Composition, formulation and method for obtaining a composition comprising blue propolis

Abstract

The present invention relates to novel biotechnological bases, enriched with blue propolis, able to dilute hydrosoluble and liposoluble compounds, for use in pharmaceutical, cosmetic, agricultural and food formulations. The technological base is obtained via mixing adequate proportions of fatty acids and amines, i.e.: mono-ethanolamine, di-ethanolamine and tri-ethanolamine generating surfactants.

Description

COMPOSITION, FORMULATION, AND PROCESS FOR OBTAINING THE COMPOSITION COMPRISING BLUE PROPOLIS Field of the Invention

[0001] The present invention relates to new biotechnological bases, enriched with blue propolis, capable of diluting water-soluble and fat-soluble compounds, for use in pharmaceutical, cosmetic, agricultural, and food formulations. Background of the invention

[0002] Propolis is a natural resin produced by bees from various botanical sources, consequently presenting a wide diversity of phytochemicals. Several varieties of propolis have been extensively studied for their phytochemical composition and biological activity. Brown, green, and red propolis are produced by Apis mellifera bees, and geopropolis, such as blue propolis, is produced by Melipona spp.

[0003] Surfactant compounds capable of solubilizing and stabilizing lipophilic molecules have numerous applications, such as in cosmetic cleaning formulations, shower gel, soap, facial cleansing lotion, bath foam, and body care formulations.

[0004] The presence of raw blue propolis extract in its composition adds applications of interest to the pharmaceutical, cosmetic, agricultural and food industries.

[0005] For applications in agricultural and sanitation areas, these compounds have the property of efficiently depositing substances present in them onto a surface through the addition of water.

[0006] Patent BR 10 2020 009939 6 deals with a natural antibacterial and antiviral biological composition with application in the hospital and home area, in particular a composition comprising ricinoleic acid from the extraction of castor oil, diethanolamine and green propolis, the process of obtaining the composition and its Use. The invention can be used to control various pathogenic microorganisms, such as bacteria belonging to the genera Staphylococcus, Pseudomonas, Enterobacter, and Bacillus; as well as hospital environment fungi (filamentous and yeast-like) and phytopathogenic fungi (Colletrotichum fragariae).

[0007] The product can be used on biotic and abiotic surfaces by spray application and then using a non-woven fabric to remove organic matter, including microorganisms. The product can also be used to disinfect medical, dental, and hospital supplies by immersion followed by a quick rinse. Due to its low toxicity, it can be used to sanitize hands and other environments, and can be used, for example, as a disinfectant soap. Brief description of the invention

[0008] The present invention discloses Beeocitrix blue, which comprises an ethanolic extract of blue propolis, fatty acids, and amine. The fatty acids are present in a range of 2.0 to 4.0% by weight, the amine is present in a range of 1.0 to 1.5%, and the ethanolic extract of blue propolis is present in a range of 0.2 to 1.0% by weight.

[0009] The fatty acid is at least one selected from the group comprising ricinoleic acid, oleic acid, stearic acid, palmitic acid and phosphoric acid.

[0010] The amine is at least one selected from the group comprising monoethanolamine, diethanolamine and triethanolamine.

[0011] Blue propolis comprises between 30 and 40% α-pinene, between 5 and 10% β-pinene, between 3 and 6% δ-cadinene, between 2 and 5% E-caryophyllene, between 2 and 5% α-cubene and between 2 and 5% γ-muurolene.

[0012] The possible formulations of Beeocitrix blue include: MEA Ricinolamide (RM1) and MEA Phosphoethanolamine (RM9); MEA Ricinolamide (RM1) and DEA Phosphoethanolamine (RD9); MEA Ricinolamide (RM1) and Phosphoric Acid (R9); Ricinoleic Acid (R1) and MEA Phosphoethanolamine (RM9); DEA Ricinolamide (RD1) and MEA Phosphoethanolamine (RM9); DEA Ricinolamide (RD1) and MEA Phosphoethanolamine (RD9). DEA (RD9); DEA Ricinolamide (RD1) and Phosphoric Acid (R9); DEA Ricinoleic Acid (R1) and Phosphoethanolamine (RD9);

[0013] The process of obtaining Beeocitrix blue comprises the steps of adding the fatty acid, heating the fatty acid to a temperature between 60 and 90°C, adding the amine under stirring, adding more fatty acid until reaching a pH between 7.0 and 8.0, cooling the mixture to 60°C until ricinolamide is formed.

[0014] In a separate beaker, add 0.1 to 0.3 g of blue propolis extract and between 2.0 and 7.0 g of ricinolamide at 50°C, mixing until homogeneous. Simultaneously, heat 50 mL of water to 40°C. After thoroughly mixing the blue propolis and ricinolamide, gradually add water to the mixture while stirring. Transfer this solution to a volumetric flask and make up to 100 mL with water. Shake gently and transfer the final composition to a bottle for storage.

