WO2014066968A1 - Composition et formulation à base d'huile de riz, et utilisations - Google Patents

Composition et formulation à base d'huile de riz, et utilisations Download PDF

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Publication number
WO2014066968A1
WO2014066968A1 PCT/BR2013/000457 BR2013000457W WO2014066968A1 WO 2014066968 A1 WO2014066968 A1 WO 2014066968A1 BR 2013000457 W BR2013000457 W BR 2013000457W WO 2014066968 A1 WO2014066968 A1 WO 2014066968A1
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day
group
groups
healing
rice
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PCT/BR2013/000457
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English (en)
Portuguese (pt)
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Paulo Eduardo Neves Ferreira Velho
Bruno Grosselli Lania
Maria Letícia Cintra
Walter Szortika Tessmann
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Univérsidade Estadual De Campinas - Unicamp
Helmut Tessmann Indústria E Comércio De Óleos Vegetais Ltda.
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Publication of WO2014066968A1 publication Critical patent/WO2014066968A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/899—Poaceae or Gramineae (Grass family), e.g. bamboo, corn or sugar cane
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like

Definitions

  • the present invention relates to a rice oil based composition. More specifically the present invention relates to a topical composition with high skin healing ability. Furthermore, further objects are their use and a formulation comprising said composition.
  • the healing process can be more simply divided into three phases: inflammatory, proliferative and remodeling (Pittman, 2007).
  • inflammatory phase after hemostatic coagulation has begun, numerous chemical mediators are released by inflammatory cells.
  • 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 lymphokines released by them influence the action of macrophages (Mandelbaum, 2003). In addition to these, this phase relies on fibronectin.
  • the proliferation phase is responsible for the closure of the lesion 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; Gentil Subscribe 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 (Gentil Subscribe et al, 1999). Matrix formation depends, in addition to inflammatory and endothelial cells, on the fibroblast responsible for the production of substances important for both debridement and physiological remodeling (Van Winkle, 1967). The proliferation of vessels that occurs at this stage is essential for the supply of 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 has about 80% of the normal tensile strength of the skin, is not bulky and is flat (Mandelbaum, 2003).
  • adiponectin adiponectin, leptin, interleukin (IL) 2, IL-4, IL-6, IL-12, tumor necrosis factor (TNF) ) -a, insulin-related growth factor (IGF) -1, interferon (IFN) -ae IFN- ⁇ .
  • IL interleukin
  • TNF tumor necrosis factor
  • IGF insulin-related growth factor
  • IFN interferon
  • 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- ⁇ (Yano, 2008). Induces a decrease in serum glucose and triglyceride levels, increases the level of glucagon.
  • 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 Lack of leptin in animals as well as in humans can lead to weight gain and also to increased 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 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 IgG1 and IgE. It suppresses the development of IFN- ⁇ -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. Together with TNF- ⁇ synergistically induces the expression of VCAM- (vascular cell adhesion molecule 1) in smooth endothelial and muscle cells, resulting in selective recruitment of eosinophils and lymphocytes at the inflammation site.
  • VCAM- vascular cell adhesion molecule 1
  • IL-4 negatively regulates the production of inflammatory mediators such as IL-1, TNF- ⁇ , and prostaglandin 2 (PGE 2 ) 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 the 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 (akhluf, 1996). ).
  • IL-6 is a cytokine that plays important roles in host defense, 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 IL-4, IL-0 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 early in the healing process and is important because it has mitogenic properties in keratinocytes as well as chemotactic to neutrophils and their infiltration to the affected area has the ability to increase local angiogenesis and increase local collagen deposition. of the wound (Lin, 2003).
  • IL-2 is a heterodimeric glycoprotein, which increases cytotoxic activity and induces IFN- ⁇ production in the natural epidermal dendritic kyler, T and T cells. It also induces IFN- ⁇ 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;
  • IL-12 promotes 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 increased gene expression, causing macrophages to produce and secrete more IFN-Y (Ishida, 2004).
  • TNF- ⁇ plays critical roles in normal host resistance to infection and growth of malignant tumors, serving as immunostimulants and as mediators of the inflammatory response. A lot of TNF production, however, has been implicated as playing a role in a number of pathological conditions, including cachexia, septic shock, and autoimmune diseases. TNF- ⁇ is produced by activation of macrophages and other cell types, including T and B cells, NK cells, 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- ⁇ is responsible for initiating the proinflammatory cascade and is responsible for healing strength and tension (Mast, 1996).
  • IGF-I also known as somatomedine C
  • IGF-I 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).
