BR102012029163A2 - COFFEE OIL BASED COMPOSITION AND FORMULATION AND USES - Google Patents

Abstract

COMPOSITION AND FORMULATION BASED ON COFFEE OIL AND USES The present invention describes a composition and its formulation based on topical coffee oil with high skin healing capacity. The composition is capable of maintaining the healing area with ideal humidity and the peripheral areas dry and protected, in addition to presenting a systemic action, factors that accelerate the healing process.

Description

COFFEE OIL BASED COMPOSITION AND FORMULATION AND USES

Field of the Invention

The present invention relates to a coffee oil based composition. More specifically, the present invention relates to a topical use composition based on roasted coffee oil with skin healing ability. Furthermore, further objects are their use and a formulation comprising said composition.

BACKGROUND OF THE INVENTION Human attempts to intervene in the healing process of

Wounds, whether accidental or intentionally promoted during surgical procedures, date back to antiquity. The incidence and prevalence of chronic ulcers is extremely high, resulting in high financial costs. Although accurate data are not found in Brazil, some 15 studies show that there is a major psychosocial and economic impact of wound chronification. They are the second cause of absence from work in Brazil (Ereno, 2003).

Many general and local variables influence this long and complex process. Among the general factors, the age, the nutritional status of the patient, the existence of underlying diseases such as diabetes, cardiocirculatory and coagulation disorders, atherosclerosis, renal dysfunction, systemic infectious conditions and systemic drug use interfere. In addition, they also interfere with the anatomical location of the lesion, race, surgical technique used, etc. (Mandelbaum, 2003).

In order for a cutaneous ulcer to heal, it occurs, the

At the cellular and molecular level, a cascade of events that work together for the repavimentation and reconstitution of injured tissue (Ortonne, 1994).

The healing process can be more simply divided into three phases: inflammatory, proliferative and remodeling (Pittman, 2007). In the inflammatory phase, after hemostatic coagulation has begun, numerous chemical mediators are released by inflammatory cells. In the first three days, the action of polymorphonuclear cells responsible for phagocytosis of bacteria predominates. The macrophage is the most important cell of this phase and will remain from the third to the tenth day. Lymphocytes appear in the lesion approximately one week later. The Iinfocins released by them have 5 influence on the action of macrophages (Mandelbaum, 2003). In addition to these, this phase relies on fibronectin. Synthesized by fibroblasts, keratinocytes, and endothelial cells, it adheres simultaneously to fibrin, collagen, and other cell types, thus acting as a glue to consolidate fibrin clot, cells, and matrix components (Mosher, 1981). The proliferation phase is responsible for closing

the injury itself. In it happens reepithelization by the migration of keratinocytes from the wound edges and attachments if they have been preserved (Dielgmann et al., 1981; Clark, 1985; Gentilhomme et al, 1999). Cytokines modulate the inflammatory process that is necessary in the healing process but can be harmful if excessive. They are also responsible for faster and more effective healing (Gentilhomme et al, 1999). In addition to inflammatory and endothelial cells, matrix formation depends on the fibroblast responsible for the production of important substances for both debridement and physiological remodeling 20 (Van Winkle, 1967). The proliferation of vessels that occurs at this stage is essential for supplying oxygen and nutrients for healing. Thus, it can be said that the proliferation phase is divided into reepithelization, fibroplasia and angiogenesis (Mandelbaum, 2003).

Remodeling is the last phase, lasting months and is responsible for increasing tensile strength and decreasing scar size. Collagen reformulations, improvement in collagen fiber components, water resorption are events that allow for a connection that increases scar strength and decreases thickness (Doillon et al., 1985). Local neovasculature decreases, and the normal healing area is about 80% of the normal tensile strength of the skin, is not bulky and is flat (Mandelbaum, 2003). Among the inflammation-related substances believed to interfere with the healing process, the following stand out: adiponectin, leptin, interleukin (IL) 2, IL-4, IL-6, IL-12, tumor necrosis factor (TNF) ) -a, insulin-related growth factor (IGF) -I, interferon (IFN) -ae IFN-γ.

Adiponectin is a protein that acts on glucose and lipid homeostasis. Circulating adiponectin levels are high, reaching approximately 0.01% of plasma proteins (Basu, 2009). It is induced during adipocyte differentiation and its secretion is stimulated by insulin. Adiponectin is an adipokine that influences systemic metabolism. It acts on glucose and fatty acid metabolism, being in some situations antagonistic to TNF-α (Yano, 2008). Induces a decrease in serum glucose and triglyceride levels, increases the level of glucagon. In the liver it promotes insulin-dependent inhibition of gluconeogenesis and has anti-fibrous action; In the 15th skeletal muscle promotes the use and oxidation of fatty acids, glucose use and lactate production. Acts as anti-inflammatory and has protective action against atherosclerosis (Berg, 2001). In the skin of diabetic patients, adiponectin acts directly on keratinocytes, regulating the expression of immunomodulatory substances produced by these cells; This indicates that adiponectin acts indirectly on other cells through keratinocyte products. Another action would be the suppression of keratinocyte proliferation and differentiation, which would be beneficial in cases of hyperkeratinization (Kaway, 2008). A study conducted in Brazil indicates that overweight has the ability to increase healing time (Nascimento, 2006).

Leptin deficiency in animals as well as humans may

lead to weight gain and also to the increase in healing time. Direct administration of leptin leads to reduced healing time, with greater reepithelization, but without interfering with angiogenesis. Another fact observed in animals is the increased expression of leptin receptor genes locally, which indicates the importance of leptin for the healing process. Leptin leads to an increase in keratinocytes at the site of application at the beginning of healing (Nascimento, 2006).

IL-2 is a pleiotropic cytokine (like most cytokines), ie it has multiple effects: a single cytokine can interact with more than one cell type, have multiple biological activities, interact with other cytokines with which it may have overlapping activities (Arai, 1990). Produced primarily by mitogenic or antigenically activated T lymphocytes. It has a key role in promoting clonal expansion of antigen-specific T cells. In addition, IL-2 is capable of mediating multiple immune responses in a variety of cell types. IL-2 stimulates thymocyte proliferation, proliferation and differentiation of activated B cells; promotes monocyte growth and differentiation and cytocidal activity; induces the growth of natural killer cells and their production of cytokines and cytolytic activity; increases the production of lymphocyte-activated killer cells (LAK) and induces oligodendrocyte proliferation and differentiation (Goldsmith, 1994). In hypertrophic scarring and keloid situations, IL-2 is found in local lymphocytes, suggesting pro-scarring action (Armour, 2007).

IL-4 is also a pleiotropic cytokine that has multiple immune response modulation activities in a variety of cell types. It is a B cell activation / differentiation factor that regulates the isotope exchange of Ig, particularly IgGI and IgE. It suppresses the development of IFN-γ-producing CD4 + T cells and regulates the differentiation of naive helper T cells in the Th2 subset that mediates the allergic and humoral immune response. Along with TNF-α synergistically induces the expression of VCAM-1 (vascular cell adhesion molecule 1) in smooth endothelial and muscle cells, resulting in selective recruitment of eosinophils and lymphocytes at the inflammation site. IL-4 negatively regulates the production of inflammatory mediators such as IL-1, TNF-α, and prostaglandin 2 (PGE2) in monocytes. Anti-tumor activity has been demonstrated both in vivo and in vitro. The cells that secrete IL-4 are basophils, CD8 + T, memory CD4 + and naive Th2, mast cells, eosinophils, and virus-activated dendritic cells. IL-4 is primarily responsible for stimulating the production of tenascin (an extracellular matrix glycoprotein, present as a thin layer in the adult papillary dermis, but which is restated in the healing process) by fibroblasts in the earlier stages of collagen deposition and cell migration (Makhluf, 1996).

