore, the use of nanotechnology can overcome these limitations, enabling the encapsulation of propolis extracts and the creation of aqueous dispersions with high solids contents and superior properties to those of pure extracts. Encapsulating molecules into nanostructures offers several advantages: improved performance and stability of active ingredients, enhanced bioavailability, solubilization of lipophilic compounds, sustained release of encapsulated active ingredients, targeting to biological targets, and reduced toxicity (Mora-Huertas et al., 2010; Daudt et al., 2013).

[0011] In addition to being easy to prepare on a large scale, lipid nanostructures, such as nanoemulsions and microemulsions, are distinguished by their biocompatibility and biodegradability, as evidenced by their greater stability and ability to encapsulate lipophilic compounds compared to, for example, liposomes (Miranda et al., 2021, Singh et al., 2017). Lipid nanostructures are versatile and can be incorporated into gels, creams, ointments, foams, and more. Lipid nanostructures can also be added to water to obtain flavored water, increasing the stability and prolonging the release of encapsulated natural compounds.

[0012] [Current situation in this technology field] Several prior art documents describe nanostructured preparations containing green or red propolis for applications in the dental and cosmetic fields, such as:

[0013] Document BR102010441-5 A2 describes a glycolic colloidal dispersion of propolis extract composed of a nonionic surfactant and a thickener, which reduces the extract's hydrophobicity and tendency to form precipitates in aqueous media, improving its miscibility without altering its structure. However, this document differs from the present invention because the colloidal dispersion is composed of a polymeric micellar system, a nanostructured system different from the nanostructured system of the present invention, which is a microemulsion. The micellar system is composed of a surfactant, propolis extract, and water, while the microemulsion is composed of a surfactant, cosurfactant, oil, propolis extract, and water. Furthermore, the aforementioned document uses a glycolic propolis extract, unlike the present invention, which uses an alcohol-free propolis extract.

[0014] Document CN103768103B describes an oil-water nanoemulsion containing 1% propolis extract, a solvent oil, a surfactant, and ultrapure water. The solvent oil is selected from the group consisting of mineral oil, vegetable oil, animal oil, or synthetic oil. The surfactant is selected from the group consisting of phospholipids, nonionic surfactants, or mixtures thereof. Furthermore, the average particle size is 15-70 nm, preferably 15-50 nm, and more preferably 15-20 nm. However, this document differs from the present invention due to the type of nanostructure and the method used. For example, the document uses a high-energy method using a high-pressure homogenizer and a high temperature (75°C), while the present invention obtains a microemulsion using a low stirring speed and temperature (25-45°C) to preserve the compounds present in the propolis extract. The microemulsion contains 5-12%, preferably 8-12%, of propolis extract. The components of the nanostructures are also clearly distinct, affecting the differences in their physicochemical and biological properties.

[0015] Document BR102015016405-0 B1 describes polymeric nanoparticles containing red propolis extract and a nanopolymer matrix composed of a coating polymer and a dispersing / stabilizing polymer. This nanoencapsulation system is a biocompatible, biodegradable binary system that protects skin cosmetic compositions from oxidative attack via the phenolic substances present in the propolis extract, while also promoting the controlled release of active ingredients. Document BR102015033031-6 A2 describes a nanoencapsulation product composition containing red propolis extract encapsulated in a biopolymer matrix that exhibits antibacterial activity, as well as a process for obtaining it, which can be used to control multidrug-resistant infections. This composition presents nanoencapsulated red propolis composed of a hydroethanolic extract of red propolis, an aqueous phase, and an organic phase. It is noted that both documents differ from the present invention due to the type of nanostructure used and its composition. In documents BR102015016405-0 and BR102015033031-6, polymer nanoparticles using biodegradable polymers such as polycaprolactone (PCL) and poly(lactic-co-glycolic acid) (PLGA) are used.

[0016] Furthermore, there are already nanostructured preparations containing pure green propolis for use in mouthwashes, as described in document WO 2013 / 163714, preparations of nanocapsules or polymer nanospheres containing green propolis extract as described in document EP 2633862 A1 or containing red propolis extract as described in documents BR102016018124-0 and WO 2018 / 023182 for use in pharmaceuticals, cosmetics, and agricultural applications, polymeric micelles containing green propolis extract for use as a liquid dispersion as described in document CN 1303899 C, and inclusion complexes of propolis extract with cyclodextrin as described in documents WO 2017 / 089842 and WO 2012 / 073051. Furthermore, document CN 101869234 describes the production of nanoemulsions using green propolis extract prepared using organic solvents (ethanol and ethyl acetate) and the stabilizer Tween®.

