Nanostructured emulsion based on extract of brassica oleracea var. capitata, anhydrous lanolin and castor oil, for the treatment of burns and their complications

Abstract

The present invention relates to a composition based on ethanolic extract of Brassica olearacea var. capitata leaves, anhydrous lanolin and castor oil, for the treatment of burns and their complications, such as infections. Emulsions made from ethanolic extract of Brassica olearacea var. capitata leaves, anhydrous lanolin and castor oil in semi-solid formulations such as creams and ointments, with 10% and 20% w / v of Brassica extract, can be used as healing agents, accelerating closure of the skin area damaged by up to 3rd degree burns, inducing the production of protective enzymes as well as the proliferation of cells and blood vessels, modulating the production of matrix components and stimulating the formation of strong scar tissue, while having no side effects due to their lack of toxicity.

Description

Nanostructured Emulsion Based on Brassica Oleracea Varietal Extract, Anhydrous Lanolin, and Castor Oil for the Treatment of Burns and Their Complications Field of application

[0001] The present invention application relates to a nanostructured emulsion based on Brassica olearacea var. capitata leaves, anhydrous lanolin, and castor oil, for use in the treatment of burns and their complications, such as infections and gangrene. The formulation described herein, in addition to being non-toxic and having no side effects, is capable of forming a protective nanometric film that protects the tissue and facilitates the controlled release of constituents, promoting the healing of tissue damaged by burns of up to 3 Sdegree, inducing the production of protective enzymes, accelerating the closure of the damaged skin area, inducing the proliferation of cells and blood vessels, modulating the production of matrix components, absorbing exudate, stimulating the formation of resistant scar tissue. State of the art

[0002] Burns are responsible for more than 265,000 deaths worldwide each year, primarily because burn wounds compromise the structure and function of the skin, exposing individuals to the risk of ulceration and infection. Therefore, the shorter the wound healing time, the better the prognosis.

[0003] Burns can be caused by heat, chemical exposure, electricity, radiation, and other factors. They are classified as 1 s degree when the damage is superficial, reaching the epidermis; 2 S degree, when it reaches the epidermis and part of the dermis; and 3 Sdegree, when it reaches the epidermis, dermis, hypodermis and muscles (R. Fekrazad, et al., Acta Cir. Bras., vol. 2, pp. 0-5, 2014). The depth of the burn is related to the time of exposure to the aggressive agent, temperature and pressure under the tissue, determining the prognosis after injury (AJ Singer, et al. J. Burn Care Res., vol. 31, pp. 646-651, 2010).

[0004] Skin repair, in turn, occurs in three phases: inflammatory, proliferative, and remodeling. In 1 â In the second phase, the release of platelet-derived mediators, bacterial byproducts, and chemoattractants promotes tissue hemostasis. Leukocytes are recruited and act to eliminate tissue debris and infectious agents. In the second phase, cells and blood vessels proliferate, leading to the formation of granulation tissue, rich in blood vessels and type III collagen. In the third phase, âIn this phase, type III collagen is replaced by type I, which is thicker and organized into fiber bundles, providing scar strength and resilience. Finally, the interaction between cells, matrix, and cytokines occurs, resulting in rapid and effective wound closure (S. Werner and M. Antsiferova, Encycl. Cell Biol., vol. 3, pp. 216-222, 2016).

[0005] Throughout this process, to protect the body from oxidative damage, antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST) are produced. These enzymes act to prevent the formation of free radicals that can damage body tissues (OM Ighodaro and OA Akinloye, Alexandria J. Med., pp. 1-7, 2017). In this context, both the decreased concentration or activity of antioxidant enzymes, combined with cellular degradation, compromise the healing process, resulting in inefficient tissue repair.

[0006] To control the healing of burns and other skin problems, the use of products based on medicinal plants has been increasing, as several phytochemical compounds can act synergistically, inhibiting the proliferation of microorganisms, preventing oxidative stress and stimulating wound closure (R. Bahramsoltani, et al. Arch. Dermatol. Res., vol. 306, pp. 601-617, 2014).

[0007] However, active chemical components of natural origin are often unstable, in addition to being in physical states that are not suitable for application, such as in the form of powders, pastes, lumps, etc. For this reason, they require the use of excipients to guarantee chemical protection, dispersion, solubilization and conveyance, which results in the need to incorporate them into formulations that allow structural limitations to be overcome in addition to allowing the establishment of an appropriate dosage for a given use.

[0008] In formulations, it is essential that additional constituents interact with the active ingredient to enhance it, protecting it from chemical oxidation (auto-oxidation, photo-oxidation, and air oxidation) and thermal degradation, thus increasing the product's shelf life. They must also promote dispersion and solubilization to facilitate transport into the target tissue and increase its residence time.

[0009] In the case of dermocosmetic formulations, the material must have viscoelastic characteristics that favor the adhesion of the active ingredients for a sufficiently long time to allow their action. Moisturizing and antimicrobial properties are also necessary to promote tissue integrity. These factors must act synergistically, which is generally achieved when the components interact with each other.

[0010] In the prior art, several herbal compositions for cutaneous use have been described and claimed. Some of these include, for example, the use of viscoelastic systems in the form of gels, ointments, or pastes, as exemplified in patents BR112019013945-1; WO201 1091492; US20170224841; WO2017089842; US6350453.

[0011] Technologies aimed at increasing the bioavailability of active agents by increasing chemical stability, vectorizing substances in target organs, protecting against fixation in unwanted tissues and organs, reducing toxic effects and contributing to the reduction of production costs can be exemplified in patents US20190169316; CN106616207; CN107156456; US20180133274.