IL-6 is a cytokine that plays important roles in the

host defense, in acute phase reactions, inflammation, hematopoiesis, bone metabolism, and cancer progression. IL-6 is essential for the transition from acute inflammation. It is secreted by various cell types and production is regulated by numerous signals such as mitogenic or antigenic stimuli, lipopolysaccharides, calcium, other cytokines, and viruses. Cytokines IL4, IL-10 and IL-13 inhibit IL-6 expression in monocytes (Hirano, 1996). Elevated IL-6 serologic levels were observed in pathological situations such as viral and bacterial infections, trauma, autoimmune diseases and inflammation. IL-6 increases at the onset of the healing process and is important, 15 as it has mitogenic properties in keratinocytes, as well as chemotactic for neutrophils and their infiltration to the affected area is able to increase local angiogenesis and increase collagen deposition in the wound site (Lin, 2003).

IL-12 is a heterodimeric glycoprotein, which increases cytotoxic activity and induces IFN-γ production in natural killer, dendritic TeT cells of the epidermis. It also induces IFN-γ production in macrophages. This cytokine, together with others of the same family (IL-23 and IL-27), is capable of promoting the development of immune response through CD4 + Th1 T cells; In response to infections, IL-12 promotes 25 Th1 cell production after IL-27 has transformed ThO into Th0 / 1; Together with IL-18, IL-12 creates memory Th1 cells from effector cells (Hamza, 2010). In scarring processes, IL-12 has its increased gene expression, causing macrophages to produce and secrete more IFN-γ (Ishida, 2004).

TNF-α plays critical roles in normal host resistance to infection and growth of malignant tumors, serving as immunostimulants and as mediators of the inflammatory response. Excessive TNF production, however, has been implicated as playing a role in a number of pathological conditions, including cachexia, septic shock, and autoimmune diseases. TNF-α is produced by activation of macrophages and other cell types, including TeB cells, NK cells, 5 endothelial cells, smooth muscle cells, and some tumor cells. In healing, the absence of receptors for this cytokine accelerates the process (Ware, 1996). In wounds, TNF-α is responsible for initiating the proinflammatory cascade and is responsible for healing strength and tension (Mast1 1996).

IGF-I, also known as somatomedine C, is a member of the insulin superfamily. It has been found to mediate hormone actions in somatic cell growth, but it has also been shown to be an important regulator of cell metabolism, differentiation and survival. IGF-I is synthesized as a preprotein that is proteolytically cleaved to generate mature protein (Humbel, 1990). IGF15 1, together with platelet-derived growth factor 2 (PDGF-2), can increase skin thickness in wound healing (Lynch, 1989), and this substance is directly involved in the regeneration of various tissue types. not just the epithelial (Chen, 2010).

IFN-ci is a glycoprotein belonging to the cytokine family 20 that participates in cell control and replication, host defense against foreign organisms such as viruses or bacteria and is a major type I interferon. It is produced by mononuclear phagocytes in response to viral infection. It has a role in inhibiting viral replication and enhances the expression of type I (MHC type 25 I) main histocompatibility complex molecules, either by autocrine or paracrine (Krause, 2005). Another form of action of IFN-α is to protect some cells against virus entry. Numerous investigations have shown that IFN is involved in cell growth regulation and immunomodulatory effect (Ferrantini, 2007).

IFN-γ, also known as type II interferon, was initially identified as a product with anti-viral activity of mitogenically activated T lymphocytes. It has a key role in host defense by performing immunoregulatory, antiproliferative, and proinflammatory activities (Boehm, 1997). Some of the most important functions are macrophage effector function stimulation, IL-12 induced Th1 differentiation, MHC class I and II expression modulation, immunoglobulin class exchange (Ig) regulation and regulation of interactions between leukocytes and endothelium. IFN-γ has also been involved in physiological roles in sensor neurons and spermatogenesis (Neumann, 1997; Kanzaki1 1998).

Both interferons have anti-scarring action, acting to inhibit collagen synthesis and cell proliferation and may increase metalloproteinases production. IFN-α may increase the production of collagenase enzyme, which increases its anti-scarring action. Thus, both must have their values increased in the final phase of healing, preventing it from progressing to hypertrophic injury (Tredget, 2000).

Studies have been conducted evaluating the potential of using

Natural products as active ingredient of healing resources. Among these, studies were carried out with oil palm (Sasidharan, 2012), thermal water (Faga, 2011), aloe (Duansak, 2003), Jatyadi Taila (Shailajan, 2011), mint (Takayama, 2011) and some types of oil such as like pine oil 20 (Tumen, 2011), rice oil (BR 10 2012 028235 6). Specifically with regard to the use of oils, it is already known from the State of the Art, for example, that those rich in essential fatty acids have antiseptic and healing actions.

Healing is a dynamic process and therefore no antiseptic or healing effect observed in vitro guarantees a favorable outcome in vivo. For example, alcohol, known to be antiseptic, is not used for healing. The fatty acid composition is not substantially altered by the process of heating the roasting of coffee beans, for example, however, the aroma and flavor compounds of the beverage are generated in this process. Just as these new substances can change the aroma and taste of the coffee powder infusion in hot water, they can potentially be involved in a different response than coffee oil in the healing process.

According to a recent review article by Ferreira et al, published in 2012 in the Journal of the School of Nursing of USP, in view of the scarcity of 5 randomized controlled trials in humans, it was not possible to generalize in clinical practice that essential fatty acids positively influence the healing process or have antimicrobial action. Thus, there is a need for research with greater methodological rigor comparing the different formulas available containing fatty acids.

Document BRPI0602842-A of 07/20/2006, for example, describes the use of green coffee oil (Coffea arabica) in cosmetic and pharmaceutical formulations for the maintenance of skin properties, where said oil acts as an activity promoting agent. of 15 regeneration; firmness and hydration of the skin; antioxidant; free radicals; anti aging; skin roughness reducer; auxiliary agent in reducing and preventing stretch marks; lipolytic agent; and anti-inflammatory agent, and these beneficial actions are obtained with coffee oil in its crude form; semi-refined, refined, fractionated, hydrogenated or interesterified pure or in mixtures with other oils and / or ingredients in preparations of cosmetic and pharmaceutical products, whose practical result gives the skin healthy characteristics.

However, although the claimed formulation has anti-inflammatory action, it cannot be stated that it could be applied effectively in the treatment of skin ulcers. This is because the healing process requires a balance between the production of inflammatory and anti-inflammatory substances and in this case, although in vitro studies may indicate some possibility of application of the product in an anti-inflammatory situation, for example, the end result in vivo may be completely different, and may even make it unfeasible to use for the aforementioned application. In the healing process, the same substance may be needed at higher concentrations at the beginning of the process and at lower doses at the end of the process to prevent, for example, a hypertrophic scar.

As will be apparent from the results of studies carried out for the embodiment of the present invention, green coffee oil or produced from raw beans does not promote a healing action in vivo, but only oil obtained from roasted coffee beans had comparable action to AGE®, based on mid-chain essential fatty acids and sunflower oil, which was used as one of the study controls. This shows that it is not any essential fatty acid, nor any coffee oil that has healing action.