[0017] Literature BR102018072871 describes a polymer-coated microemulsion formulation containing green propolis extract or its isolate, which has a different composition from that of the present invention. The aqueous phase is composed of water, polyethylene glycol, and bile salts, and the oil phase is composed of olive oil or sunflower oil and saturated fatty acids. Lecithin was used as a surfactant along with the bile salts. Unlike the present invention, which utilizes a spontaneous formation process under low agitation as a single step, the microemulsion containing this extract was obtained under high agitation. Furthermore, after microemulsion formation, the inventors coated the nanostructures with chitosan or albumin to obtain nanostructures different from those of the present invention. Changing the composition of nanostructures can significantly alter their physicochemical properties and, consequently, their biological effects and interactions with living organisms (Ridolfo et al., 2021; Albanese et al., 2012).

[0018] It is recognized that the cited documents differ from the invention presented herein in the type of nanostructures, chemical composition and physicochemical properties. Furthermore, the present invention may be the first to provide a nanostructured lipid system that encapsulates a high content of total solids of propolis extract, in particular green propolis or Brazilian red propolis extract, obtained in a single step under low agitation and without the use of organic solvents.

[0019] Thus, the present invention provides an innovation in the form of a scalable nanostructured lipid system in microemulsions for aqueous formulation applications in pharmaceutical, food, cosmetic and veterinary fields with antioxidant, antibacterial, anti-inflammatory, antitumor and antiviral activity, including activity against the SARS-CoV-2 virus that causes COVID-19, which avoids the use of organic solvents and contains a high content of propolis extract (Nano-AP), in particular but not limited to, green propolis or Brazilian red propolis extract. [Summary of the Invention]

[0020] The present invention aims to propose nanostructured aqueous formulations (Nano-AP) containing a high content of propolis extract, in particular green propolis extract (Nano-APV) or Brazilian red propolis extract (Nano-APVB), for their application in pharmaceutical, food, cosmetic and veterinary formulations. The biological activities present in propolis extract contribute to its antioxidant, antibacterial, anti-inflammatory, antitumor and antiviral effects, which are enhanced by encapsulating the extract in microemulsions. [Brief explanation of the figure]

[0021] For a complete and comprehensive visualization of the object of the present invention, reference is made to the following figures:

[0022] Figure 1 shows the particle size distribution and polydispersity (PDI) analysis of A) Nano-APV at 30 days (red curve) and after 400 days (green curve), and B) Nano-APBV at 1 day (red curve) and after 150 days (green curve), demonstrating the high stability of aqueous formulations containing high extract contents.

[0023] FIG. 2 shows a graphical representation of the chromatogram obtained by HPLC analysis of the aqueous Nano-APV formulation.

[0024] FIG. 3 shows a graphical representation of the chromatogram obtained by HPLC analysis of the commercial aqueous extract 1.

[0025] FIG. 4 shows a graphical representation of the chromatogram obtained by HPLC analysis of the commercial aqueous extract 2.

[0026] Figure 5 shows a graph of the antioxidant potential of pure green propolis extract and Nano-APV measured by the DPPH radical reduction method, demonstrating the high antioxidant activity of Nano-APV.

[0027] Figure 6 graphs the cytotoxicity of pure green propolis extract and Nano-APV against human colon adenocarcinoma cells (Caco-2), showing that Nano-APV has a superior cytotoxic effect against tumor cells than the pure extract.

[0028] Figure 7 shows the graph of the antiviral effect of pure green propolis extract and Nano-APV on Caco-2 cells infected with SARS-CoV-2 virus, showing that Nano-APV has a better effect on SARS-CoV-2 virus than the pure extract. [Detailed Description of the Invention]

[0029] The present invention provides a nanostructured lipid formulation containing high solids content green propolis extract or Brazilian red propolis extract, which can be used as an aqueous dispersion or added to a base (semi-solid or liquid) and can be used in the pharmaceutical, cosmetic, food or veterinary fields. The formulation comprises the following ingredients: an oily phase consisting of 0.5-20%, preferably 2-10%, of oil (lipid), 5-12%, preferably 8-12%, of propolis extract, and optionally 8-18%, preferably 10-16%, of lipophilic surfactants; an aqueous phase consisting of 12 to 35%, preferably 16 to 30%, of a hydrophilic surfactant and qsp of water having a pH between 6 and 9, preferably between 7.0 and 8.5;

[0030] The aqueous phase and the oil phase are mixed under stirring at a temperature of 25 to 50°C, 400 to 1500 rpm, preferably 600 to 1000 rpm, and 25 to 35°C.