[0012] On the INPI website, a search for emulsion formulations containing extracts using the keywords "ointment" and "extract" and searching the abstract yielded 42 applications. A similar search on the USPTO using the keywords "ointment" and "natural extract" yielded 211 applications. On Google Patents, using the same keywords, the number was much higher, around 13,845 results, demonstrating that this strategy is already recognized in the state of the art.

[0013] Brassica oleracea Var. Capitata is a plant of the Brassicaceae or herbaceous cruciferous family, with upper leaves arranged closely together, forming a compact head. It is believed to have evolved through natural selection from other cabbage-like species of the Acephala group, originating in the region around the Mediterranean Sea, around 100 AD. Phytochemical components of Brassica, such as glucosinolates, sulforaphanes, tannins (phenolic acids), and flavonoids, are already known for their healing potential and antimicrobial properties (I. Hassini, et al. J. Sci. Food Agric., vol. 97, pp. 2291-2299, 2017).

[0014] In the technology based on Brassica oleracea Var. Capitata (BR1020160063078), the process for obtaining its extract was claimed, combining various solvents and methods to enhance the extraction effect. The same technology also claimed compositions based on leaf extracts in the form of emulsions, gels, and emulsions for the healing of various wounds.

[0015] However, as mentioned above, wounds resulting from 3-degree burns S present a high degree of injury, inevitably resulting in infectious processes, which in turn require extreme care to avoid complications. This means that formulations aimed at healing such wounds require a more robust composition, with multifunctional characteristics, which can be achieved by using substances with complementary biological activity, such as antimicrobials, cell stimulants, emollients, moisturizers and antioxidants; in addition to vehicles with greater penetrating action, such as a combination of surfactants.

[0016] Anhydrous lanolin is an example of an additive with emollient, lubricating, protective, and healing properties used in dermocosmetic formulations. Anhydrous lanolin is a substance produced by the cebaceous glands of sheep and is responsible for the natural protection of their epithelium. It has a waxy appearance due to the presence of a complex mixture of macromolecules, especially high-molecular-weight lipid compounds, whose approximate composition is: 12% sterols, 46% cholesterol esters, 10% monoesters, 21% diesters, and 11% free fatty alcohols (Thewlis, J. Lanolinna: nuovi orízzonti, Cosmetic news XIX, 106, 27, 1996). It is commonly extracted from sheep wool by fractionation methods such as leaching with a surfactant solution, followed by centrifugation, or by extraction with supercritical CO2 (W02002100990A1 ) or ethanol / methanol in supercritical conditions (W02002100990A1 ).

[0017] Although it is cited only as an excipient, as in technologies WO201 2174622A1 , WO1994001588A1 , WO2014094101 A1 , it has been cited in the state of the art, lubricating and healing activity in fragile tissues, as reported in technology WO2011 103648A1 ; or even, being part of compositions aimed at reinforcing the protective activity of the skin (US3666857, W02002069912A1 , US3052608, US4279262, US2758152, US2758125).

[0018] Another notable adjuvant capable of enhancing formulations is castor oil, also known as castor oil. Castor oil is a mixture of constituents, composed of approximately 95% ricinoleic acid and the remainder of linoleic, oleic, and palmitic acids. In many technologies, it is cited and claimed only as a carrier (CA2079255A1, KR102133381 B1).

[0019] Castor oil has a high penetration capacity and is used to reduce inflammation, protect wound surfaces, preserve moisture, remove sepsis and promote healing of wounds and wound surfaces (CN105412207A). It has antifungal activity (Takano, EH et al. Defesa Fitosanitária Cienc. Rural, 37 (5), 2007), as well as hair growth stimulating activity (Miranda, TF, 2018. http: / / dspace.fasf.edu. br / handle / 123456789 / 52) (US5827510A, CN106726902A, US9757321 B2).

[0020] A new search in the USPTO database (http: / / patft.uspto.gov / netahtml / PTO / search-bool.html) using the operator “aclm / ” and the keywords “aclm / (ointment)” and “aclm / (brassica)” retrieved some results, but none of them with the characteristics described in this technology. A similar search was conducted at the INPI, and only three results were retrieved (BR1020160063078, PI1005858-3, PI04015274), all belonging to the owner of this technology, but with preparation mechanisms, adjuvants, and excipients completely different from those proposed in this formulation. When the search was performed considering the keywords “castor oil” and “anhydrous lanolin,” no results were retrieved in the cited databases.

[0021] Currently, there are a wide variety of emulsions, which can be classified according to their penetrability or degree of contact with underlying tissue layers. Epidermal emulsions have low or no penetrating power; endodermal emulsions are capable of penetrating the epidermis, acting on the deeper tissue layers without the active ingredients reaching the bloodstream; diadermal emulsions penetrate so deeply that they can reach the bloodstream. In the case of burn wounds with epithelial destruction, tissue regeneration requires interaction of the components with the bloodstream, which explains the presence of distinct chemical constituents with diadermal characteristics (high penetrability).

[0022] Therefore, the subject matter of this application refers to a new dermocosmetic formulation (new emulsion), with multifunctional characteristics, resulting from the combination of at least three active ingredients, in addition to its manufacturing process and use for healing wounds resulting from burns of up to 3 o degree. The new emulsion can be obtained by combining Brassica extract with anhydrous lanolin and castor oil, in addition to constituents that, although known in the state of the art, have not yet been cited or claimed in combination with Brassica extract, either for the purpose of healing any wounds or for healing burns of 3 o degree. Among the other constituents, the secondary active ingredients and excipients described below stand out:

[0023] Ethoxylated sorbitan ester (ESE): These are a group of nonionic surfactants widely used in cosmetics, detergents, pharmaceuticals, agrochemical formulations, and food. As surfactants, they have emulsifying properties and are capable of forming micellar nanodomains, which act as controlled-release devices for active ingredients. The use of this additional surfactant provides more effective penetrating action in damaged tissue (PYLER, EJ, Baking science & technology, Kansas 3rd ed. v.1, 1988).