[0015] Throughout this process, the formation of ethanolamine and diethanolamine residues is reduced, eliminating the toxicity of the composition.

[0016] The fatty acid must be added in a higher molar proportion than the amine.

[0017] The amine must be added under stirring of up to 60 RPM and controlled temperature between 30 and 45°C. Description of the figures

[0018] The present invention will be described in more detail after the presentation of the figures, which contain a preferred embodiment. The Figures show:

[0019] Figure 1 shows a graph where each peak and number represents a compound present and quantified in the propolis sample.

[0020] Figure 2 shows a gas chromatogram of the crude blue propolis extract.

[0021] Figure 3 reveals the reaction of fatty acids with ethanolamine.

[0022] Figure 4 indicates the products formed from the formulations presented in Table 3.

[0023] Figure 5 shows the products formed from the reaction of fatty acids with ethanolamine in a 5% dilution in water.

[0024] Figure 6 shows the products formed from the mixture of the reaction product of fatty acids and ethanolamine with 2% blue propolis extract.

[0025] Figure 7 shows the 5% dilution in water of the products formed from the mixture of the reaction product of fatty acids and ethanolamine with 2% propolis extract.

[0026] Figure 8 shows the products formed with propolis extract and their respective dilutions in water. Detailed description of the invention

[0027] Before describing the invention in detail, it should be understood that this invention is not limited to the specific component parts of the objects described, as such objects may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It should be noted that, as used in the specification and appended claims, the singular forms "um," "uma," "o," and "a" include singular and / or plural referents, unless the context clearly indicates otherwise. Furthermore, it should be understood that, where parameter ranges delimited by numerical values ​​are provided, the ranges are deemed to include such limiting values.

[0028] It should also be understood that the embodiments disclosed herein should not be construed as individual embodiments that are not related to each other. Features discussed in connection with one embodiment should also be disclosed in connection with other embodiments shown herein. If, in a case, a specific feature is not disclosed in connection with one embodiment but with another, one skilled in the art would understand that this does not necessarily mean that said feature is not intended to be disclosed in connection with said other embodiment. The skilled in the art you would understand that the essence of this request is to disclose the said characteristic also for the other modality, but that just for the purposes of clarity and to keep the present descriptive report in a manageable volume, this was not done.

[0029] The term “Beeocitrix blue”, used interchangeably in this description, refers to a composition comprising the ethanolic extract of blue propolis, at least one fatty acid and at least one amine.

[0030] The present invention relates to biotechnological bases formulated from a mixture of fatty acids and ethanolamines in appropriate proportions, enriched with crude blue propolis extract. These compounds have applications in the development of new pharmaceutical, cosmetic, chemical, and food products, as the incorporated propolis extracts add properties of interest to various fields. Furthermore, the present invention features non-toxic, stable compositions characterized by their easy preparation and high yield, ensuring the complete solubilization of hydrophilic and lipophilic chemical substances.

[0031] Blue propolis is a type of propolis previously unknown in the literature, with little known composition and pharmaceutical properties. Its main botanical source is the plant Schinus terebinthifolius Raddi, a member of the Anacardiaceae family, native to southern Brazil. This type of propolis is produced by Mandaçaia bees (Melipona quadrifasciata), a species of stingless bee native to Brazil. Recognized as the largest of the bees in this subspecies, Mandaçaia bees have robust bodies, four yellow stripes on their abdomens, and excellent temperature control, enabling them to withstand colder, more humid climates. They are found in the regions of São Paulo, Santa Catarina, and Rio Grande do Sul.

[0032] Among the properties of blue propolis, we highlight its insolubility in water, solubility in organic solvents and its composition, which consists of a complex mixture of resins, waxes, essential oils, and pollen.

[0033] Chromatographic and spectrophotometric analyses show a high concentration of terpenes and terpenoids. The chromatogram (Figure 2) revealed that the volatile fraction of blue propolis comprises peaks with higher intensity, indicating the major compounds, with high concentrations of α-pinene (33.04%), β-pinene (6.39%), δ-cadinene (4.82%), ε-caryophyllene (3.88%), α-cubebene (3.18%), and γ-muurolene (3.05%). The non-volatile phase of blue propolis includes Naphthgeranine A, Picfeltarraegenin IV, Milbemectin A4, Naphthgeranine A, among others. The following tables describe the compounds identified in the volatile and non-volatile phases of blue propolis, with the main compounds highlighted. Table 1. Phytochemicals identified by mass spectrometry of the crude extract of “blue propolis”, retention time, and peak area Peak Retention Time (min) Peak Area Identified Compound 21 23.86 0.42 Cubebene <β-> Table 2. Phytochemicals identified by positive mode mass spectrometry of blue propolis crude extract, retention time and peak area Mass Formula Molecular Formula Exact retention time METLIN (hr) 1 2.57 --- 3.32 --- 3 381 3.72 --- 4 173 4.09 --- 275 4.33 --- 133 4.75 --- 599 4.68 --- C 24 H 23 N4O Acetic acid--4,5-dimethoxy-2-[(4- W 35 H 45 O6diff ppm 0.9 C36H41N4O2 diff ppm 1.6 14 561 8.32 561.3221 --- C 23 H 49 N2O 13 diff ppm 2.0 C33H43N3O5 diff ppm 3.2 C 38 H 43 NO3diff ppm 3.9 1,2,3-trimethoxy-4-(2,3,4- trimethoxyphenyl)benzene C18H23O6 Formula: C 18 H 22 The diff ppm 1.26[M+H]+ m / z 335.1489 M 334.1416 C 2,2',4,4',6,6'-Hexamethox 5 335 8.55 335.1491 16 HNO ybiphenyl 1 21 3 5Formula: C 0 18H 22O diff ppm 3.6 pH equal to 4.6 (± 0.04), before removing the alcohol to form the soft propolis extract. Its blue color can vary from light blue to navy blue, depending on its geographic origin and handling.