Thus, the same substance may be anti-inflammatory and have no pro-healing action and it is necessary to compare and inventive work to obtain a product whose action in vivo proves to be beneficial in the healing process as a whole.

Unlike oil extracted from green or raw coffee (Coffea arabica),

where most of the research is related to the pharmaceutical and cosmetic sector, the oil extracted from roasted coffee, obtained directly by pressing the beans, is best known for its use in the food sector, mainly as candy, candy and truffle fillings. of 20 liqueurs, flavor enhancer in instant coffee, ice coffee, cappuccinos, various desserts, ice cream, puddings, sweets and milk based preparations. There are no previous studies evaluating the action of green coffee oil or roasted coffee oil on healing and the anti-inflammatory action of raw coffee oil has been evaluated mainly in in vitro studies.

Study by Wagemaler, T.AL; Carvalho, C.R.L; Maia, N.B; Baggio,

S; Guerreiro Filho, O., entitled "Sun protection factor and composition of Iipid fraction of coffee beans" and published in the journal Industrial Crops and Products, 33 (2011) 469-473, showed that the main agent involved in photoprotection is caveol, as described in patent application 30 221105532784 of 06/13/2011 by inventor Maia, NB The authors of this application try to find new applications for coffee oil and the procritic action can only be observed when using oil obtained from roasted beans of Coffea arabica.

As mentioned, no in vivo studies on the action of coffee oil, whether green (roasted) or roasted, related to healing processes have been found in the literature.

In the light of the available information and somewhat contrary to the state of the art, the present invention relates to a coffee oil based composition, including roasted coffee beans, for the treatment of skin ulcers. The topical use of this composition influences the systemic production - and not only at the site of application - of healing-related substances, suggesting that its use has a beneficial systemic healing action. Additionally, the proposed composition has advantages in several respects. First, the characteristics of an effective wound care product should include ease of removal and comfort for the patient, good value for money, keeping the healing area moist and dry peripheral areas and protected, ease of application and adaptability (conformation to various parts of the body). Another differential of the process described here is related to the systemic action that the technology has, where the 20 serum levels of some substances have been altered, as well as being able to accelerate the healing process in relation to the type of collagen deposited in the site, being more effective than other products commonly used for this purpose.

Brief Description of the Invention

The present invention is an oil based composition.

of topical coffee with high skin healing capacity. Furthermore, the present invention also relates to the use and formulation of said composition.

BRIEF DESCRIPTION OF THE FIGURES The structure and operation of the present invention, together with further advantages thereof, may be better understood by reference to the accompanying figures and the following description:

Figure 1 shows the unhealed area of wounds (%) over 10 days of treatment of Group 1 animals (Witness).

Figure 2 shows the unhealed wound area (%) over 10 days of treatment of Group 2 animals (AGE).

Figure 3 shows the unhealed wound area (%) over 10 days of treatment of Group 3 animals (Crude Coffee Oil).

Figure 4 shows the unhealed wound area (%) over 10 days of treatment of Group 3 animals (Roasted Coffee Oil).

Figure 5 presents a graph regarding cellularity on the control side of the 4 groups.

Figure 6 presents a graph regarding the cellularity on the treated side in the 4 groups.

Brief description of the attachment

Annex 1 exemplifies the appearance of injuries on the fourth day of treatment in one of the visual evaluations performed.

Detailed Description of the Invention

The present invention describes a topical healing coffee oil based composition.

The main examples of products which may be prepared from the composition object of the present invention are:

- lotion;

- cream;

- ointment;

- Post;

- sprays;

- folders; - Suspensions and

- Stickers.

The composition of the present invention comprises the following components:

- coffee oil;

- Pharmaceutically acceptable vehicle.

In addition to these components, the composition of the present invention may further comprise optional components to provide some desirable feature not achieved with the aforementioned components such as emollient, humectant, chelating agent, thickening system, antioxidant component, preservative system, colorants and flavorings.

The components used in the composition of the present invention as well as other components optionally added in the formulation will be described in more detail below. For each product prepared the concentrations and components may vary.

Feedstock

Coffee used as an oil extraction raw material may be of any type of beverage market classification: soft, hard or rinded and of any type of grain size or sieve used for grain size separation.

The roasting required for the process is the same industrial roasting used for the production of grain intended for consumption as a beverage, capable of generating the substances that promote the aroma and flavor characteristic of the beverage made by infusion with hot water.

The oil extraction process can be either by compression to

cold as used in the developed work, as by extraction with simple solvents or in supercritical state. However, there are differences in the amount of essential fatty acids found in different coffee species. This was observed in the aforementioned study "Sun protection factor and composition of 30 Iipid fraction of free coffee beans" in which one of the authors of the present invention (Maia, N.B.) was one of the authors. Thus, studies carried out for the embodiment of the present invention make it possible to ensure that Coffea arabica roasted grain oil obtained by cold pressing showed healing action in vivo. For the embodiment of the present invention, the coffee used as raw material was purchased directly from the producer and later sent for roasting and oil extraction. This procedure was necessary because the commercialization system and scale used by the roasters are much larger than the 12 bags volume needed to obtain the oils used in the tests performed.

In addition to the use of blends to blend, coffee offered in the normal market has no control over batches purchased by roasters, does not guarantee the harvest of the product (post harvest time), requires a minimum purchase of 20 bags and do not guarantee or specify storage conditions. To have a control of these factors, it was decided to buy from the producer and later industrialization.

As the minimum volume for oil extraction in the equipment

used was six bags, 12 bags were already benefited, being 6 sent to a roasting and used to obtain material for the treatments with roasted coffee oil and roasted coffee filter sludge. The others were directed directly to the extraction and produced crude coffee oil and filter dregs.

The roasting of the coffee was done in a commercial roaster (LiIIa (B)) with water cooling and forced ventilation to six bags to ensure a homogeneous raw material volume, without contamination of other batches previously or subsequently processed in the industry's silos.

The roasting treatment used was the conventional one applied to the production of coffee for internal drink, with a yield of 0.79 of raw / roasted coffee, which resulted in a total of 285kg of roasted coffee for extraction of roasted coffee oil.

Extraction and Filtering of Oils The coffee oil employed in the present invention can be obtained by pressing the beans. Both cold compression extraction as used in the work carried out, such as extraction with simple or supercritical solvents, are potentially capable of produce material suitable for treatment, provided that both the oil with the appropriate fatty acid composition and the substances, for example those responsible for the characteristic aroma and taste, generated in the roasting process are obtained. The oil added to a pharmacologically acceptable carrier in a concentration ranging from 0.001% to 99.999%, preferably 70% may be used as a healing agent.

The dosage of coffee oil to be used depends on many factors such as the causative agent, the age, weight and clinical condition of the patient as well as the experience and judgment of the physician or practitioner administering the therapy. The effective amount of coffee oil provides the patient improvement detectable by a qualified practitioner. The dosage range varies with the compound used, the route of administration and the potency of the particular compound.

However, for the embodiment of the present invention, both types of coffee (raw and roasted) were sent for extraction in an industrial press. The equipment used was an “Expeller” type press (Piratininga®) adapted for extracting oil from both raw and roasted coffee beans. The machine has been retrofitted to receive suitable stainless steel hoppers, drip trays and vessels to obtain oil for use in the experiment, without contamination of other raw materials or extracted oils, 25 yet ensuring a quality product that can be repeated later. industrialization.