[0024] Sorbitan monooleate: also known as "Tween 80" or "Span 80," is an anionic surfactant of the fatty acid ester class, commonly used in the cosmetics industry as an important element for homogenizing aqueous ingredients. In addition to acting as a thickening agent (consistency enhancer), it spontaneously forms a protective film useful for reducing water loss when used in moisturizing creams. It also has emollient and solubilizing action, as it is a surfactant (Front. Microbiol., 22 November 2016 | https: / / doi.org / 10.3389 / fmicb.2016.01878). It is also cited in the prior art as having anti-biofilm activity against P. aeruginosa and S. aureus.

[0025] Vaseline: is a liquid paraffin completely insoluble in water, oily in nature, clear, colorless, non-fluorescent, odorless. Because it is a mixture, it has an undefined melting point, with a softening around 36 °C and completing the transition to the liquid state above 60 °C. Its main characteristic is related to the rheological effect (consistency donor) and the reduction of the hydrophilicity of the formulation, increasing its adhesion to the epithelium.

[0026] Polyol fatty acid esters: are moisturizing and emollient agents, used in cosmetics and pharmaceutical formulas, used as a softener of formulations and stabilizer against thermal shocks and crystallization, as described in technologies PI 051 1493-4 and PI 9504838-3.

[0027] Carbamide: A water-soluble adduct of hydrogen peroxide and urea, it is used as a whitening, antiseptic, and antiseptic agent. It promotes whitening of the formulation and provides an antiseptic effect.

[0028] The combination of these constituents, previously unheard of in the state of the art, results in a robust formulation whose viscoelastic rheological characteristic is due to the presence of nanostructures capable of dispersing, emulsifying, and generating rheological consistency. These nanostructures are capable of retaining the active ingredient, hydrating the skin, inducing antioxidant enzymes, and enhancing the regeneration of burns. S degree, inducing cell proliferation, blood vessel growth and collagen. Brief description of the figures Figure 01: Thermogravimetric analysis (TGA) of the pure Base, Base / Carbamide, Base / Brassica and Final Formulation in the temperature range between 20 and 160 °C. Figure 02: Differential Thermal Analysis (DTA) of the pure Base, Base / Carbamide, Base / Brassica and Final Formulation in the temperature range between 20 and 160 °C. Figure 03: Oscillatory Rheological Analysis as a function of the deformation rate, from 0.01% to 100%, at 25 °C, o = 1 Hz; for Pure Base, Base / Carbamide, Base / Brassica and Final Formulation. Figure 04: Oscillatory Rheological Analysis as a function of the deformation frequency from 1 to 125 Hz, at 25 °C, with y% = 0.2% (kept within the LVE); for Pure Base, Base / Carbamide, Base / Brassica and Final Formulation. Figure 05: Stationary Thermorheological Analysis as a function of temperature, cp = 0.1 s-1 , for Pure Base, Base / Carbamide, Base / Brassica and Final Formulation. Figure 06: Average values of hydrodynamic diameters <dh>obtained from the dispersion of 50 jiL of ethanolic solutions of the extracts (0.0033 g / mL) and supramolecular phytocomplexes (0.0066 g / mL) in milliQ water. Experiments carried out at 25 °C, scattering angle of 90°. Figure 07: Electrical conductivity of aqueous suspensions obtained from the dispersion of 50 jiL of ethanolic solutions of the extracts (0.0033 g / mL) and supramolecular phytocomplexes (0.0066 g / mL) in milliQ water. Experiments carried out at 25 °C and alternating DDP of 10 mV. Figure 8: Zeta potential of aqueous suspensions obtained from the dispersion of 50 µL of ethanolic solutions of the extracts (0.0033 g / mL) and supramolecular phytocomplexes (0.0066 g / mL) in milliQ water. Experiments carried out at 25 °C and alternating PDD of 10 mV. Figure 9: Proportion of cells (A), blood vessels (B and C) and mast cells (D, E and F) in the scar tissue of rats treated with Brassica extract. Figure 10: Proportion of type I collagen fibers (A), type III collagen fibers (B) and elastic fibers (C) in the scar tissue of rats treated with B. oleracea extract. Figure 11: Levels of superoxide dismutase (SOD) (A), catalase (CAT) (B) and glutathione estransferase (GST) (C) in scar tissue of rats treated with B. oleracea extract. Detailed description of the invention

[0001] The present invention application consists of two main steps (I and II) to obtain the final product.

[0002] Stage I: is based on obtaining ethanolic extracts from Brassica oleracea var. capitata leaves. To do this, Brassica oleracea var. capitata leaves must be collected and selected, sanitized by immersion in 2% sodium hypochlorite in distilled water for 2 minutes, and washed in autoclaved water for 2-20 minutes. Extraction must be done by static maceration with 90-95% ethyl alcohol (ethanol) as the extraction solvent. Every week, replace with an equal volume of solvent for remaceration. The suspension is subjected to ultrasound, vacuum filtered, and heated to 55°C. S C for 24 h to remove the solvent and, consequently, concentrate the extract.

[0003] Stage II: called formulation, consists of obtaining a viscoelastic, nanostructured semi-solid formulation by combining the extract with the other constituents. To do this, in a main tank, the constituents are mixed as follows:

[0004] a) In the main tank, considering the final mass of the finished product, the hydrophobic fraction of the composition is prepared, adding under constant stirring between 400 and 800 rpm, and heating at a temperature ranging from 60 to 90 °C; the hydrophobic constituents of the formulation: petroleum jelly (from 5% to 10% by weight), anhydrous lanolin (from 5% to 15% by weight), hydrogenated castor oil (from 2% to 10% by weight), polyol fatty acid esters (from 5% to 10% by weight).