  • IGF-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- ⁇ is a cytokine family glycoprotein 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 enhancing the expression of type I (MHC type I) main histocompatibility complex molecules, either by autocrine or paracrine activity (Krause, 2005). Another form of action of IFN- ⁇ 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- ⁇ also known as type II interferon
  • type II interferon was initially identified as a product with anti-viral activity of mitogenically activated T lymphocytes.
  • Some of the most important functions are macrophage effector function stimulation, IL-12 induced Th1 differentiation, modulation of MHC molecule class I and II expression, regulation of immunoglobulin (Ig) class exchange and regulation of interactions between leukocytes and endothelium.
  • IFN- ⁇ has also been involved in physiological roles in sensory neurons and spermatogenesis (Neumann, 1997; Kanzaki, 1998).
  • Both interferons have anti-scarring action, inhibiting collagen synthesis and cell proliferation and may increase the production of metalloproteinases.
  • IFN- ⁇ may increase the production of collagenase enzyme, which increases its anti-scarring action.
  • both must have their values increased in the final phase of healing, preventing it from progressing to hypertrophic injury (Tredget, 2000).
  • a study conducted with rice bran oil demonstrates that it has the ability to increase the immune response of mice, such as proliferation of B lymphocytes and cytokines such as IL-2 and TNF- ⁇ , as well as decreased IL-4 and IgE ( Sierra, 2005).
  • a study with rice bran oil isolated oryzanol indicates that this phytochemical has the ability to increase cellular and humoral immune activity, such as delayed hypersensitive response, induces antibody production by B and T lymphocytes, increases hematopoiesis and induced phagocytosis. by macrophages (Ghatak, 2012).
  • Rice bran oil is also reported to be a natural source of squalene (an intermediate triterpene in cholesterol biosynthesis) which is the major component of skin surface polyunsaturated lipids and has advantages for the skin as emollient and antioxidant as well as for hydration and anti-tumor activities, having dermocosmetic development potential (Huang, 2009). Another study indicates that consuming about 18 grams of modified rice bran milk for 5 weeks has the ability to lower serum cholesterol and also LDL cholesterol in patients with type 2 diabetes (Lai, 2011). South Korean researchers report that a brown rice diet (present in many drinks and teas and marketed as a type of "sprouted brown rice") suppresses weight gain and fat accumulation in the liver and epididymal adipocytes and improved profiles.
  • squalene an intermediate triterpene in cholesterol biosynthesis
  • Paepe Patent et al., 2002
  • collaborators describe the use of rice starch in two formulations for the treatment of dermal diseases.
  • the use of water-suspended rice starch was used to increase the barrier function of damaged but not ulcerated skin.
  • the document does not evaluate the use of rice oil, nor the action on healing.
  • US20020182260 of 11/03/2002 deals with an anti-inflammatory product containing at least one animal, mineral or vegetable oil or wax and among the vegetable source options they cite rice bran.
  • the inventors cite the use of any oil of mineral, animal and vegetable origin, including rice oil, which may have an immunomodulatory action used at any concentration and conveyed in any formulation.
  • the inventive work is limited to the method for obtaining compositions containing an inflammation-causing therapeutic agent and an immunomodulatory concentrate.
  • the inventors themselves mention previous patents that have anti-inflammatory substances in their composition and do not consider that in the healing process the benefit of the use of a given product is often due to the balance of pro and anti-healing action of that product. Since inflammation is needed in healing, it can be detrimental to the process if too much. The action of TNF-alpha in the healing process makes this reality clear.
  • This cytokine may have pro or anti-healing action depending on the concentration or phase of the healing process in which it is used, as studied by Mast as early as 1996.
  • Adiponectin for example, has an anti-healing action which is desirable at the stage. end of the healing process called remodeling in order to prevent keloid formation.
  • the same inflammatory product may have anti-inflammatory 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.
  • US20070207222 of 28/02/2007 describes a composition that includes a wax for treating anti-inflammatory diseases, among the wax options they cited rice.
  • the therapeutic substance is the tar used for topical treatment of dermatological diseases. responsive to tar.
  • the inventors cite rice wax as a substance that can be added to the tar solution and rice starch as a possible absorbent to be added to the tar.
  • WO2004093569 of 11/02/2004 describes a honey and rice flour based composition, which in the document is detached for absorbing wound fluids, keeping honey longer in place. It is just like the above, with no mention of the use of oil as an active ingredient.
  • the process described in the present invention has advantages in several respects.
  • 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 and protected, ease of application and adaptability (conformation to different parts of the body).