The six coffee bags for each type of raw coffee and the six roasted coffee generated respectively 23 and 38kg of crude oil. This volume was compatible with the minimum required volume of 20kg to operate the press type filter. The hand-closing horizontal filter press is specially built for high pressure filtration and clear oil, in addition to separating the filter sludge with high non-saponifiable material of interest for use in treatments. Table 1 shows the yields of the extraction process performed.

Table 1. Yields from the extraction process of coffee beans (C. arabica) crude and roasted by expeller press

Toasted Crude Oil grains (kg) 360 285 Crude oil obtained (kg) 24 20 Oil yield (%) 6.67 7.02 Filtering slurry (kg) 1.3 1.8 Sterilization of oils

The material processed in the extraction and filtration was packed in sealed glass vials with identified samples that were sent for gamma sterilization testing to a company that uses as a 60Co radioisotope emitter. The different materials (crude and roasted coffee oil and coffee grounds) properly identified were submitted to doses 3; 5; 7; 10; 12 and 15 KGy.

After irradiation, a contamination study was performed on 15 microorganism culture plates. Aliquots of 20μΙ_ of material (oil or sludge) obtained at the factory filtration to count colony forming units on Blood Agar, Chocolate Agar, BHI (Brain Heart Infusio) Broth and Sabouraud, especially for fungi. All samples tested inhibited the growth of microorganisms.

For this reason, it was decided to use sterilized oils with the

lower radiation dose (3KGy) for application in animal wound treatment.

Oil Analysis

To characterize the proportion of fatty acids in the different extracted and irradiated oils, an aliquot of the lipid extract containing approximately 400 mg of lipids was dried on a rotary evaporator. Transmethylation was performed according to the method of Hartman & Lago (1973), using ammonium chloride and sulfuric acid in methanol solution as esterifying agent.

Gas chromatography was performed on a Varian model 3900 gas chromatograph equipped with an automatic sampler; split injector, ratio 75: 1; CP-SIL 88 capillary column (100 m x 0.25 mm i.d., 0.20 μηι film); flame ionization detector (FID) and a workstation for data acquisition.

Chromatographic conditions: programmed column temperature, initial temperature 120 ° C / 2min, heating from 120 ° C to 220 ° C on a scale of 2.2 ° C / min and from 220 to 235 ° C on a scale of 1.5 ° C / min, remaining at 235 ° C for 15 minutes; carrier gas, hydrogen at a flow rate of 1 mL / min; make-up gas, nitrogen at 30 mL / min; injector temperature, 270 ° C; detector temperature, 3100C; injection volume 1 μί.

The identification of fatty acids was performed by

comparison of retention time of sample and standard fatty acids and co-chromatography.

Quantification was performed by area normalization and the results were expressed in g / 100g of sample.

Table 2 shows the data on the fatty acid composition of oils as a function of grain roasting and gamma ray treatments. No significant differences were observed as a function of the application of any of the gamma rays applied.

Table 2: Composition (%) of fatty acids of raw and roasted coffee oils subjected to different doses of gamma radiation for sterilization purposes.

_ Roasted Coffee_ _ Raw Coffee_

Dose Range Rays Dose Range Rays

(K (K

0 3 5 7 10 12 15 Mé 0 3 5 7 10 12 15 Mé Acid 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0.1 C14 C16 31 31 31 31 31 31 31 31. 30 30 30 30 30 30 30 30, C17 0, 0, 0, 0, 0, 0, 0.1 0, 0, 0, 0, 0, 0, 0 , 1 C18 8.8, 8, 8, 8, 8, 8, 8.6 9, 9, 9, 9, 9, 9, 9, 9.3 C18: 1 10 10 10 10 10 10 10 10 10, 9, 9 , 9, 9, 9, 9, 9, 9.2 C18: 2 38 38 38 38 38 37 38 38, 40 39 40 40 40 39 39 40, C18: 3 1, 1, 1, 1, 1, 1, 1, 1.2 1, 1, 1, 1, 1, 1, 1, 1.1 C20 3, 3, 3, 3, 3, 3, 3.2, 3, 3, 3, 3, 3, 3.3, C21: 1.0, 0, 0, 0, 1, 0, 0, 0.4 0.4, 0, 0, 0, 0, 0, 0.2 C20: 3, 0, 0 , 0, 0, 0, 0, 0, 0.2 0, 0, 0, 0, 0, 0, 0, 0.2 C22 0, 0, 0, 0, 0, 0, 0, 0.6 0 , 0, 0, 0, 0, 0, 0, 0.8 NI (not 0, 0, 0, 0, 0, 0, 0.2 0, 0, 0, 0, 0, 0, 0.1 C24 0, 0, 0, 0, 0, 0, 0.2, 0, 0, 0, 0, 0, 0, 0, 0.2 Acids Totals Saturated 44 44 44 44 44 44 44 44. 44 44 44 44 44 44 44 44, Monoin 11 11 11 11 11 10 11 11, 9, 9, 9, 9, 9, 9, 9, 9.4 Polyinsat 39 39 39 39 39 39 39 39, 41 41 41 41 41 41 41 41, omega 6 38 38 38 38 38 38 38 38, 40 40 40 40 40 40 40 40 40, omega 3 1, 1, 1, 1, 1, 1, 1, 1,2 1, 1, 1 , 1, 1, 1, 1, 1.1 trans 0 0 0 0 0 0 0 0.0 0 0 0 0 0 0 0 0 0.0 Nl (not 0, 0, o, o, Of o, 0 0.0 o, 0, o, o, o, 0, 0.0 Pharmaceutically acceptable vehicle:

Local absorption and effectiveness of the present invention may be enhanced by using as appropriate carrier an appropriate amount of medium chain essential fatty acids or other chemical compounds known to facilitate absorption and delivery of the active compounds of the present invention used as a carrier for the preparation. coffee oil. This will be the preferred carrier of the composition of the present invention by a suitable percentage (q.s.p.) to achieve 100% of the formula based on the total weight of the composition. Still other pharmaceutically acceptable carriers may be used as saline and buffers.

In addition to these components, the composition of the present invention may further comprise optional components such as:

- chelating agent such as ethylenediaminetetraacetic acid (EDTA) and

their salts;

- pH adjusting agent such as triethanolamine or hydroxides

inorganic;

Preservative such as parabens, organic acids, imidazolidinines, diazolidines, phenolic alcohols; - Emollient such as alcohols and fatty acids, esters, ethers, mono-, di- or triglycerides, natural or synthetic hydrocarbons or organic carbonates and combinations thereof.

Humectant such as glycols, preferably glycerin, propylene glycol, butylene glycol and combinations thereof.

Bacteriostatic agent such as methyl hydroxybenzoate, propyl hydroxybenzoate, chlorocresol, benzalkonium chloride and the like.

- Active ingredients such as antibiotics, anesthetics, pain killers and scaling. Examples of representative drugs within these classes

include gentamicin, benzoyl peroxide, glucocorticoids, hydrocortisone, vitamin D3, methotrexate, cyclosporine, retinoids.

- dyes;

- Flavoring.

The composition described in the present invention may be prepared employing any process known in the prior art. Coffee oil extracted by pressing the beans may be mixed under sterile conditions with a pharmacologically acceptable carrier, and with any preservative, buffer or propellant as required. Topical preparations can be made by combining coffee oil with conventional pharmaceutical diluents and commonly used carriers in dry, liquid, cream and aerosol topical formulations. Ointments and creams may, for example, be formulated with an aqueous or lipid base with the addition of suitable thickening or gelling agents. Ointments, creams, pastes and gels may also contain excipients such as vegetable and animal fats, oils, waxes, paraffins, starch, cellulose derivatives, talc, zinc oxide, silicones, polyethylene glycol, silicic acid among others. Powders and sprays may contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, or a mixture of these substances. Additionally, sprays may contain propellants such as chlorofluorohydrocarbon, butane and propane.