[0005] b) When ensuring the fusion and mixing of all the constituents, ethanolic extract of Brassica oleracea var. capitata is added (in a proportion of 10% to 30% by weight) until completely homogenized;

[0006] c) In a secondary tank, considering the final mass of the finished product, the hydrophilic fraction of the composition is prepared in the presence of emulsifying agents, adding demineralized water (15% to 35% by weight), cetostearyl alcohol surfactant (from 2.5% to 10% by weight), ethoxylated sorbitan monooleate (from 5% to 10% by weight), carbamide (from 1% to 5% by weight).

[0007] d) After mixing and homogenizing the agents of the hydrophilic fraction, the preservatives (methylparaben, propylparaben and butylated hydroxytoluene (from 1% to 10% by weight each) are added.

[0008] e) Finally, with both hydrophilic and hydrophobic fractions, both at a temperature close to 30-40 °C, the hydrophobic fraction is poured into the hydrophilic fraction, at a constant flow rate, maintaining agitation between 400 and 800 rpm, until the product is completely homogenized, which can then be cooled to room temperature and packaged.

[0009] The present technology does not consist of mere dilution of the constituents in the vehicle, since interaction between the constituents of the formulation was demonstrated, as well as the fact that the formulation as a whole showed effectiveness according to results that can be understood through the examples described below.

[0010] To facilitate descriptions in the non-limiting examples below, formulations containing 10% Brassica (called PB1) and 20% PB2 were produced, keeping the concentrations of the other constituents constant.

[0011] For the purpose of proving the interactions between the Brassica components and the emulsion components from a physicochemical point of view, in order to prove the inventive activity of the technology in the examples below, the following compositions were also prepared: Base (control, i.e., the pure base), Base / carbamide (without Brassica) and Base / Brassica (without carbamide). EXAMPLE 1: Thermal stability of the formulation by thermogravimetric analysis (TGA) and differential thermal analysis (DTA).

[0012] Thermogravimetric analysis (TGA) and differential thermal analysis (DTA) are analytical tools already well described in the state of the art, which allow Monitoring the thermal behavior of materials as a function of temperature. Both techniques complement each other, allowing us to identify phenomena resulting from heating, such as dehydration, decomposition, melting, etc. When using these techniques to compare the thermal behavior of a formulation with that of its precursors, changes in the decomposition profile in TGA or the heat flow profiles in DTA are indicative of intermolecular interactions between the constituent species in the formulation.

[0013] The TGA (Figure 1) and DTA (Figure 2) analyses were performed in an oxidizing atmosphere (air), with a flow rate of 300 mL / min and a heating rate of 20 °C / min, in the temperature range of 20 °C to 160 °C. For comparison purposes, the samples analyzed were: Base (control), Base / Carbamide, Base / Brassica and PB2. During the experiments, porcelain crucibles and an approximate mass of 3 mg for each sample were used.

[0014] In Figure 1, the TGA curves for all materials showed relatively smooth mass loss in the range of 20 to « 60 S C, attributed to dehydration and loss of volatile substances. The mass loss percentages were 12.6% for the base, 14.7% for the Base / Carbamide sample, 11.1% for the Base / Brassica sample, and only 9% for PB2. It is important to note that from 20 to « 60 S C encompasses the range of use, handling, and transportation of the compound. Given the data obtained, considering that PB2 presented the lowest mass loss, it is possible to conclude that the product has superior thermal stability compared to the others, resulting from the interaction between the constituents.

[0015] In the DTA curve, an endothermic heat flow is observed for the Base, with a maximum at 54 S C. For the Base / Carbamide sample, a broad exothermic peak of 51 is observed. S C to 54 S C while for the Base / Brassica sample no significant heat flow is observed in this temperature range. On the other hand, in the range from 51 to 54 S C, the PB2 formulation presented an intermediate peak between Base / Carbamide and Base / Brassica.

[0016] It is important to highlight that the urea present in carbamide does not present any thermodecomposition in this temperature range, as reported in state of the art (Maik Eichelbaum, et al. 2010, V 9, p 90-97, Applied Catalysis B: Environmental). Therefore, this thermal compensation effect can be attributed to the interaction of Brassica components with Carbamide.

[0017] From « 60 S C, it is observed that the samples undergo successive mass losses via distinct mechanisms, since the curves do not overlap. Furthermore, PB2 presented higher mass percentage values than the other samples throughout the temperature range examined, demonstrating greater thermal stability resulting from interactions between the components, which in turn does not result from the additive effect of the simple combination between the species.

[0018] In DTA, a strong endothermic peak is observed for all four samples, in the range of 75 to 100 S C, attributed to the decomposition of part of the components or desorption of small molecules. For each sample, the values were: 93.5 S C for pure Base, 79.2 S C for Base / Carbamide, 78.5 S C for Base / Brassica and 87.8 S C for PB2. Although specific assignment is not possible due to the complexity of the samples, it is clear once again that the combination of species increases the thermal stability of the sample, also in this other temperature range, resulting from a non-obvious effect of combination between species. EXAMPLE 2: Rheological Characterization of the Formulation

[0019] The influence of the composition on the mechanical response of the PB2 formulation was evaluated by oscillatory rheological analysis and thermorheological analysis, in comparison with the Base, Base / Carbamide and Base / Brassica. For the characterization, a Dynamic Hybrid Rheometer (DHR1; TA Instruments®) with parallel plate geometry (stainless steel, diameter 40.0 mm, DIN standard) was used, coupled to a Peltier plate for temperature control.