[0035] Phytochemical characterization showed that blue propolis has a total phenol content of 2.85 mg EAG / ml (± 0.04), flavonoids 11.30 mg EQ / ml (± 0.05), solids total solubles 0.40 g / ml (± 0.001) and other components that give it antioxidant activity (48.23 ± 1.05%).

[0036] From the values ​​obtained for phenolic compounds and flavonoids in the aqueous and ethanolic propolis solutions, it is observed that the flavonoid content is 69.35 ± 0.38 mg / g in the ethanolic propolis compared to 23.67 ± 2.14 mg / g in the aqueous propolis. This reduction in values ​​is also observed in phenols, with 98.74 ± 0.96 mg / g in the ethanolic propolis extract compared to 36.57 ± 0.35 mg / g in the aqueous extract. In addition to these quantitative differences, there are also qualitative differences, since the polarity of compounds that are soluble in an ethanolic solution is often not soluble in aqueous solutions, resulting in precipitation or even denaturing.

[0037] This process offers the possibility of preparing aqueous solutions with propolis components, as well as essential oils and other lipophilic substances, which would not normally be soluble in such solutions, without losing the active ingredients that act in the products developed from this process, opening up a spectrum of new applications in the most diverse areas of knowledge, including, but not limited to, human, animal and plant health.

[0038] Aiming to reduce the formation of toxic ethanolamine and diethanolamine residues, this invention presents the result of new formulations obtained by the reaction of various fatty acids with ethanolamines, without the addition of a catalyst. The proportions of reagents used are presented in Table 2, adopting two proportions for each acid aiming at the total neutralization of the ethanolamine used in each formulation. Table 3 – Proportions (v / v) of fatty acids and ethanolamine AR and DEA AO and DEA AP and DEA AE and DEA AF and DEA Proportion with 75.84:24.16 74.78:25.22 73.24:26.76 74.78:25.22 54.92:45.08

[0039] In a preferred embodiment of the present invention, the fatty acid must be added in a molar proportion greater than that of the amine.

[0040] All reactions were conducted over 3 hours on a magnetic stirrer (speed 1) and heater (speed 5) in an Erlenmeyer flask with a volume at least twice that of the total reagent volume, covered with perforated aluminum foil to allow vapors to escape during the reaction. All acids were heated to nearly 100°C before the ethanolamine additions to prevent solidification and reduce viscosity, allowing for better stirring. At the end of the reaction, the product was diluted 5% in water for analysis of solubility, foam stability, and pH.

[0041] Ricinoleic Acid (RA) and Diethanolamine (DEA): Viscous liquid product, liquid-phase reaction for all temperatures. Characteristic odor. Amber color. Good and rapid dilution in water. Good foam stability. Good reaction and solubility with propolis extract, showing no separation when diluted in water. Stoichiometric ratio: Mass-to-mass ratio (RA:DEA): 73.95:26.05 pH: 8.2 Proportion with excess acid aiming for maximum conversion of 90%: Mass-to-mass ratio (RA:DEA): 75.84:24.16 pH: 8.2

[0042] Oleic Acid (OA) and Diethanolamine (DEA): Pasty product, reaction in gelatinous phase at low temperatures, should be conducted close to 100°C to maintain the liquid phase and consequently better homogenization. Neutral odor in the finished product, slightly herbal. Ocher color. Good dilution in water, however slow, presenting some separation. Very good foam stability. Good reaction and solubility with propolis extract, forming a pasty product and presenting separation when diluted in water. Stoichiometric ratio: Mass-to-mass ratio (AO:DEA): 72.87:27.13 pH: 9.3 Proportion with excess acid aiming for maximum conversion of 90%: Mass-to-mass ratio (AO:DEA): 74.78:25.22 pH: 9.2

[0043] A 1:1 mixture of ricinolamide and oleic acid amide forms a pasty product.