Example 1: In vivo tests For the tests were used isogenic rats of Rattus norvegicus albinus IinhagemNTacUnibiSD (SpragueDawIey) species, with an average age of 7 weeks and a weight between 220 to 270g. The experiment was approved by the Animal Research Ethics Committee of the State University of Campinas, according to Federal Law 6.638.

After the cage adaptation period, the animals were prepared to perform the wounds and began to receive daily treatments with the different types of oil, separated into "groups" as shown in Table 3 below. In each group of animals they were sub-divided to be sacrificed on days 2, 4 and 10 after the first treatment application.

The change in periods designed for sacrifice of two, seven, and

14 days, because a faster than expected healing process was clinically observed in the periods of less than 4 and 10 days, the three phases of healing could be more appropriately evaluated.

Respecting the direction of the head to the tail, in all animals four wounds were made on the left side, designated "A", "B", "C" and "D" and those on the right side another four designated "E". "," F "," G "and" H ". The left side always received the product of "Control" or "Witness" and the right the "Treatment".

Table 3 schematically shows the distribution of wounds and treatments received by each group of animals.

Table 3. Treatments applied in each study group.

Left Side (Control) Right Side (Treatment) Group 1 Occlusive Dressing with Sterile Gauze Mineral Oil (Nujol®) Group 2 Mineral Oil (Nujol®) Essential Fatty Acids Group 3 Mineral Oil (Nujol®) AGE® Raw Coffee Oil Group 4 Mineral Oil (Nujol®) Roasted Coffee Oil To receive the specified treatment and avoid stress, each

The animal was taken to a separate room from the others where it was anesthetized with a specific dose for its mass class with ketamine 50mg.kg'1, xylazine 7.0 mg.kg'1 and diazepam 2.0 mg.kg'1. After being completely anesthetized the paravertebral and scapular regions were completely trichotomized and disinfected with a 2% solution of chlorhexidine diglyconate for asepsis of the area where the wounds would be produced.

In the trichotomized area 4 wounds were made on the right side and

another 4 on the left side, 1 cm bristle along the spine. For a better homogeneity of the wounds between the 8 wounds of each animal and among all animals studied, a 6mm diameter disposable circular scalpel was used for a disc-shaped cut across the entire depth of the skin. The disc-shaped cutaneous fragment was lifted by forceps and excised with scissors.

Immediately after the injuries were produced, the animal received the first application of the wound-specific product as stipulated in Table 3, which was covered with gauze on each side of the animal to prevent contamination of the different oils applied to the same animal. . The gauze was attached by micropore-type adhesive and reinforced with external adhesive. The wounds were cleaned daily with saline, the treatments repeated with the respective oils and the bandages replaced until the day determined for the sacrifice. For the daily dressings 20 the animals were not anesthetized so that there was no interference with hepatic metabolism, since all animals accepted all procedures without aggression and were tolerant to manipulation. The back and each wound of each animal were photographed with camera and digital magnifying glass, respectively, for later evaluation.

After a draw for sacrifice on days 2, 4 or 10, the animals in which

had been produced the wounds were again anesthetized with thiopental (85.0 mg.kg'1), photographically documented and decapitated with guillotine. Immediately thereafter, blood was collected for dosing of inflammation-related substances by the Elisa technique.

The skin of the trichotomized areas of the dorsal region was excised.

to the fascia, separating the proximal halves, with two wounds on each side for 10% formal fixation until slide preparation for histopathological evaluation.

New biopsies with circular blades of the same diameter were made at the four points of the anterior biopsies in the caudal portion of excised skin 5. Each of the four dermis or skin fragments removed was immediately identified and placed in a container containing liquid nitrogen and cryopreserved at -80 ° C at the end of the experiment until preparation for tissue real time PCR of apoptosis-associated substances. . The visual assessment made during the daily manipulation of each

of the animals, for treatment renewal, photographic registration and dressing change, indicated a significant improvement of the Group 4 animals treated with roasted coffee oil. From the second or third day a more effective wound healing could be observed. An example of the appearance of injuries on the fourth day of treatment can be seen in Annex 1.

On the sacrificial day of each animal, the animal wounds were photographed at constant focal length to maintain the same scale on all images. Unhealed areas were quantified in pixel number. Photo scales of excised specimens were corrected for area determination using the “ImageTooI v.3 UTHSCSA - The University of Texas Health Science Center in San Antonio” program. In Attachments 2, 3, 4, and 5 are photos of all parts. Tables 4, 5 and 6 show the areas obtained and partial analysis of the regressions of the injuries.

Table 4. Pixel areas of wounds treated for 2 days.

D2 D2 D2 D2 D2 D2 D2 D2 G1 2240 G2 2758 G3 2720 G4 1153 G1 3635 G2 4928 G3 2161 G4 957 G1 1556 G2 4044 G3 5815 G4 1577 G1 2355 G2 4180 G3 6424 G4 1391 G1 4758 G2 3601 G3 0 G4 0 G4 0 G2 7246 G3 3039 G4 269 G1 6115 G2 7893 G3 2177 G4 2058 _G1 GM

31

yàl

31

Gl

CH

31

CM

G1

31

31

Gl

D4

G1

G1

G1

G1

G1

G1

G1

G1

G1

G1

G1

G1

G1

G1

G1

G1

G1

G1

G1

G1

D1I

G1

G1

22/35

3886

3522

6922

5448

4907

3251

4152

4236

6495

1713

2416

3661

1544

G2

G2

G2

G2

G2

G2

G2

G2

G2

G2

G2

G2

G2

3996

5101

3976

4577

4172

4128

3270

3343

5676

235

3573

2953

4127

G3

G3

G3

G3

G3

G3

G3

G3

G3

G3

G3

G3

G3

4434

2536

3692

4676

3343

2774

4665

3355

4906

4391

5809

6417

G4

G4

G4

G4

G4

G4

G4

G4

G4

G4

G4

G4

G4

2881

198

1490

2490

3214

3278

1355

1360

1607

313

2311

2060

3609

Table 5. Areas in

jixel of wounds treated by 4 c

Yeah.

D4

D4

D4

D4

D4

D4

D4

7248

G2

12988

G3

4939

G4

791

1068

G2

7809

G3

2974

G4

4381

4842

G2

7099

G3

3016

G4

2307

3085

G2

5894

G3

3513

G4

2419

17033

G2

6980

G3

3140

G4

2076

3394

G2

1578

G3

3033

G4

3512

5882

G2

12355

G3

2985

G4

6348

5573

G2

8151

G3

2203

G4

6460

16062

G2

3060

G3

3014

G4

3818

G2

1301

G3

2333

G4

5585

4162

G2

10792

G3

3882

G4

6676

5349

G2

3874

G3

3807

G4

7480

4986

G2

2593

G3

2935

G4

5136

3533

G2

5331

G3

2973

G4

5547

4013

G2

7097

G3

3022

G4

5564

4013

G2

5227

G3

3724

G4

4654

G2

11235

G3

3088

G4

4756

G2

6222

G3

2902

G4

2886

G2

5015

G3

3578

G4

3418

G2

11205

G3

3944

G4

5779

Table 6. Areas in

jixel of wounds treated for 10 days.