[0020] The rheological experiments in oscillatory mode were carried out as follows: 1) Oscillatory sweep of deformation amplitude, from 0.01% to 100%, at 25 S C, keeping the angular frequency constant (1 Hz) in order to determine the linear viscoelasticity (LVE) region and the yield stress (TO). 2) After determining the maximum deformation limit in the storage modulus (G'), the deformation amplitude was fixed at 0.2% (kept within the LVE) and the oscillation frequency varied from 1 to 125 Hz, at 25 S C. It is important to emphasize that G' is a measure of the rigidity of the material (solid behavior), that is, the higher G', the more rigid the formulation.

[0021] For the purpose of determining the LVE as well as the yield stress of the four formulations, a graph of (G') as a function of the shear stress (T) was initially constructed, which allows a comparative analysis of the degree of rigidity of the formulations and, consequently, of the interactions.

[0022] As demonstrated in Figure 3, the G' values within the LVE followed the order: Base > Base / Brassica > Base / Carbamide > PB2, demonstrating that the combined presence of carbamide and Brassica extract generates the non-obvious effect of reducing stiffness, when compared to the pure Base. This effect is due in part to the presence of carbamide, which has emollient capacity. However, the mutual interaction between the components enhances the reduction in stiffness, making PB2 more fluid.

[0023] All samples presented both LVE and O, due to the fact that the base components are the majority in relation to the others. The presence of LVE and TO demonstrate semi-solid viscoelastic behavior. However, both the Base and Base / Brassica presented higher yield stress values (practically identical, being TO = 37.4 Pa for Base and TO = 38.5 Pa for Base / Brassica). The presence of brassica, therefore, does not cause a significant effect on the yield stress when compared to the Base. On the other hand, in the presence of carbamide, it is observed that the TO value suffered a reduction, also attributed to the emollient character of carbamide, which reduces the solid behavior (elasticity) of the sample. However, sample PB2, because it contains carbamide and brassica, presented intermediate behavior between the Base and Base / Carbamide.

[0024] The G' and TO data show that both the incorporation of brassica and carbamide have effects on reducing the stiffness of the samples, although the effect of carbamide (of an emollient nature) is more expressive.

[0025] Oscillatory rheological analyses, as a function of shear frequency, allowed the determination of the complex viscosity n* (Pa.s), which has the same unit (Pa.s) and the same physical meaning as the stationary viscosity r|, both being measures of resistance to flow. In practice, the difference between them lies in the measurement method.

[0026] Figure 4 shows the values of r|* em function of shear frequency where it is possible to observe that the incorporation of both brassica and carbamide has effects similar to those observed in the oscillatory analysis as a function of amplitude, with viscosity magnitudes in the order: Base > Base / Brassica > Brassica / Carbamide > PB2; confirming the synergistic effect of Brassica and Carbamide in reducing flow resistance.

[0027] Finally, thermorheological experiments were carried out, sweeping the temperature from 10 to 50 S C, at low shear rate (0.1 s -1 ), in order to obtain information about the effect of temperature on the mechanical response of the formulations. As observed in Figure 5, a significant effect of temperature is observed, so that in 50 S C, all interaction effects are canceled out by the high temperature, since the viscosity values become equal.

[0028] At low temperature (10 S C), the orders of viscosity values are changed: Base > PB2 > Base / Brassica > Base / Carbamide. However, in the temperature range between 20-30 S C, which in turn is the main range of practical use of the product, the magnitude of the viscosity values is the same as that observed in oscillatory rheology as a function of frequency: Base > Base / Brassica > Brassica / Carbamide > PB2.

[0029] However, the mechanism by which viscosity drops varies from sample to sample, due to intermolecular interactions. For PB2, the drop in viscosity is more pronounced than the other samples, in the range of 10 to 25 S C. However, from « 25 S C to 40 S C, there is a range of low viscosity variation, which is advantageous because it is expected that there will be no major changes in sample consistency due to weathering during transport, handling, or storage. For the Pure Base, a region of viscosimetric stability is only observed above 35 S C, while for Base / Brassica and Base / Carbamide, there is no viscosimetric stability plateau. EXAMPLE 3: Evaluation of the nanostructuring of the formulation in the presence of water.

[0030] When a semi-solid formulation is applied to a surface, it is common for a microlayer to form that acts to protect the epithelium from the elements, increasing hydration (emollient action) and favoring the controlled release of constituents into the environment.

[0031] Because the formulation is composed of hydrophilic (carbamide, polyol fatty acid esters), amphiphilic (ethoxylated sorbitan ester, tween 80, and other emulsion constituents), and hydrophobic (Brassica extract, petrolatum, lanolin, and castor oil) agents, when exposed to excess water, for example, during cleaning and bathing procedures, the formation of nanostructures containing components of the formulation is common. These structures can attach to the surface of the epithelium, thus reducing the drag caused by excess water, generating a residual effect on the surface, even after washing.

[0032] To investigate the effect of excess water on the formation of nanoaggregates, the samples were dispersed in milli-Q® water, at a concentration of 3.33 g / L (0.01 g of emulsion in 3 ml of water), subjected to vigorous agitation in ultrasound, for 2 minutes and then subjected to zeta potential, electrical conductivity and particle size analyses.

[0033] Zeta potential and electrical conductivity measurements were performed on a zetasizer NANO-ZS-90 photometric module using a DTS-1070 polyethylene capillary cuvette. Size measurements were made in the form of mean hydrodynamic diameter. <dh>, a square polyethylene cuvette DTS0012 was used.