  • Another differential of the process described here is related to the systemic action that the technology has, where the 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 others. products commonly used for this purpose.
  • the present invention is a topical rice oil based composition with high skin healing ability. Furthermore, the present invention also relates to the use and formulation of said composition.
  • FIG. 1 shows the unhealed area of the wounds (%) over 10 days of treatment of group 1 animals (Control).
  • - Figure 2 shows the unhealed wound area (%) over 10 days of treatment of Group 2 animals (AGE).
  • FIG. 3 shows the unhealed area of wounds (%) over 10 days of treatment of Group 3 animals (Rice Oil).
  • FIG. 5 presents a graph regarding the cellularity on the treated side in the three groups.
  • Annex 1 shows the aspect of the side injuries subjected to the treatments for four days.
  • the present invention discloses a topical rice oil healing composition.
  • the main examples of products which may be prepared from the composition object of the present invention are:
  • composition of the present invention comprises the following components:
  • composition of the present invention may further comprise optional components to provide some desirable characteristic not achieved with the aforementioned components such as emollient, humectant, chelating agent, thickening system, antioxidant component, preservative system, colorants and flavorings.
  • optional components to provide some desirable characteristic not achieved with the aforementioned components such as emollient, humectant, chelating agent, thickening system, antioxidant component, preservative system, colorants and flavorings.
  • compositions of the present invention as well as other components optionally added in the formulation will be described in more detail below.
  • concentrations and components may vary.
  • the rice oil employed is obtained by the process described in patent application BR1020120087189, and added in concentration ranging from 0.001% to 99.999%, preferably 70%.
  • the dosage of rice oil, extracted through the patent BR 10 2012 0087189, 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 professional. who administers the therapy.
  • the effective amount of rice 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.
  • Local absorption and effectiveness of the present invention can be improved 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 oil. of rice.
  • 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.
  • composition of the present invention may further comprise optional components such as: - Chelating agent such as ethylenediaminetetraacetic acid (EDTA) and its salts;
  • EDTA ethylenediaminetetraacetic acid
  • PH adjusting agent such as triethanolamine or inorganic hydroxides
  • 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.
  • Representative drugs within these classes include gentamicin, benzoyl peroxide, glucocorticoids, hydrocortisone, vitamin D3, methotrexate, cyclosporine, retinoids.
  • composition described in the present invention may be prepared employing any process known in the prior art.
  • Rice oil extracted through BR 10 2012 0087189 may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservative, buffer or propellant as required.
  • Topical preparations can be made by combining rice oil extracted through BR 10 2012 0087189 with conventional pharmaceutical diluents and vehicles commonly used 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.
  • 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 1 below. In each group of animals were subdivided to be sacrificed on days 2, 4 and 10 after the first treatment application.
  • Table 1 schematically shows the distribution of wounds and treatments received by each group of animals. Table 1. Treatments applied in each study group.
  • each 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 7.0mg. Kg " 1 and diazepam 2 0.0 mg. kg "1. After it is completely anesthetized and the paravertebral completamete scapular regions were shaved and disinfected was made with a 2% solution of chlorhexidine digluconate for disinfection of the area where the wound would be produced.
  • the animal Immediately after the wounds were produced, the animal received the first application of the wound-specific product as stipulated in table 1, 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.
  • 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.
  • 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 the preparation of the slides for histopathological evaluation.
  • New biopsies with circular slides of the same diameter were made at the four points of the anterior biopsies in the caudal portion of the excised skin.
  • Each of the four dermis or skin fragments removed were immediately identified and placed in a container containing liquid nitrogen and cryopreserved at -80 ° C at the end of the experiment until preparation for the tissue real time PCR of the inflammation-associated substances. .
  • Figures 1, 2 and 3 show the curves of the second degree polynomial regression, the percentage of unhealed wound area in the animals of groups 1, 2 and 3.
  • the X-axis shows the number of days of treatment and the number of days.
  • Y is the percentage of the unhealed wound area, considering that on day zero all wounds had the same area (100%).
  • the red vertical bars represent when the average healing curve reached 75% wound and 25% healed and the black bars represent when 50% of the area was healed.
  • the wounds of the proximal portion were initially stored in 90 ° alcohol for 24 hours and then in 10% 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 part was stained by the hematoxylin and eosin (HE) method and contained in paraffin block for microtome work and slide assembly.
  • HE hematoxylin and eosin
  • 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.
  • cycloid there are 30 half circles. Each nucleus that touches 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 you are 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).
  • the objective evaluation showed a lower cellularity in rice oil treated ulcers compared to the contralateral side in which ulcers were treated with mineral oil which documented a better healing with this oil.