D10

40

116

D10

G2

G2

D10

134

D10

G3

G3

D10

D10

G4

G4

D10

55 G1 127 G2 0 G3 0 G4 0 G1 185 G2 0 G3 71 G4 0 G1 0 G2 0 G3 0 G4 0 G1 7 G2 0 G3 582 G4 0 G1 0 G2 840 G3 1688 G4 0 G1 24 G2 846 G3 5056 G4 0 G1 39 G2 0 G3 0 G4 0 G1 196 G2 0 G3 0 G4 0144 G1 211 G2 226 G3 0 G4 0 G1 418 G2 142 G3 291 G4 238 G1 0 G2 0 G3 24 G4 0 G1 85 G2 1147 G3 3295 G4 0 G1 58 G2 255 G3 4231 G4 0 G1 62 G2 361 G3 19 G4 0 G1 174 G2 198 G3 0 G4 0 G1 0 G3 0 G4 0 G1 16 G2 120 G3 340 G4 0 G1 18 G2 154 G3 677 G4 216 It was determined for each treated wound on the right side of the animal, the reduction relative to its topographic opposite on the left side, untreated. Thus, it can be proportionally quantified how much of the treated wound remained unhealed after the start of the experiment, in relation to the injuries of the same animal and in the same transverse plane. It was considered that on day zero 100%.

Figures 1, 2, 3 and 4 respectively show the curves of the second degree polynomial regression of the percentage of unhealed wound area in the animals of the Group (control), Group 2 (AGE), Group 3 (Coffee Oil). Crude) and Group 4 (Roasted Coffee Oil). X-axis shows the number of days of treatment and Y-axis the percentage of the unhealed area, considering that on day zero all injuries were in the same area (100%).

The red vertical bars represent when the curve of

Average healing reached 75% wound and 25% healed and black bars when 50% of the area was healed. Figure 1 shows that 25% and 50% healing was achieved at 4.7 and 7.7 days after the start of treatment.

The results of calculating the derivatives of the regression polynomial equations are presented in Table 7. Parameter (a) of the first degree equations 5 represents the maximum point of the second degree regressions and indicates the time required in days for the curve to reach the zero value, ie the total wound healing. So the farther from zero, the lower the initial healing effect of the treatment. Thus, it can be observed that the treatment with AGE had a better initial effect than all the others. The parameter (b) indicates the slope of the line, in this case with values

negative, is the graphical representation of the area without healing. The more inclined, the faster the healing rate. The slope of the curves represents the accumulated effects over time, corroborating with clinical observations that animals treated with roasted coffee oil showed a significant improvement after the fourth day of treatment.

Table 7. Derivatives of the second degree polynomial regression equations (y '= a + bx) of the four treatments studied.

Treatment Coefficients ab G1 (witness) -5.9 0.422 G2 (AGE) -1.6 0.738 G3 (Raw Coffee) -2.8 0.622 G4 (Roasted Coffee) -3.7 0.986 Table 8 summarizes the days needed to if 20 to 25% and 50% of initial wound healing is achieved and the second degree derivative of the curves. Since the numbers analyzed correspond to the percentage of the area without healing, the negative value of the second degree derivative of polynomial function represents the healing acceleration (% of healing divided by the time square) of each treatment. Table 8. Number of days for healing of 25% and 50% of the treated areas and value of the second degree derivative.

Scarred Area Treatment - δ “25% 50% G1 (witness) 4.72 7.7 0.422 G2 (AGE) 7.2 10.0 0.738 G3 (Raw Coffee) 4.9 8.5 0.622 G4 (Roasted Coffee) 3 .9 6.7 0.986 δ healing acceleration

The animals responded to the treatments with different healing accelerations. In increasing order the Witness treatment, followed by Raw Coffee Oil, AGE and Roasted Coffee Oil.

There was a clear clinical difference in healing on the side that was only cleaned with saline and the side treated with mineral oil in the animals of group 1. This observation reinforces the knowledge described since 1945 that healing is more effective while maintaining the humid environment (Field & Kerstein, 1994). The animals of group two (AGE) and group three (crude coffee oil) presented clinically comparable healing process. Rats treated with roasted coffee oil showed the best recovery over the 10 day treatment period, and it was possible to observe improvement from the third day.

The results found with the measurement of ulcer areas showed a faster healing rate with increasing AGE, crude coffee oil and roasted coffee oil. The healing area, however, was sometimes difficult to measure by the presence of scabs that did not detach with gentle cleaning. Persistent crusts were not removed because objective histological evaluation was the most relevant parameter.

Example 2: Histological Analysis

The preparation of the slides for histological analysis was performed as follows: the proximal wounds were initially stored in alcohol and later in formaldehyde in individual bottles properly identified for the preparation of slides for histological analysis. After extraction of each wound from a longitudinal section covering the entire remaining wound or scar, the piece was stained by the Hematoxylin and Eosin (HE) method and contained in paraffin block for microtome work and mounting of the slide.

The blades were subjectively and objectively analyzed to estimate the wound healing rate blindly, that is, without the evaluators knowing the identification of the blade they were examining.

1) Subjective histological evaluation

Subjective histological evaluation was done blindly, that is, the dermatopathologist who participated in the project made the evaluation without knowing the identification of the slides she was examining.

On day 2 (inflammatory phase) it can be observed that the reepithelialization 15 had already started, that is, the epidermis began to proliferate to cover the wound (the faster the healing, the greater the extent of the wound that already had reepithelization), but , at this stage of the process, it was not justified to assess the extent of the wound that was already reepithelized, because all animals were beginning this process. The vessels were very congested; The greater the number of dilated vessels at this stage, the better for healing to occur. A subjective assessment was made: 0 = mild; 1 = evident. In the next phase, they were no longer as dilated, but their cells began to proliferate and were evaluated in the item “cellularity”.

The very immature dermal collagen tissue began to be observed, little cellular, with much basophilic fundamental substance. It is known that for this phase, the more young connective tissue, the earlier the healing. The cells of this connective tissue may already begin to appear at this stage of healing and the more present, the better the healing process. Cellularity was also estimated subjectively: 0 = very low; 1 = low; 2 = evident; The percentage of young collagen present in the operative cavity was also subjectively estimated. Although there is no histological evaluation on day 3, it can be inferred by evaluating the animals on day 4 that in this phase there was a significant increase in cellularity. These were endothelial cells (from newly formed vessels that bring nutrition to help heal) and collagen-producing cells (fibroblasts and myofibroblasts). Collagen may be seen in greater quantity on day 4 in those with earlier healing.

By day 4 the proliferative phase, reepithelialization, was already well advanced, with a larger extension of the reepithelialization area. Healing was evaluated: (0 = incipient; 1 = advanced; 2 = complete or almost complete). At this stage there was still a significant number of cells, but,

In animals at an earlier stage of healing, the cellularity that began on day 3 begins to decrease as, as collagen is produced, it inhibits cell production (called contact inhibition), and in animals in these animals with earlier healing already had 15 mature collagen. Those with less effective healing had more cells and less mature collagen (observed as thicker acidophilic fibers) and quite immature collagen, as on day 2. The cavity formed in the experiment was already partially filled; however, not the central area. It was subjectively estimated the percentage of fill 20 relative to adjacent normal skin (in 5). As for the tissue that filled the wound and contained cells, fibrin, young collagen and mature collagen; The amount of mature collagen fibers was subjectively estimated (0 = no amount; 1 = few fibers; 2 = more evident fibers; 3 = several fibers; 4 = numerous fibers).