[0031] The preparation of the formulation comprises the following steps: a) preparing an oil phase containing 0.5-20% oil and 5-12% propolis extract, said oil phase may or may not contain a lipophilic surfactant (8-18%); b) preparing an aqueous phase containing 10-35% of a hydrophilic surfactant and water having a pH between 6 and 10; c) pouring the aqueous phase into the oily phase at a temperature of 25-45°C under stirring at 400-1500 rpm; d) keeping the system under stirring for 2 to 24 hours.

[0032] The lipid is selected from the group consisting of soybean oil, capric / caprylic triglyceride, Capryol® 90, linseed oil, canola oil, grapeseed oil, corn oil, wheat germ oil, and palm oil, preferably linseed oil, canola oil, and soybean oil.

[0033] The surfactant is selected from the group consisting of polyoxyethylene-polyoxypropylene-polyoxyethylene copolymers (Pluronic® F68 or 407), Kolliphor® HS15 or polysorbates (Tween® 80, Tween® 60, Tween® 20) or sorbitan monooleate (Span® 60, Span® 80, Span® 20), preferably Kolliphor® HS15, polysorbate 80 and sorbitan monooleate 80.

[0034] [Example of the invention] Variations in the ratio between the components of the lipid nanostructures as well as the type of propolis extract lead to differences in the physicochemical properties of the resulting nanostructures:

[0035] [Example 1] Nanostructured aqueous formulations (Nano-APV) containing green propolis extract were prepared by mixing an aqueous phase consisting of water with a pH between 6 and 9, preferably between 7 and 8, and 16-30%, preferably 26%, of a surfactant (e.g., Kolliphor® HS15) with an oil phase consisting of 2-12%, preferably 2.4%, linseed oil enriched with omegas 3, 6, and 9, and 5%-11%, preferably 11%, of green propolis extract, under mechanical stirring (600-800 rpm) at 30-35°C.

[0036] This formulation (Nano-APV), containing a high solids content of green propolis extract, exhibited an average diameter between 120 and 189 nm and a polydispersity index (PdI) of 0.224–0.353 (Figure 1A). The diameter of the Nano-APV nanostructures favors skin penetration of active ingredients, making them interesting for the cosmetics sector (Lemos et al., 2018; Pivetta et al., 2019). Furthermore, this dispersion exhibited a low polydispersity index, making it suitable for pharmaceutical, food, cosmetic, and veterinary formulations. Its reduced composition and diameter favor increased absorption of the extracted compounds, making it interesting for oral products (Ali and Kolter, 2019). The formulation exhibited high physical stability (>1 year), and the same diameter profile of the nanostructures was observed for 400 days (Figure 1A).

[0037] [Example 2] The potential of nanostructured lipid systems for the high-content encapsulation of different propolis extracts was evaluated, and Brazilian red propolis extract (Nano-APVB) was encapsulated.

[0038] Nano-APVB was prepared by mixing an aqueous phase consisting of water at pH 7.5, 16-30%, preferably 20% surfactant (e.g., Kolliphor® HS15), with an oil phase consisting of 2-12%, preferably 3.5% linseed oil enriched with omegas 3, 6, and 9, and 6-11%, preferably 6% Brazilian red propolis extract, under mechanical stirring (600-800 rpm) at 30-35°C.

[0039] Nanostructured aqueous formulations containing Brazilian red propolis extract had diameters between 15 and 50 nm, polydispersity indices (PdI) between 0.05 and 0.157, and were highly stable (Figure 1B).

[0040] [Example 3] Flavored water was prepared using the Nano-APV aqueous dispersion obtained in Example 1. The composition of the water consisted of 50-500 mg / L, preferably 150-300 mg / L, of Nano-APV, and contained 0.04-0.10% sodium sorbate as a preservative. The pH of the water was adjusted to between 5 and 8, preferably between 5 and 6.5, and then the flavored water was filtered through a 0.22 μm filter. The antioxidant activity, evaluated by the DPPH radical reduction method, was found to have an EC of between 8 μg / mL and 15 μg / mL. 50 showed.