  • the ulcers treated with rice oil and later with AGE had better healing than when only mineral oil and saline were used in the ulcers of group 1 animals.
  • the results found in the rice oil treatment suggest a possible systemic action of the oil because The control side cell count of the rice oil group animals was lower than that of group 1.
  • Qiazol reagent method Qiagen, USA
  • the High CapacitycDNA Reverse Transcription Kit Applied Biosystems, Foster City, CA, USA
  • 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.
  • TaqMan TM assays were used for all genes, namely: adiponectin (Rn00595250_m1), leptin (Rn00565158_m1), IL-2 (Rn00587673_m1), IL-4 (Rn01456866_m1), IL-6 (Rn014) 12 (Rn00575112_m1), IFN- ⁇ (Rn01400027_g1), IFN-Y (Rn00594078_m1), IGF-1 (Rn00710306_m1) and TNF- ⁇ (Rn00562055_m1).
  • the GAPDRaX gene (TaqMan TM - AppliedBiosystems), Partnumber 4352338E, was chosen as endogenous reaction control. It 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.
  • the target gene system for example adiponectin and leptin
  • GAPD rat was validated with the endogenous control GAPD rat.
  • Gene amplification efficiencies were found to be close to 100%. This step is essential for endogenous control to be used to normalize the relative expression values of the gene of interest.
  • a scatter plot was constructed, which aims to define the range of concentrations for which the system is efficient.
  • 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.
  • a trend line for these values was obtained, which has a straight line equation that allows to verify the slope value of this straight line.
  • 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 validated (the system is 100% efficient at these concentrations).
  • sample concentration validated as efficient for the adiponectin, leptin and GAPD ⁇ o genes is 40.0 ng of cDNA.
  • the methods for relative quantification of gene expression allow quantifying differences in the level of expression 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.
  • two analyzes were made: in one analysis, a sample was chosen as a 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 used for all groups presented the median value among the three measurements performed for each treatment day (two, four or ten days). In the other, each treated wound sample was calibrated with a control sample from the animal itself; thus each sample had a different and specific calibrator.
  • Table 2 Summaries of the RQ values found for the different cytokines evaluated in groups 1, 2 and 3, in relation to group 1, by Real Time PCR.
  • the objective of this experiment is to evaluate the existence of differences between the study groups and the days after the injury, in which the animals.
  • An analysis of variance (ANAVA) was used considering a two-factor experiment and then the Scott-Knott mean test at 5% significance was applied.
  • Group 3 has the highest amounts of IL-6 compared to the control group.
  • Group means test: in relation to the animal as its own control, only group 3 was statistically different and inferior to the other groups.
  • Group means test: The test indicates that groups 1 and 3 are statistically equal and lower in quantity than group 2.
  • adiponectin rice oil, in the local expression of this substance, can be seen that since day 2 its amount is lower than that expressed in the control and AGE groups. By analyzing the PCR results, it is possible to verify that the values increase on days 4 and 10. As adiponectin is anti-inflammatory, it is ideal that its levels are low in the onset of healing and increase near the end of the healing process (Berg, 2001), as seen in rice oil (adiponectin levels, although lower than those in the other groups, tend to increase along the healing process). Statistical analyzes for real time PCR show that in none of the groups and in none of the days did the animals present different amounts of adiponectin in relation to group 1 and in relation to the control side itself.
  • leptin the animals treated with AGE had its value increased on days 4 and 10 in relation to control group. According to the literature review, it is interesting that the amount of leptin is increased at the beginning of healing, since it favors the proliferation of keratinocytes (Nascimento, 2006), having been found a value three times higher in the group treated with keratin oil. rice in relation to the control group.
  • the amounts of leptin when day is 4, the amounts of leptin (compared to group 1) are higher in group 2 and when day is 10, the largest amounts found were in group 3. When the side was compared treatment and control of each animal, statistically no differences were found.
  • IL-2 produced by neutrophils and responsible for keratinocyte and cocagen proliferation (Armour, 2007), is interesting that it is found at the beginning of the healing process and its level decreases to prevent hypertrophic scarring.
  • the IL-2 levels of the rice oil treated animals were higher on day 2 and 4 compared to control and AGE groups, a result visualized by both the Elisa method and real time PCR; locally, the gene expression detected on day 4 was higher than the control group, but lower than the rice oil treated group.
  • group 3 presented larger amounts, since groups 1 and 2 are statistically equal.
  • the amount of IL-2 compared to the contralateral wound is higher in group 3 and the Scott-Knott test indicated that on days 2 and 4
  • the amounts of IL-2 are statistically equal and higher than the amounts found in the blood of the animals evaluated on day 10.