[0034] Initially, the measures of <dh>were performed to determine the average size of the nanostructures dispersed in water. As observed in Figure 6, the hydrodynamic diameter values were in the 400 nm range, except for the Base / Carbamide system, which presented a size of 800 nm (close to the micrometer). The presence of nanoaggregates in all systems corroborates the hypothesis of nanostructure formation due to the presence of emulsifying species. There was no significant difference (p < 0.05) between the sizes of the aggregates formed from Base / Brassica and PB2 in relation to the pure base. However, the higher values presented for the Base / Carbamide formulation, with a significant difference at p < 0.05, were attributed to the precipitating action of carbamide. These data confirm that the components of the brassica extract interact with carbamide to reduce its precipitating effect.

[0035] In Figure 7, when analyzing the electrical conductivity values of the composition formed by pure Base, it can be seen that the electrical conductivity values of Base / Carbamide, Base / Brassica, and PB2 are much lower than those of pure Base. This suggests that these compositions have a lower ionization potential, are more hydrophobic, and, consequently, have a greater affinity for the epithelium. This result once again corroborates the effect of interaction between the constituents, favoring interaction with the biological environment.

[0036] Finally, Figure 8 shows the zeta potential data for the different formulations. It is important to emphasize that the intensity of the zeta potential of a nanoparticle is related to its colloidal stability, since electrostatic repulsion helps to prevent nanoparticle coagulation (Fernandez-Nieves and Nieves 1999; Plaza, de Vicente et al. 2001; Douroumis, Fatouros et al. 2007; Nascimento, Goulart et al. 2014). It is reported in the state of technique that zeta potential values greater than |30| mV indicate colloidal stability. This means that the lower the zeta potential modulus, the greater the material's hydrophobicity.

[0037] According to the data presented in Figure 8, all formulations have a tendency to precipitate. However, it can be observed that for the pure Base, the zeta potential is more negative, corroborating the hypothesis of greater hydrophilicity and greater ease of dragging by water (p < 0.05). In the presence of the other constituents, the zeta potential value gradually decreases in the order presented in the figure, so that PB2 has the lowest zeta potential value, indicating greater hydrophobicity and, consequently, greater affinity for the epithelium. EXAMPLE 4: Assessment of the wound contraction index through application of the healing composition.

[0038] The possibility of applying the new use of the composition for specific treatment of wounds of 3 S degree, was proven by evaluating the wound contraction index in a biological model of in vivo healing. For this, the compositions were applied to male Wistar rats, as described below.

[0039] Initially, male Wistar rats - Rattus norvegicus (3 months old; weight 335.4 ± 16 g); were housed in individual cages, cleaned daily and maintained under ambient conditions (T = 22 ± 2 °C, humidity: 60 to 70% and light / dark cycle: 12 / 12 h). Water and food were provided ad libitum. The procedures were approved by the Ethics Committee (CEUA / UFV) (registration no. 90 / 2017). Aiming to reduce the number of animals used, formulations containing only Base / Carbamide or Base / Brassica were not used from then on.

[0040] The rats were anesthetized by intraperitoneal injection of Pentobarbital (70 mg / kg) before the burns were applied. The 3 S degree were induced according to the procedure described in the state of the art (A. Partoazar, et al., Drug Res. (Stuttg)., vol. 66, pp. 660-665, 2016.), and then measured with an analog caliper.

[0041] The animals were randomly separated into five groups (n = 5 / group): (group 1 - SAL) - animals with wounds treated with 0.9% saline solution; (group 2 - OV) - animals with wounds treated with pure Base; (group 3 - SS) - animals with wounds treated with 1% silver sulfadiazine (positive control); (group 4 - PB1) - animals with wounds treated with the Final Formulation containing 10% Brassica; (group 5 - PB2) - animals with wounds treated with the Final Formulation containing 20% Brassica. The daily amount of emulsion applied was 0.1 g to each wound.

[0042] Wounds were cleaned daily with 0.9% saline before emulsion application. All treatments were initiated 24 h after wounding and repeated once daily for 8 days. Regenerating tissue samples were collected on days zero (wound 0 = F0), four (wound 1 = F1), and eight (wound 2 = F2), repeating the anesthetic previously reported for sample collection (Pentobarbital 70 mg / kg). The animals were sacrificed at the end of the procedure by cardiac puncture after anesthesia.

[0043] Once the procedures were carried out, the healing process was assessed on the last day of the experiment by measuring the wound area, which in turn was calculated using the formula A = nr 2 , where r = radius and II « 3.14. The wound contraction index (WCI) was calculated by the equation: 100

[0044] The results showed that the wound area was smaller in the 4 s (F1 ) and 8 S (F2) days in the PB1 and PB2-treated groups compared with the other groups. The wound contraction index was higher in the PB1 and PB2-treated groups compared with the other groups on day 8 (F2) (Table 1), showing a significant reduction in the burn area. Table 1 - Different concentrations after 4 and 8 days. SAL: 0.9% saline; SS: 1% silver sulfadiazine (control), PB1: B. oleracea extract (10%); and PB2: B. oleracea extract (20%). F0 = intact tissue; F1, F2 = scar tissue after 4 and 8 days, respectively. Data are represented as mean ± standard deviation of the mean. *Statistical difference between treatments: SAL, Base, and SS (Student-Newman-Keuls test). RWC: Wound contraction rate (mm 2 / day). EXAMPLE 7: Histological Analysis

[0045] To corroborate the healing process, tissue samples were collected from the wounds and fixed in a 10% formaldehyde solution buffered in 0.1 M sodium phosphate (pH 7.2), dehydrated in ethyl alcohol, diaphanized in xylene, and immersed in paraffin for histological analysis. Histological sections (4 μm) were obtained by a Leica Multicut® rotary microtome 2045 (Reichert-Jung Products, Germany) and stained with hematoxylin.