[0044] Palmitic Acid (PA) and Diethanolamine (DEA): Plastic product, reaction in the gelatinous phase at low temperatures, should be conducted near 100°C to maintain the liquid phase and consequently better homogenization. Crayon odor in the finished product. Beige color. Good dilution in water, but slow. Excellent foam stability, forming a creamy product with high hydration. Good reaction and solubility with propolis extract, forming a plastic product and presenting no separation when diluted in water. Stoichiometric ratio: • Mass-to-mass ratio (PA:DEA): 70.92:29.08 • pH: 8.8 Proportion with excess acid aiming for maximum conversion of 90%: • Mass-to-mass ratio (PA:DEA): 73.24:26.76 • pH: 9.8

[0045] A 1:1 mixture of ricinolamide with palmitic or stearic acid amide forms a product with a texture similar to vegetable butter (lip balm).

[0046] Stearic Acid (EA) and Diethanolamine (DEA): Plastic product, reaction in gelatinous phase at low temperatures, should be conducted near 100°C to maintain the liquid phase and consequently better homogenization. Resin / tallow odor in the finished product. Beige color. Dilution is good in water, but slow, producing a creamy product. Good foam stability. Good reaction and solubility with propolis extract, forming a dry product and not presenting separation when diluted in water. Stoichiometric ratio: • Mass-to-mass ratio (EA:DEA): 73.01:26.99 • pH: 9.2 Proportion with excess acid aiming for maximum conversion of 90%: • Mass-to-mass ratio (EA:DEA): 74.78:25.22 • pH: 9.3

[0047] Phosphoric Acid (PA) 85% in water and Diethanolamine (DEA): Liquid product, reaction with high heat generation, the amine must be introduced gradually to avoid accidents. Neutral odor in the finished product. Slightly greenish coloration. Good and rapid dilution in water, but presents phase separation. No foaming. Low reaction and solubility with propolis extract, presenting separation when diluted in water. Stoichiometric ratio: • Mass-to-mass ratio (PA:DEA): 52.30:47.70 • pH: 2.9 Proportion with excess acid aiming for maximum conversion of 90%: • Mass-to-mass ratio (AF:DEA): 54.92:45.08 • pH: 2.3 Beeocitrix blue preparation process

[0048] Before preparing Beeocitrix, the ricinolamide solution must be prepared. To do this, the fatty acid is added to a beaker and heated until it reaches a temperature between 60 and 90°C. The amine is gradually added to this beaker, stirring at 20 to 60 rpm. The reaction between these two compounds is exothermic, releasing heat. Therefore, when it reaches approximately 90°C, stirring must be reduced to control the temperature. After stabilizing the pH, temperature, and viscosity, the ricinolamide is ready.

[0049] To prepare the solution, initially weigh between 0.1 and 0.3 g of propolis into a 100 mL beaker. Next, weigh 2.0 to 7.0 g of ricinolamide into a 50 mL beaker. The ricinolamide is then heated to 50°C and then poured into the 100 mL beaker containing the propolis. The two components are gently mixed with a glass rod until homogeneous. In parallel, water heated to 40°C is added to the 50 mL beaker, filling it almost completely.

[0050] After the ricinolamide and propolis are completely mixed, water from the 50 mL beaker is gradually added to the initial mixture, while gently stirring to ensure homogeneity. The mixture is then transferred to a 100 mL volumetric flask. To ensure complete transfer, more water is added to the 50 mL beaker and this water is poured into the 100 mL flask using a Pasteur pipette. The final volume is adjusted in the flask until the meniscus reaches the 100 mL mark. Finally, the solution is gently stirred, transferred to a labeled bottle, and stored properly.

[0051] An effective strategy used in this process and product patent to increase the stability of propolis during industrial processing is to work at lower temperatures (40 – 60°C) during dissolution in ricinoleamide – DEA and later in other formulations. High temperatures are commonly used to increase the solubility of propolis resin, but they can also accelerate the degradation of its bioactive components. By employing low-temperature techniques, the structural integrity of sensitive compounds can be maintained, preventing thermal degradation and preserving the full spectrum of beneficial properties, as well as creating new property standards. For example, cold extraction methods using solvents at controlled temperatures can help retain the antioxidant, antimicrobial, and anti-inflammatory activities of propolis. Another bonus of dissolving propolis with ricinoleamide-DEA is the inert photoprotective effect of this ethanolamide salt, protecting the bioactive compounds from UV radiation, acting as a preservative, and preventing propolis from absorbing this radiation, as it does naturally as a photoprotector in hives.

[0052] In addition to preserving bioactivity, low-temperature processes are also advantageous for ensuring product consistency and shelf life. Heat-induced degradation can lead to variations in the chemical composition of propolis-based products, making it difficult to standardize their quality and efficacy. By minimizing thermal exposure, manufacturers can produce more stable formulations with consistent levels of essential bioactive compounds, such as artepillin C. This not only improves the reliability of propolis products but also increases their safety and efficacy when used for extended periods. Finally, GC / MS analyses indicate that the reaction occurs without ethanolamine residues in the final product, as its molecular ion was not detected in the analysis, ensuring a safe and environmentally friendly surfactant. Example 1. Formulation from the reaction of phosphoric acid, ricinoleic acid, and their derivatives.