By day 10 (remodeling phase) the animals already had the

complete reepithelialization. Some still had remnants of the young collagen (the same as day 2) which has not yet been replaced by the mature, thick fiber. Much of the cavity formed in the experiment is already filled; however, the central area was still depressed relative to adjacent normal skin. Fill was estimated as a percentage. Regarding the tissue that was filling the surgical wound, in this phase more and less cellular areas were observed, in inverse proportion to the number of mature collagen fibers. A percentage estimate of the amount of mature collagen fibers that occupied the tissue filling the wound was made.

Subjective evaluation showed no statistical difference between the groups, however, on the second day of the experiment, better healing was observed in the animals that used crude coffee oil and roasted coffee oil in the parameters observed on the second day. On the fourth day, findings 10 suggested lower cellularity in the three groups: AGE, raw coffee oil, and roasted coffee oil, but the filling appeared to be higher in the animals in the untreated group. On day 10 of the experiment the filling was better with roasted coffee oil.

2) Objective histological evaluation Objective evaluation was performed under a microscope with a

cycloid for cell counting. The number of cells in the cuts responsible for wound healing was evaluated. For this, the cell nuclei that touched the cycloid half circles were quantified.

In the cycloid there are 30 half circles. Each core that touched one of the

cycloids or half circles was counted. This is a comparative method that shows whether healing is developed (adult, composed of fibrocytes) or if it is still young (composed of fibroblasts). In the “adult phase” the number of nuclei is small; In the “young phase”, the number of nuclei is higher. Thus, if a sample has many nuclei, it can be said that the tissue is young (it is still at the beginning of the healing process).

Objective analysis was based on cellularity count, as previously described, and is the most important parameter in the analysis of the effect of treatments on healing.

The lowest cellularity was observed in ulcers treated with

AGE and roasted coffee oil when compared to the contralateral side on which ulcers were treated with mineral oil which documented better healing with these oils. Ulcers treated with crude coffee oil had worse healing than when only mineral oil and saline were used in the ulcers of group 1 animals.

Example 3: Quantitative PCR (qPCR) - Real time PCR (analysis of inflammatory cytokines in scar tissue)

Real-Time PCR tissue evaluation was performed to try to understand the local action of the product used and serological evaluation to evaluate possible systemic action of the product used on the dermis. The evaluated inflammatory cytokines have different actions on the

healing process and some have more intense action in the early stages of healing. For this reason the dosages, besides the histological evaluation, were also made in the different healing phases: D2, inflammatory phase; D4, proliferative phase and D10, beginning of remodeling phase that extends for months.

Total RNA was extracted from frozen skin samples at -80 ° C by Qiazol reagent method (Qiagen, USA). For cDNA production, we used the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA), with the final 20 cDNA concentration being 3.0 Dg. This cDNA was diluted to the concentration required for the efficient amplification of each gene. This efficiency was verified according to the method described below.

Real-time PCR reactions were performed using the TaqMan ™ system (Applied Biosystems), which consists of a pair of primers and a fluorophore labeled probe. For the adiponectin and leptin genes, TaqMan ™ assays (Applied Biosystems) Rn00595250_m1 and Rn00565158_m1 kits, respectively, were used.

The GAPD Rat gene (TaqMan ™ - Applied Biosystems), Part number 4352338E, was chosen as the endogenous control of the reaction, which serves to normalize the expression of the gene of interest in the different samples. The GAPD probe is labeled with VIC fluorophore, while target primers are labeled with FAM fluorophore.

Before starting the relative quantification experiments of the expression of any gene, the validation of the target gene system, 5 in this case, adiponectin and leptin, was performed with the endogenous control GAPD rat. We found that the amplification efficiencies of the genes were close to 100%. This step is essential for endogenous control to be used to normalize the relative expression values of the gene of interest.

Validation of the efficiency of the genes of interest consisted of amplifying both the gene of interest primers and the endogenous control primers of triplicate cDNAs of different concentrations (serial dilutions) of a randomly selected sample. Then, a standard curve was constructed from the log concentration of the samples by the Ct [Threshold Cycle: cycle in which each amplification curve crosses the detection threshold (which is arbitrarily defined]. In this curve, the slope values of the curve and the reliability of the replicas (R2) were obtained. Thus, the efficiency of a system was calculated by the formula: E = io ('1 / s / ope) -1. For the adiponectin, leptin and GAPD gene validation plate, triplicates of a rat hepatocyte cDNA sample were made at 7 different concentrations (5x serial dilutions).

After calculating the amplification efficiencies of each gene of interest and the endogenous control, a scatter plot was constructed, which aims to define the range of concentrations for which the system is efficient. For the construction of the graph, the same logarithm values of the concentration of the samples on the X axis were used and the difference between the means of the endogenous control Cts and the means of the gene of interest for each concentration on the Y axis. if a trend line for these values, which has a line equation 30 in which it is possible to verify the slope value of this line. For a system to be considered efficient, the slope value must be less than 0.1 (the closer to zero this value is, the smaller the slope of the curve and, therefore, the more constant is the difference between the mean Cts of the gene of interest and endogenous control). The points on the graph corresponding to the concentrations that are closest to the trend line are considered validated (the system has 100% efficiency at these concentrations).

The sample concentration validated as efficient for the adiponectin, leptin and GAPD genes was 40.0 ng cDNA.

For the relative quantification of the genes under study, real time PCR reactions were performed in triplicate from: 6.25μΙ_ TaqMan Universal PCR Master Mix (Applied Biosystmes, Foster City, CA, USA) 2x, 0.625μΙ_ da primer and probe solution, 1.625μΙ_ of water and 4, cDNA OpL (40ng cDNA), and in the negative control, 4.0 μΙ of water was added instead of cDNA. The cycling conditions used were: 50 ° C for 2 15 minutes, 95 ° C for 10 minutes and 40 cycles of 95 ° C for 15 seconds and 60 ° C for

1 minute. Relative gene expression values were obtained by analyzing the results in the 7500 System SDS Software program (Applied Biosystems, Foster City, CA, USA).

Results obtained by real-time PCR dosing of inflammatory cytokines are described below.

The values obtained were the calculated data of RQ, which is the Relative Quantification.

The methods for Relative Quantification of Gene Expression allow quantifying differences in the expression level of a specific gene (target) between different samples. Data production is expressed as a change or difference in the number of times in expression levels. For the study, a sample was chosen as calibrator and an endogenous gene was also chosen to normalize the results. The standardized results obtained are always relative to the calibrator data. The calibrator sample 30 used for all groups presented the median value between the three measurements performed for each treatment day (two, four or ten days).

The results showed that adiponectin was tissue increased in D4 and D10 in group 3 when compared to the 5 control group and increased in group 4 when compared to the contralateral side. IGF-1 was increased on days 2 and 4 in group 3 when compared to the control group and both in group 3 and group 2 when compared to the contralateral side. IL-6 showed better tissue results in group 3 and group 2 when compared to control and group 3 and group 4 in D2 when compared to the contralateral side. IFN-alpha showed better results in the early days (2 and 4) in group 2 and group

4 and, on day 10, in group 3 when compared to the control group. In comparison with the contralateral wound, group 2 presented better results. IFN-gamma showed better results in groups 2 and 4 compared to the 15 control group and group 2 compared to contralateral wounds. IL-12, which as IFN has anti-healing action, had lower levels in group 4 compared to control group and group 2 compared to contralateral wounds.