[0046] Histological images were viewed and captured using a BX-60® optical microscope (Olympus, São Paulo, Brazil) coupled to a QColor-3® digital camera (Olympus, São Paulo, Brazil). Five images were obtained per random section, with a resolution of 2592 x 1944 pixels and 200x magnification. Fibroblasts and blood vessels were counted in digitized images with a grid of 300 intersections, and elastic and collagen fibers with a grid of 256 intersections (Media Cybernetcs®, Silver Spring, USA). Mast cells were analyzed at 400x magnification in 10 random histological sections to obtain a total area (TA) of 3.11 x 106 pm 2 . The number of mast cells per unit of histological area was calculated according to the formula QA = Z mast cells / TA.

[0047] Figure 9 shows photomicrographs of mast cells in scar tissue treated with B. oleracea extract (20%) (toluidine blue, E = 40x magnification and F = 100x magnification). F1 , F2 = scar tissue after four and eight days, respectively. SAL: 0.9% saline; OV: Pure Base; SS: 1% silver sulfadiazine, PB1: B. oleracea extract (10%); and PB2: B. oleracea extract (20%). Arrows show blood vessels in scar tissue treated with B. oleracea extract (20%). Data are represented as mean ± standard deviation of the mean, a, b, c, d represent the statistical difference between treatments: SAL, OV, SS, and PB1, respectively (p < 0.05) (Student-Newman-Keuls test).

[0048] The test showed an increase in the number of cells in the wounds treated with PB1 on day four (F1) compared to the SAL, SS, and PB2 groups. On day 8 (F2), cellularity was higher in the groups treated with PB1 and PB2 compared to SAL and SS. The number of blood vessels was higher in PB1 compared to SAL, OV, and SS on day 4. On day 8, the number of blood vessels was higher in the PB2 group when compared to SAL and SS (Figure 9 - A, B). The number of mast cells was higher in the wounds treated with PB2 compared to the SAL, OV, and SS groups on day 4. On day 8, the number of mast cells was higher after treatment with PB1 and PB2 than the SAL, OV, and SS groups (Figure 9-C).

[0049] Figure 10 shows F1, F2 = scar tissue after four and eight days, respectively. D, E, F: photomicrograph of a histological section of skin treated with 20% B. oleracea extract (PB2) on day 8. D and E: type I (white arrow) and type III (white star) collagen fibers, Sirius red staining (20x magnification). F: elastic fibers (black arrow), Verhoeff staining (40x magnification). SAL: 0.9% saline; OV: Pure base; SS: 1% silver sulfadiazine, PB1: B. oleracea extract (10%); and PB2: B. oleracea extract (20%). Data represented as mean ± standard deviation of the mean, a, b, c, d represent the statistical difference between treatments: SAL, OV, SS and PB1, respectively (p < 0.05) (Student-Newman-Keuls test).

[0050] The test shows that the proportion of collagen III fibers was higher in the groups treated with OV and SS compared to SAL, PB1 and PB2 on day 4. On day 8, the proportion of these fibers was lower in the groups treated with PB1 and PB2 when compared to the other groups. The group treated with silver sulfadiazine (SS) presented a lower proportion when compared to SAL (Figure 10-A).

[0051] The proportion of type I collagen fibers was higher after PB2 treatment than all other groups on day 4. On day 8, this proportion was higher in the groups treated with PB1, PB2, and SS compared to SAL and OV (Figure 10-B). Furthermore, the proportion of fibers in the group treated with the highest dose of PB2 was higher than in the group receiving the lowest dose of PB1. Figure 10-D shows a histological section of the skin treated with PB2 evidencing the intense presence of type I collagen fibers (arrow). EXAMPLE 8: Measurement of tissue-protective enzymes

[0052] The effect of applying samples PB1 and PB2 to burn-injured tissue was assessed by measuring the enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione stransferase (GST). Measuring these enzymes is important because they are primarily responsible for the healing process from a molecular perspective, as they protect the tissue against oxidative damage caused by reactive oxygen species. For this purpose, tissue fragments were collected and frozen (-196 S C) and stored at -80 S C. The samples were homogenized in PBS buffer and centrifuged at 5 S W.

[0053] SOD activity was determined by a method described in the state of the art (Dieterich, U. Bieligk, et al. Circulation, vol. 101, no. 1, pp. 33-39, 2000). CAT activity was determined by a method described in the state of the art (H. Aebi, "Catalase in vitro,” Methods Enzymol., vol. 105, pp. 121-126, 1984). GST activity was measured using a method described in the state of the art (W.H. Habig, et al. J. Biol. Chem., vol. 249, pp. 7130-7139, 1974).

[0054] Figure 1 1 shows F1 , F2 = scar tissue after four and eight days, respectively. SAL: 0.9% saline; OV: Pure base; SS: 1% silver sulfadiazine, PB1: B. oleracea extract (10%); and PB2: B. oleracea extract (20%). Data are represented as mean ± standard deviation of the mean, a, b, c, d represent the statistical difference between treatments: SAL, OV, SS, and PB1, respectively (p < 0.05) (Student-Newman-Keuls).

[0055] The test shows that SOD enzyme levels were higher after PB2 treatment compared to all other groups on days 4 and 8 (Figure 11-A). CAT enzyme levels were higher in PB1 and PB2 treatment on day 8 compared to SAL, OV, and SS groups (Figure 11-B). GST values were higher in both PB1 and PB2 treated groups compared to SAL and OV on day 8.