[0053] In an attempt to develop a new pharmaceutical base with adequate stability and physical and chemical properties, the reactions of ricinoleic acid and its derived amides with phosphoric acid and its respective derived amines were tested without the addition of any catalyst. T01 – Ricinolamide MEA (RM1) and Phosphoethanolamine MEA (RM9):

[0054] The reaction product presented a heterogeneous mixture with a lower phase of greater volume, diverging in the densities and proportions of the reagents, and also presenting turbidity. Both phases solubilized the propolis extract, following RM1 but differing from RM9. In the dilutions, the upper phase solubilized the propolis extract in water as occurs with RM1, but presented turbidity, unlike Ricinolamide MEA, and the lower phase presented turbidity and solubilized the propolis extract in water, unlike what occurs with Phosphoethanolamine MEA. T02 – Ricinolamide MEA (RM1) and Phosphoethanolamine DEA (RD9):

[0055] The reaction product presented a heterogeneous mixture with a larger upper phase, converging with the density and proportions of the reactants. Both phases solubilized the propolis extract, following RM1 but differing from RD9. In the dilutions, the upper phase solubilized the propolis extract in water as occurs with RM1, but presented turbidity, unlike Ricinolamide MEA. The lower phase did not solubilize the propolis extract in water as did RD9, but presented turbidity, unlike what occurs with Phosphoethanolamine DEA. T03 – Ricinolamide MEA (RM1) and Phosphoric Acid (R9):

[0056] The reaction product presented a heterogeneous mixture with a larger upper phase, converging with the density and proportions of the reactants. Both phases solubilized the propolis extract, following RM1 but differing from R9. In the dilutions, the upper phase did not solubilize the propolis extract in water, unlike what would occur with RM1. The lower phase, however, did not solubilize the propolis extract, as occurred with R9. T04 – Ricinoleic Acid (R1) and MEA Phosphoethanolamine (RM9):

[0057] The reaction product presented a heterogeneous mixture with a lower phase of greater volume, diverging from the densities and proportions of the reagents, and also presenting turbidity. Both phases solubilized the propolis extract, following R1 but differing from RM9. In the dilutions, the upper phase did not solubilize the propolis extract in water. same way as ricinoleic acid. However, the lower phase did not solubilize the propolis extract like RM9, but presented turbidity, unlike the phosphoethanolamine of MEA. T05 – Ricinolamide of DEA (RD1) and Phosphoethanolamine of MEA (RM9):

[0058] The reaction product presented a heterogeneous mixture with a larger upper phase, converging with the density and proportions of the reactants. Both phases solubilized the propolis extract, following RD1 but differing from RM9. In the dilutions, the upper phase solubilized the propolis extract in water as occurs with RD1, but presented turbidity, unlike DEA ricinolamide. The lower phase solubilized the propolis extract in water and presented turbidity, unlike what would occur with RM9. T06 – DEA ricinolamide (RD1) and DEA phosphoethanolamine (RD9):

[0059] The reaction product presented a heterogeneous mixture with a larger upper phase, converging with the density and proportions of the reactants. Both phases solubilized the propolis extract, following RD1 but differing from RD9. In the dilutions, the upper phase did not solubilize the propolis extract in water and presented a different turbidity than that observed with RD1. The lower phase did not solubilize the propolis extract as R9 did, but presented a different turbidity than that of DEA phosphoethanolamine. T07 – DEA ricinolamide (RD1) and phosphoric acid (R9):

[0060] The reaction product presented a heterogeneous mixture with a larger upper phase, converging with the density and proportions of the reactants. Both phases solubilized the propolis extract, following RD1 but differing from R9. In the dilutions, the upper phase did not solubilize the propolis extract in water, unlike what would occur with RD1. The lower phase did not solubilize the propolis extract as R9 did, but presented a different turbidity than phosphoric acid. T08 – Ricinoleic Acid (R1) and DEA Phosphoethanolamine (RD9):

[0061] The reaction product presented a heterogeneous mixture with an upper phase of greater volume, converging with the density and proportions of the reactants. Both phases solubilized the propolis extract, following R1 but differing from RD9. In the dilutions, the upper phase did not solubilize the propolis extract in water as occurs with ricinoleic acid. However, the lower phase did not solubilize the propolis extract as RD9 did.

[0062] The reagent proportions used followed the values ​​in Table 3. All reactions were carried out over 1 hour under manual stirring. Table 4 – Reagent proportions Reagents Proportion

[0063] Observed and that all reactions showed phase separation visibly corresponding to the proportion and density of the reactants. However, in T01 and T04, a lower phase of greater volume with some turbidity is observed, diverging from the proportion and density of the reactants since ricinolamide (RM1) and ricinoleic acid (R1) are less dense and were added in greater volume than phosphoethanolamine (RM9). Obtaining water-soluble formulations from the bases obtained by mixing fatty acids and ethanolamines

[0064] It was observed that the solutions presented different degrees of solubilization of propolis in water, with solutions T05 and T06 presenting the best results, with the smallest fractions (supernatant – S, precipitate – I) remaining most of the propolis dispersed in the aqueous solution. The results of this analysis are seen in Figure 4.