[0041] [Example 4] Topical formulations were prepared using the aqueous dispersions of Nano-APV obtained in Example 1 or Nano-APVB obtained in Example 2. To obtain the topical formulations, thickeners and / or adhesives were added to the dispersions of Nano-APV or Nano-APVB. The topical formulations consisted of 98% Nano-APV and 2% quaternized guar gum or 2% hydroxyethyl cellulose, or a 2:1 mixture of thickeners, guar gum and hydroxypropyl methylcellulose (2-9%). The formulations exhibited pseudoplastic rheological behavior, with a decrease in viscosity with increasing shear rate (n<1).

[0042] Analysis of the phytochemical profile of aqueous formulations of green propolis extract (Nano-APV) The phytochemical profile of an aqueous formulation of green propolis extract was evaluated by HPLC to assess key biomarkers (Figure 2). The phytochemical profile was compared with two commercial preparations of aqueous green propolis (Figures 3 and 4).

[0043] In the commercially available aqueous extract, we were unable to identify coumaric acid and artepelin C, compounds closely related to the pharmacological activity of propolis. However, high concentrations of these compounds were quantified in the Nano-APV formulation (Table 1). Baccharin was detected in all samples, but concentrations in the Nano-APV formulation were 4-15 times higher than those quantified in the commercial aqueous product. The same profile was observed for dorupanin and dimethoxykaempferol (Table 1). Thus, by encapsulating green propolis extract in lipid nanostructures, important propolis biomarkers were preserved, exhibiting concentrations similar to those in hydroalcoholic extracts, which are not found in commercially available aqueous green propolis products. It is particularly noteworthy that this new technology makes it possible to produce a commercially available aqueous solution containing all important propolis markers at the expected concentrations, thus ensuring enhanced pharmacological activity through nanostructuring. [Table 1]

[0044] biological activity Nano-APV exhibited superior biological activity to pure green propolis extract, including antioxidant activity (Figure 5), suppressing DPPH radical (EC 50 ) was reduced by 50% (pure propolis extract (EC 50 = 29.09 μg / mL), three times lower than the concentration indicated by the white nanostructures. The oil, like the white nanostructures, also showed no antioxidant activity, indicating that the formulation enhances the antioxidant activity of the propolis extract by reducing the nanostructure diameter and by self-assembly of selected components.

[0045] Furthermore, Nano-APV exhibited dose-dependent in vitro antitumor activity (Figure 6). Its cytotoxicity in human colon adenocarcinoma cells (Caco-2) was superior to that of pure propolis extract, with an IC of 40.87 ± 0.034 μg / mL. 50(2.5-fold lower than the 101.9 ± 0.027 μg / mL observed for pure green propolis extract). The white nanostructures showed an IC of greater than 150 μg / mL. 50 The nano-APV formulation showed low cytotoxicity in normal Vero cells, and the oil was not toxic in the range of concentrations evaluated. Thus, the nanostructured formulation, due to its composition and nanometer size, enhanced the antitumor activity of propolis. Furthermore, the Nano-APV formulation showed low toxicity (IC 50 = 184 μg / mL), demonstrating selective effects on colon adenocarcinoma tumor cells.

[0046] Nano-APV also demonstrated dose-dependent in vitro antiviral activity, reducing the viral load of SARS-CoV-2 by 87%, indicating its potential in the treatment of COVID-19. The antiviral activity of Nano-APV was superior to that of pure green propolis extract, which only reduced viral load by 21% (Figure 7). White nanostructures showed no antiviral activity, demonstrating that Nano-APV formulations enhance the antiviral effect of propolis through self-assembly of selected nanostructure components with decreasing diameter.

[0047] Thus, the aqueous formulation Nano-APV with a high total solids content exhibited high stability, retained important propolis biomarkers, and improved the biological and pharmacological effects of propolis extract, including antioxidant, antitumor, and antiviral activities in vitro. Furthermore, the nanostructured lipid system was able to encapsulate a high content of Brazilian red propolis extract, demonstrating its versatility in encapsulating propolis extracts. Thus, the present invention offers an innovative nanostructured lipid formulation of microemulsions obtained without the use of organic solvents as an encapsulation system for high-content propolis extracts in aqueous media, which can enhance the pharmacological effects of propolis and can be used as an aqueous dispersion or incorporated into semi-solid or liquid formulations for cosmetics, pharmaceuticals, food, and / or veterinary medicine.

[0048] Therefore, Nano-AP formulations utilize the resources of nanotechnology to enhance the biological effects of the bioactivity of propolis extracts, and furthermore, they exhibit the properties of improved permeability and absorption due to their composition and diameter, higher stability, and the aqueous dispersion form that allows them to be easily incorporated into other pharmaceutical, cosmetic, or food formulations.