[0056] Given the results demonstrated in the tests, it was proven that the administration of the emulsion induces an increase in the main enzymes responsible for the protection of tissue proteins and lipids (SOD, CAT and GST) against reactive oxygen species.< / dh> < / dh> < / dh>

Claims

CLAIMS 1. “PROCESS FOR OBTAINING A NANOSTRUCTURED EMULSION BASED ON BRASSICA OLERACEA VAR. CAPITATA LEAVES, ANHYDROUS LANOLIN AND CASTOR OIL”, characterized by the process of obtaining the emulsion being obtained from the following steps: a) In the main tank, considering the final mass of the finished product, the hydrophobic fraction of the composition is prepared, adding under constant stirring between 400 and 800 rpm, and heating at a temperature ranging from 60 to 90 °C; the hydrophobic constituents of the formulation: petroleum jelly, anhydrous lanolin, hydrogenated castor oil, polyol fatty acid esters; b) Once the fusion and mixing of all the constituents is ensured, ethanolic extract of Brassica oleracea var.capitata ethanolic extract until complete homogenization; c) In a secondary tank, considering the final mass of the finished product, the hydrophilic fraction of the composition is prepared in the presence of emulsifying agents, adding under constant stirring between 400 and 800 rpm and heating at a temperature ranging from 40 to 80 °C; demineralized water, cetostearyl alcohol surfactant, ethoxylated sorbitan monooleate, carbamide; d) After mixing and homogenizing the agents of the hydrophilic fraction, the preservatives (methylparaben, propylparaben and butylated hydroxytoluene (from 1% to 10% by weight each) are added; e) Finally, with both hydrophilic and hydrophobic fractions, with both at a temperature close to 30-40 °C, the hydrophobic fraction is poured into the hydrophilic fraction, at a constant flow rate, maintaining agitation between 400 and 800 rpm, until the product is completely homogenized, which can then be cooled to room temperature and bottled.

2. “NANOSTRUCTURED EMULSION, BASED ON BRASSICA OLERACEA VAR. CAPITATA EXTRACT, ANHYDROUS LANOLIN AND CASTOR OIL”, according to claim 1, characterized in that the hydrophobic fraction contains petroleum jelly (from 5% to 10% by weight), anhydrous lanolin (from 5% to 15% by weight), hydrogenated castor oil (from 2% to 10% by weight), polyol fatty acid esters (from 5% to 10% by weight), ethanolic extract of Brassica oleracea var. capitata (in the proportion of 10% to 30% by weight).

3. “NANOSTRUCTURED EMULSION, BASED ON BRASSICA OLERACEA VAR. CAPITATA EXTRACT, ANHYDROUS LANOLIN AND CASTOR OIL”, according to claim 1, characterized in that the hydrophilic fraction contains demineralized water (15% to 35% by weight), cetostearyl alcohol surfactant (from 2.5% to 10% by weight), ethoxylated sorbitan monooleate (from 5% to 10% by weight), carbamide (from 1% to 5% by weight).

4. “NANOSTRUCTURED EMULSION, BASED ON BRASSICA OLERACEA VAR. CAPITATA EXTRACT, ANHYDROUS LANOLIN AND CASTOR OIL”, according to claim 1, characterized by containing the preservatives methylparaben, propylparaben and butylated hydroxytoluene (from 1% to 10% by weight each).

5. “NANOSTRUCTURED EMULSION, BASED ON BRASSICA OLERACEA VAR. CAPITATA EXTRACT, ANHYDROUS LANOLIN AND CASTOR OIL”, according to claim 1, characterized by containing the hydrophilic, hydrophobic and preservative fractions, described in claims 2-4.

6. “NANOSTRUCTURED EMULSION, BASED ON BRASSICA OLERACEA VAR. CAPITATA EXTRACT, ANHYDROUS LANOLIN AND CASTOR OIL”, according to claims 1-5, characterized in that the combination of the hydrophilic, hydrophobic and preservative constituents results in an increase in the thermal stability of the formulation in the range of 30 to 170 °C.

7. “NANOSTRUCTURED EMULSION, BASED ON BRASSICA OLERACEA VAR. CAPITATA EXTRACT, ANHYDROUS LANOLIN AND CASTOR OIL”, according to claims 1-5, characterized by: a) presenting viscoelastic behavior with non-zero yield stress; b) the interaction of the carbamide with the Brassica extract generates a stiffness reduction effect, when compared with the pure Base; c) the presence of carbamide causes an emollient effect; d) the interaction of the components of the Brassica extract with the urea results in a reduction in the viscosity of the formulation; e) the interaction of the components of the Brassica extract with the carbamide generates greater rheological stability (low oscillation in viscosity) in the range of 20 to 45 °C.

8. “NANOSTRUCTURED EMULSION, BASED ON BRASSICA OLERACEA VAR. CAPITATA EXTRACT, ANHYDROUS LANOLIN AND CASTOR OIL”, according to claims 1-5, characterized by: a) formulation PB1 or PB2 self-emulsifies in the presence of water, forming nanostructures of 356 ± 11 nm and zeta potential of -1.289 ± 0.021 mV; b) interaction of the Brassica extract with the carbamide reduces the size and zeta potential values of the nanostructures.

9. “USE OF NANOSTRUCTURED EMULSION, BASED ON BRASSICA OLERACEA VAR. CAPITATA EXTRACT, ANHYDROUS LANOLIN AND CASTOR OIL”, according to claims 1-8, characterized by being for the prevention of topical antioxidant stress induced in wounds caused by burns of 3 S degree.

10. “USE OF NANOSTRUCTURED EMULSION, BASED ON BRASSICA OLERACEA VAR. CAPITATA EXTRACT, ANHYDROUS LANOLIN AND CASTOR OIL”, according to claims 1-8, characterized by stimulating the proliferation of blood vessels and collagen fibers in wounds caused by burns of 3 S degree. 1 1. “USE OF NANOSTRUCTURED EMULSION, BASED ON EXTRACT OF BRASSICA OLERACEA VAR. CAPITATA, ANHYDRO LANOLIN AND OIL CASTOR OIL”, according to claims 1-8, characterized by stimulating the acceleration and homogenization of cellular growth of fibroblasts and mast cells in burn wounds of 3 S degree.

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