[0065] The products generated from the reactions between fatty acids demonstrated a high buffering capacity, for the most part, since even after the addition of acidic pH propolis, minimal variations were observed.

[0066] An increase in the amount of fatty acids at the beginning of the reaction can correct this pH, reducing the values ​​to around 7.5. Pharmacokinetic properties of the active components present in the new biotechnological bases enriched with blue propolis

[0067] The pharmaceutically interesting properties of the constituents of blue propolis, present in all new biotechnological bases that have this extract in their composition, were predicted through theoretical calculations in relation to their absorption, distribution, metabolization and excretion (ADME) capacity.

[0068] In pharmacokinetic studies, intestinal absorption (AGI), skin permeability rate (Kp), and the substance's potential to cross the blood-brain barrier (BBB) ​​were evaluated.

[0069] The evaluated compounds α-pinene and β-pinene demonstrated potential BBB crossing capabilities, indicating a high probability of action in the central nervous system (CNS). However, the compounds exhibit low potential for gastrointestinal tract penetration, which may be related to the calculated TPSA results.

[0070] Metabolism results were assessed using the following criteria: whether the substances undergo phase 1 metabolism and whether they inhibit any enzyme in the cytochrome P450 (CYP) complex. Substances that inhibit two or more CYPs, particularly CYP3A4 and CYP2C9, can interfere with the metabolism of a large number of drugs and other substances, potentially contributing to increased toxicity.

[0071] For substances that inhibit only one CYP, there may be a reduction in the number of drugs that have a pharmacokinetic interaction with this complex. Substances Non-inhibiting and non-inducing CYP compounds are considered ideal substances because they do not interfere with the metabolism of other drugs. Considering this information, the compounds α-pinene and β-pinene demonstrated the best results, although they have potential for inhibition of the CYP2C9 isoenzyme, this is the only interaction found. All other compounds exhibit more than one unfavorable interaction, including the CYP2C9 isoenzyme of the cytochrome P450 complex. Additionally, the results did not indicate any alerts for PAINS (Pan Assay Interference Compounds), an index that shows the tendency of these compounds to react nonspecifically with numerous biological targets, suggesting a high possibility of selectivity. Example 2. In silico prediction: Toxicological profile of the active components present in the new biotechnological bases enriched with blue propolis. Theoretically calculated acute in vivo toxicity.

[0072] Table 5 shows an in silico prediction of LD50 values ​​for mice with five administration methods (oral, intravenous, intraperitoneal, subcutaneous, and inhalation). This prediction was made based on information on ~10,000 chemical structures with data on acute rat toxicity represented by LD50 values ​​(log10 (mmol / kg)). Therefore, the lower the calculated value, the lower the toxic potential of the compound. Table 5. Acute toxicity (LD50) in rats and in vivo toxicity classification predicted by theoretical calculations within the model applicability domain. Compound Predicted acute in vivo toxicity in mg / kg γ-muurolene 378.30 40.27 2,065.00 404.50 - aeas aç o ape o ea; - aeas aç oae osa; a - a of oral administration; SC - Subcutaneous route of administration Skin Toxicity

[0073] Skin sensitization is an immunological response to certain chemicals that manifests as an inflammatory skin reaction. This inflammatory response is mediated by delayed-type T cells, which cause allergic contact dermatitis. The Organization for Economic Cooperation and Development (OECD) summarized the adverse outcome pathway for skin sensitization as having four main events (KE): KE1 is the covalent binding of a chemical to skin protein; KE2 is the activation of epidermal keratinocytes; KE3 is the activation, maturation, and mobilization of dendritic cells; and KE4 is dendritic cell-mediated antigen presentation to naive T cells, followed by proliferation / activation of allergen-specific T cells.

[0074] The calculation method applied in this study achieves balanced accuracy, sensitivity, and specificity of up to 89–100%. The predicted results from the in chemico, in vitro, in vivo, and in silico prediction models are presented in Table 6.