TNF-alpha and leptin showed better results in group 3 when compared to the control group. Leptin was increased in group 2 compared to the contralateral wound.

IL-2 was increased in group 3 as compared to the control group and contralateral wounds. This cytokine was also increased in group 4 when compared to the control group. No significant variations were observed regarding IL-4.

Example 4: Elisa (analysis of inflammatory cytokines in serum)

This assay employs the quantitative sandwich immunoassay technique. A monoclonal antibody specific for the target substance is pre-added to a microplate at the factory. Samples, controls and standards are pipetted into the wells and if the test substance is present it will be bound by the immobilized antibody. After washing the unbound substances, a substance-specific enzyme-linked polyclonal antibody is added to the wells. After washing to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells. The reaction enzyme produces a blue product that turns yellow when the Stop Solution is added. The color measurement intensity is in proportion to the amount of target substance bound in the initial step. The sample values are then read from the standard curve.

For the reactions performed in this project, commercial R&D Systems (Minneapolis, USA) Quantikine ELISA kits were used for the substances IL-6, IGF-1 and TNF-alpha; For leptin and adiponectin substances commercial kits provided by Millipore (St. Charles, Missouri, USA) were used.

The plates of the 5 kits were analyzed on the Bio Rad 680 Microplate Reader, BioRad Laboratories (Hercules, CA, USA) at 490 and 570nm wavelengths as recommended by the manufacturers.

The quantifications of inflammatory cytokines dosed by Elisa technique are summarized in the Table below:

Table 9: Summary of values found in cytokine dosages by Elisa method

Leptin Control AGE Raw Roasted Coffee Day 2 0.26 0.47 0.71 0.76 Day 4 0.74 0.43 0.51 Day 10 0.74 0.76 0.8 0.57 IGF- 1 AGE Control Roasted Raw Coffee Day 2 594.56 602.21 1029.02 730.48 Day 4 767.18 884.84 924.57 813.58 Day 10 786.54 960.43 616.76 812.59 IL- 6 AGE Control Roasted Raw Coffee Day 2 280,1067 255,5352 243,6248 270,7462 Day 4 256,3285 259,4808 251,1673 273,7785 Day 10 276,9472 222,9879 225,3636 269,0089 Adiponec Control AGE Roasted Raw Coffee Day 2 8,97218 11,11608 9,766461 7,92819 Day 4 8,337174 8,712682 9,493863 8,137195 Day 10 116,52638 113,46616 I 8,367467 I 11,89413

TNF-alpha AGE Control Roasted Raw Coffee Day 2 30.22 26.90 21.30 20.19 Day 4 18.62 15.01 17.36 16.02 Day 10 18.39 23.61 20.75 16.03 Systematically, a favorable difference can be observed for group 4 with IL-6, IFN-alpha (in the early days, 2 and 4), IFN-gamma, leptin, and IL-2, IL-12 (in the first days). 10).

The cytokines that favored healing in group 3 were the

IGF-1, INF-alpha and IL-12 (on day 10). Adiponectin was systemically favorable for healing that occurred in group 2. In this group IL-12 was elevated in this group, acting favorably in the early days (2 and 4).

IL-4 was elevated in group 1, favoring healing. Example 5: Statistical interpretation of evaluations of inflammatory (Real-Time PCR) and systemic (Elisa) scar tissue cytokines

In PCR, which evaluates the amount of cytokines in the tissue, it was possible to determine a statistically significant difference favorable to group 3 (of raw coffee oil) when compared to the control group for cytokines: 15 IGF-1 and leptin (only on day 4). . When compared to the contralateral wound, the cytokines found at levels favorable for healing in group 3 were INF-alpha and gamma and leptin (on day 4).

For group 4 (roasted coffee oil) the amount of cytokines in the tissue with statistically significant difference was observed when only adiponectin and INF-gamma were compared when compared to the control group and IL-12 when compared to the contralateral wound.

AGE favorably interfered with tissue production of IGF-1, IFN-alpha and IL-12 when compared to the control group and ITN-alpha when compared to the contralateral wound.

Systematically, by the blood dosage by Elisa, it was observed

favorable and statistically significant difference between IL-2, IFN-alpha and gamma in group 4, adiponectin in groups 2 and 4, TNF-alpha in group 2 and IL-12 in groups 2 and 3.

Thus, it was found that roasted coffee oil was more effective than mineral oil and raw coffee oil in promoting wound healing in rats. This was observed in the clinic, ulcer area evaluation and histological evaluation.

Roasted coffee oil produced tissue healing action with significant statistical difference when dosing adiponectin and INF-gamma and the systemic dosage of adiponectin, IFN-alpha and gamma and IL-2 suggesting the importance of these cytokines in the healing process.

This systemic effect signals a possible drug use of coffee oil, since many dermatoses and other diseases are related to the high production of TNF-α, for example, such as psoriasis and various rheumatological diseases. This systemic effect was particularly evident on the second day of the experiment.

Locally these oils stimulated or inhibited the production of several of the studied cytokines. The increase in IGF-1, IL-6 and TNF-a production on the tenth day was in agreement with both coffee oils.

In view of the results obtained, the presence of an agent with healing action, unidentified, present or with greater effectiveness in roasted coffee oil, is evident in comparison with other treatments. Therefore, the use of coffee oil based compositions systemically in the treatment of inflammatory diseases should be the focus of further studies, as there is clear potential for the use of these substances in the treatment of other dermatoses and other systemic diseases that require oral use. or parenteral of medications.

Claims (15)

1. Composition comprising the following components: - Oil obtained from roasted coffee beans; - Pharmaceutically acceptable vehicle. Composition according to Claim 1, characterized in that it comprises coffee oil with a concentration ranging from 0.001% to 99.999%, preferably 70%. Composition according to Claim 1, characterized in that the coffee oil is preferably extracted from coffee of the species Coffea arabica. Composition according to Claim 1, characterized in that the coffee oil comprises any coffee classified for drink. Composition according to Claim 1, characterized in that the coffee oil comprises any type of grain size or sieve used for size separation. Composition according to Claim 1, characterized in that the oil obtained from roasted coffee beans comprises a roasting process capable of generating the flavor-promoting substances and the characteristic flavor of the coffee beverage infused with water. hot. Composition according to Claim 6, characterized in that the roasting process comprises the same process used for the production of beverage grains. Composition according to Claim 1, characterized in that the coffee oil comprises the crude oil; filtered, clarified, semi-refined; refined; fractionated; hydrogenated; pure interesterified; and mixed with other oils and / or ingredients. Composition according to Claim 8, characterized in that the coffee oil preferably comprises the oil in its filtered or semi-refined form. Composition according to Claim 1, characterized in that the pharmaceutically acceptable carrier is selected from medium chain triglycerides (TCM), saline or buffers, preferably medium chain triglycerides (TCM) by a suitable percentage (qsp) to achieve 100%. % of formula based on total weight of composition. Use of the composition according to claims 1 to 10, characterized in that it has application in skin healing. 12. Use of coffee oil characterized by being applied in the production of formulations for skin healing. A formulation comprising the composition of claims 1 to 10 in a pharmaceutically acceptable carrier and optional components selected from chelating agents, pH adjusting agents, preservatives, emollients, humectants, bacteriostatic agents, active ingredients, colorings and flavorings. Use of the formulation according to claim 13, characterized in that it has cutaneous application. Use of the composition described in claims 1 to 10 for the preparation of a skin healing medicament.