[0049] Aldana-Mejia, JA; Ccana-Ccapatinta, GV; Squarisi, IS; Nascimento, S.; Tanimoto, MH; Ribeiro, VP; Arruda, C.; Nicolella, H.; Esperandim, T.; Ribeiro, AB; de Freitas, KS; da Silva, LHD; Ozelin, SD; Oliveira, LTS; Melo, ALA; Tavares, DC; Bastos, JK Nonclinical Toxicological Studies of Brazilian Red Propolis and Its Primary Botanical Source Dalbergia ecastaphyllum. Chemical Research in Toxicology, 34, 1024-1033, 2021.

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[0067] Albanese A, Tang PS, Chan WCW. The Effect of Nanoparticle Size, Shape, and Surface Chemistry on Biological Systems. Annual Review of Biomedical Engineering 14, 1-16, 2012 [Brief explanation of the drawings]

[0068] [Figure 1] Figure 1 shows the particle size distribution and polydispersity (PDI) analysis of A) Nano-APV at 30 days (red curve) and after 400 days (green curve), and B) Nano-APBV at 1 day (red curve) and after 150 days (green curve), demonstrating the high stability of aqueous formulations containing high extract contents. [Figure 2] FIG. 2 shows a graphical representation of the chromatogram obtained by HPLC analysis of the aqueous Nano-APV formulation. [Figure 3]FIG. 3 shows a graphical representation of the chromatogram obtained from the HPLC analysis of the commercial aqueous extract 1. [Figure 4] FIG. 4 shows a graphical representation of the chromatogram obtained from the HPLC analysis of the commercial aqueous extract 2. [Figure 5] Figure 5 shows a graph of the antioxidant potential of pure green propolis extract and Nano-APV measured by the DPPH radical reduction method, demonstrating the high antioxidant activity of Nano-APV. [Figure 6] Figure 6 graphs the cytotoxicity of pure green propolis extract and Nano-APV against human colon adenocarcinoma cells (Caco-2), showing that Nano-APV has a superior cytotoxic effect against tumor cells than the pure extract. [Figure 7] Figure 7 shows the graph of the antiviral effect of pure green propolis extract and Nano-APV on Caco-2 cells infected with SARS-CoV-2 virus, showing that Nano-APV has a better effect on SARS-CoV-2 virus than the pure extract.

Claims

1. 1. A nanostructured lipid formulation of propolis extract, characterized in that it comprises: an oily phase consisting of 0.5-20%, preferably 2-10%, of oil (lipid), 5-12%, preferably 8-12%, of propolis extract, and optionally 8-18%, preferably 10-16%, of lipophilic surfactants; an aqueous phase consisting of 12-35%, preferably 16-30%, of a hydrophilic surfactant and qsp of water having a pH between 6 and 9, preferably between 7.0 and 8.5; wherein the mixing of the aqueous phase with the oily phase is carried out at a temperature of 25 to 50°C, preferably 25 to 35°C, under stirring at 400 to 1500 rpm, preferably 600 to 1000 rpm.

2. 2. The formulation of claim 1, wherein the lipid is selected from the group consisting of soybean oil, capric / caprylic triglyceride, Capryol® 90, linseed oil, canola oil, grapeseed oil, corn oil, wheat germ oil and palm oil, preferably linseed oil, canola oil and soybean oil.

3. 2. The formulation of claim 1, wherein the surfactant is selected from the group consisting of polyoxyethylene-polyoxypropylene-polyoxyethylene copolymers, mainly Pluronic® F68 or 407, Kolliphor® HS 15, or polysorbates, mainly Tween® 80, Tween® 60, Tween® 20, or sorbitan monooleates, mainly Span® 60, Span® 80, Span® 20, preferably Kolliphor® HS 15, polysorbate 80 and sorbitan monooleate 80.

4. 4. Use of a formulation according to any one of claims 1 to 3, wherein said formulation is in liquid form or is added to a semi-solid or liquid base to obtain a product having antioxidant, antibacterial, anti-inflammatory, antitumor and antiviral activity.

5. 5. Use of a formulation according to claim 4, wherein said formulation is for application in the fields of pharmacy, food, cosmetics and veterinary medicine.

6. 5. Use of the preparation according to claim 4, wherein said preparation is also for adding to water to obtain flavored water.

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