[0075] Computational analyses using in vivo models evaluate antigen-specific T cell proliferation, histocompatibility complex representation, T cell activation and proliferation, and inflammatory capacity after allergen exposure. Furthermore, the HRIPT (Human Repeated Insult Correction Test) history is considered. and HMT (human maximization trial). The in silico results are based on the Bayesian model, which is a consensus model that integrates predictions from all other trials for an integrative qualitative risk assessment (QRA) of skin sensitization based on the weight of evidence (WoE). Table 6 - Prediction of skin toxicity and confidence level in the applicability domain of models Composite Models

[0076] DPRA: Direct Peptide Reactivity Assay; h-CLAT: Human Cell Line Activation Test; LLNA: Local Lymph Node Assay; HRIP: Human Repeat Insult Patch Testing; HMT: Human Maximization Test; Bayesian: A low-confidence in silico prediction indicates that two or more individual predictions disagree with the Bayesian model; a high-confidence in silico prediction indicates that the individual prediction agrees with the Bayesian model. Predicted biological targets for the active components present in the new biotechnological bases enriched with blue propolis

[0077] In silico biological target hunting is a technology that allows for the prediction of therapeutic applications of designed compounds based on their chemical structure, using information from chemical and biological databases. It provides a comparative analysis of recent studies through the similarity of the new molecule to other known ones. Targets are classified according to a score that combines the 2D and 3D similarity values ​​with the most similar known active substance, with the query molecule. Inhibition or stimulation of the genes indicated as targets can result in important medicinal properties that the substances may exhibit.

[0078] The pharmacological targets indicated in the study are: cannabinoid receptor type 2 (CNR2), muscarinic receptor M2 (CHRM2), α-estrogen receptor (ERS1) and peroxisome proliferator-activated receptor alpha (PPARA).

[0079] CNR2 receptors may have potential therapeutic roles in the treatment of neurodegenerative disorders such as Alzheimer's disease. Specifically, agonist agents have been shown to induce macrophages to remove native beta-amyloid protein from frozen human tissue. In patients with Alzheimer's disease, beta-amyloid proteins form aggregates known as senile plaques, which disrupt neural function. Alterations in endocannabinoid levels and / or CNR2 receptor expression have been reported in nearly every disease affecting humans, ranging from cardiovascular, gastrointestinal, liver, kidney, neurodegenerative, psychiatric, bone, skin, autoimmune, and lung diseases to pain and cancer.

[0080] M2 muscarinic receptors are located in the heart, where they act to slow the heart rate to normal sinus rhythm after negative stimulatory actions of the parasympathetic nervous system, decreasing the rate of depolarization. They also reduce the contractile forces of the atrial heart muscle and slow the conduction velocity of the atrioventricular node.

[0081] The estrogen receptor is a ligand-activated transcription factor composed of several domains important for hormone binding, DNA binding, and transcriptional activation. ERα plays an important role in the physiological development and function of a variety of organ systems to varying degrees, including the reproductive, central nervous, skeletal, and cardiovascular systems. Thus, ERα is widely expressed throughout the body, including the uterus and ovary, male reproductive organs, mammary gland, bone, heart, hypothalamus, pituitary gland, liver, lung, kidney, spleen, and adipose tissue.

[0082] PPARAs are ligand-dependent transcription factors and are important in the regulation of triglyceride levels and energy homeostasis. Example 3. Acute oral toxicity and dermal toxicity of Beeocitrix blue in vivo

[0083] Blue propolis also has applications in healthcare. Acute oral and dermal toxicity tests evaluate the effects of chemical substances after a single exposure, using laboratory animals and following standardized protocols. In the oral test, the substance is administered directly into the stomach of animals such as rats, in escalating doses, to determine the 50% lethal dose (LD50), which is the dose required to cause death in half of the test population. The animals are monitored for up to 14 days to record signs of toxicity, behavioral changes, or mortality. In the dermal test, the substance is applied directly to the skin, usually of rabbits or rats, after scraping a specific area to expose the skin. The goal is to evaluate local reactions, such as irritation or inflammation, as well as possible systemic effects, in order to calculate the cutaneous LD50.

[0084] Blue propolis ethanol extract has been classified as Category 5 of the GHS classification, which corresponds to the least severe level of acute toxicity. This category indicates that the substance has low toxicity, with the potential to cause only mild effects in cases of accidental exposure at very high doses.

[0085] The parameters evaluated include the LD50 (Lethal Dose 50%), which is the dose required to cause the death of 50% of a test population. The results demonstrated: • Acute oral toxicity: LD50 >5000 mg / kg, indicating safety at high doses. • Acute dermal toxicity: cutaneous LD50 >2000 mg / kg, demonstrating low toxicity via the cutaneous route.

[0086] These data reinforce the safety profile of blue propolis ethanol extract under normal conditions of use. Example 4. Acute oral toxicity and acute inhalation toxicity of Beeocitrix blue in vivo

[0087] The results of the acute oral toxicity test classified Beeocitrix blue as GHS Category 5, meaning the substance was classified as having low toxicity. The oral LD50 was determined to be greater than 5000 mg / kg, indicating that the substance has low toxicity if swallowed.

[0088] In addition, an acute inhalation toxicity test was performed, which also classified Beeocitrix blue as belonging to GHS Category 5, with an LD50 greater than 2000 mg / kg, demonstrating low inhalation toxicity. Example 5. Pharmacological properties of formulations obtained and enriched with Beeocitrix blue. Microbiological activity.

[0089] The methodology used in the tests was disk diffusion, with evaluation of the halos in mm from the edge of the disk. Inoculums were standardized at 0.5 on the Macfarland scale, and each disk received 10 uL of the treatments from the formulations obtained. Table 7 shows the percentages of blue propolis used in each formulation considered for this test. Table 7 – Treatments used in the antimicrobial test for the new biotechnological bases enriched with blue propolis 1. 30% ethanolic extract (Residue from 1st aqueous extraction)

[0090] The use of ricinoleamide to dissolve the blue propolis extract, treatments 3 and 5, drastically altered the antibacterial activity profile, with an increase in mycobactericidal activity in treatment 3. Treatment five consisted of extracting the bioactives from the propolis without the use of ethanol, but using ricinoleamide as a solvent / extractor at a temperature of 25°C for 5 days (Table 8). Table 8 – Results of the inhibitory capacity for the new biotechnological bases enriched with blue propolis Microorganism Treatments E Example 6. Corneal opacity and permeability tests

[0091] In tests carried out with the item Beeocitrix Blue in its pure form, non-uniform opacity and increased corneal permeability were observed, resulting in a Corneal Injury Index (LIS) of 140.58. These results indicate a potential for interaction with ocular tissues, however, it was not possible to determine the ocular hazard classification based on the Bovine Corneal Opacity and Permeability Test (BCOP) method, in accordance with OECD 437 (2023) guidelines.

[0092] According to the methodology used, the BCOP classifies substances into two specific categories, in accordance with the Globally Harmonized System (UN GHS): Category 1 – Eye corrosive or severe irritant substances. Uncategorized – Non-irritating substances.

[0093] Because the results for Beeocitrix Blue did not clearly fall into any of these categories, it was classified as "No Prediction," indicating the need for further testing to determine the potential for ocular harm and its definitive classification under the UN GHS.

Claims

MODIFIED CLAIMS Received by the International Secretariat on 6 May 2025 (06.05.2025) 1. Composition characterized by the fact that it comprises: from 2.0% to 4.0%, by weight, of at least one fatty acid selected from the group comprising ricinoleic acid, oleic acid, stearic acid, palmitic acid and phosphoric acid; from 1.0% to 1.5%, by weight, of at least one amine selected from the group comprising monoethanolamine, diethanolamine and triethanolamine; from 0.2% to 1.0%, by weight, of ethanolic extract of propolis, whose volatile phase comprises: from 30% to 40% of a-pinene; from 5% to 10% of p-pinene; from 3% to 6% of 5-cadinene; from 2% to 5% of E-caryophyllene; from 2% to 5% of a-cubebene; from 2% to 5% of y-muurolene; and whose non-volatile phase comprises at least one compound selected from the group including Naphthgeranine A, Picfeltarraegenin IV and Milbemectin A4.

2. Formulation of the composition, as defined in any of the preceding claims, characterized in that the formulation may be as follows: T01: MEA Ricinolamide (RM1) and MEA Phosphoethanolamine (RM9); T02: Ricinolamide from MEA (RM1) and Phosphoethanolamine from DEA (RD9); T03: Ricinolamide MEA (RM1) and Phosphoric acid (R9); T04: Ricinoleic Acid (Rl) and MEA Phosphoethanolamine (RM9); T05: DEA ricinolamide (RD1) and MEA phosphoethanolamine (RM9); T06: DEA Ricinolamide (RD1) and DEA Phosphoethanolamine (RD9); T07: DEA Ricinolamide (RD1) and Phosphoric Acid (R9); T08: Ricinoleic Acid (Rl) and DEA Phosphoethanolamine (RD9).

3. Process for obtaining the composition, characterized by the fact that it comprises the following steps: a. addition of the fatty acid; b. heating the fatty acid to a temperature between 60 and 90°C; c. adding the amine under stirring; d. adding more fatty acids until reaching a pH between 7.0 and 8.0; e. cooling to 60°C until ricinolamide is formed; f. in a separate beaker, add between 0.1 and 0.3 g of the blue propolis extract; g. adding between 2.0 and 7.0 g of ricinolamide at 50°C to the beaker with blue propolis; gently mix the two components with a glass rod, until reaching homogeneity; h. in parallel, in a beaker, heat 50 ml of water to 40°C; i. after completely mixing the blue propolis with the ricinolamide, gradually add water to this solution under stirring; j. this mixture is transferred to a volumetric flask, completing the solution with water, until it reaches 100 mL; and k. shake gently and transfer to a bottle for storage. During the process, there is a reduction in the formation of ethanolamine and diethanolamine residues, eliminating the toxicity of the composition.

4. Process, according to claim 3, characterized by the fact that the fatty acid must be added in a molar proportion greater than that of the amine.

5. Process, according to claim 3, characterized by the fact that the amine must be added under stirring of up to 60 RPM.

6. Process, according to claim 3, characterized by the fact that the amine must be added at a temperature within the range between 30 and 45°C.

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