  • IL-4 was not detectable by real time PCR.
  • IL-6 in the local analysis, shows a decrease in the value expressed on day 2 and increase on day 10.
  • IL-6 is an important cytokine at the beginning of the healing process, where the highest levels, both local as systemically, they should be found.
  • the total absence of IL-6 in gene knockout mice healing time is three times longer (Lin, 2003).
  • the group presented the smallest amount, comparing with groups 1 and 2 (statistically equal and with higher amount of IL-6). ), which indicates greater systemic action of group 3, since the difference in the amounts of IL-6 in the treated wound and the control wound was the smallest of all groups.
  • group 3 presented the highest amounts of IL-6 given a significant increase in D10. Observing the values obtained, it is noted that the levels of IL-6 were lower than the values found in D2 and D4 and there was a great increase already in the remodeling phase. Perhaps, if a subsequent dosage had been done, there would have been a systemic elevation of the levels of this cytokine, considering that the elevation in the application site should be prior to the systemic one.
  • IL-12 rice oil showed decreased local expression on days 2 and 4 compared to the control group. This decrease in IL-12 is desirable also in other inflammatory diseases such as psoriasis, for example.
  • the AGE group showed an increase in local gene expression every day.
  • the role of IL-12 in healing is to stimulate the production of interferons by macrophages, and its increase is important (Ishida, 2004), which should occur in the final stages of healing.
  • the real time PCR statistical analysis in relation to the control group allows us to say that there is statistical difference between the groups only on day 4, where the quantities found in group 2 are superior to those in groups 1 and 3 (statistically equal). In relation to the animal itself, only group 2 presented statistically higher quantities compared to groups 1 and 3.
  • IGF-1 is elevated in the early phase (day 2) of the healing process, which can be treated with rice oil or AGE, a result observed in real time PCR. From day 4, rice oil decreases in the amount of IGF-1 until day 10 and AGE continues to increase on day 4 and falls on day 10. As this substance indirectly acts on healing (it increases the function of other As PDGF-2) is related to the increase in skin thickness, it is expected that the circulating amount is not high in the second and third stages of healing, as a high amount of this substance may lead to the development of hypertrophic scarring (Chen , 2010).
  • IFN- ⁇ when compared to local expression, when treated with rice oil showed an increase on day 2 and AGE treated showed an increase on days 4 and 10, compared to the control group.
  • group 2 is the one with the highest amounts of IFN- ⁇ .
  • the test results were all not significant due to the large variability caused by the large amount of undetected values. Looking at the data set, only for group 1 there is a greater number of data and in the other groups few samples detected the substance.
  • This assay employs the quantitative sandwich immunoassay technique.
  • a target substance-specific monoclonal antibody 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.
  • a substance-specific enzyme-linked polyclonal antibody is added to the wells.
  • a substrate solution is added to the wells.
  • the reaction enzyme produces a blue product that turns yellow when "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.
  • the plates from the 10 kits were analyzed on the BioRad 680 Microplate Reader, BioRadLaboratories (Hercules, CA, USA) at 490 and 570nm wavelengths as recommended by the manufacturers.
  • the objective of the statistical analysis of this test was to evaluate the existence of differences between the study groups and the days after the injury in which the animals were evaluated.
  • An analysis of variance (ANAVA) was used considering a two-factor experiment and then the Scott-Knott mean test at 5% significance was applied.
  • the Scott-Knott test was applied to identify differences between groups and days.
  • group 3 has lower amounts than groups 1 and 2 (statistically higher and equal).
  • Mean Day Test According to the above test, adiponectin values found on day 10 were higher than those found on days 2 and 4.
  • IL-12 amounts are higher on day 2, while values found on days 4 and 10 are lower and statistically equal.
  • the Scott-Knott test was applied to identify differences between groups and days.
  • adiponectin As for rice oil there was a systemic decrease in the amount present in the serum in relation to the control group and AGE; from day 2 the decrease is small, but on days 4 and 10 there is a significant decrease. As for AGE, there is a systemic increase in circulating quantity on days 2 and 4, decreasing on day 10.
  • the Scott-Knott test indicated that groups 1 and 2 are statistically equal and the amounts of adiponectin found in the animals' blood is higher. than the amount found in the blood of animals in group 3. In this group, only animals that were analyzed on day 10 had a higher amount of adiponectin than the other days.
  • Leptin had its systemic value increased compared to the group in both the AGE and rice oil-treated groups on days 2 and 10 (on both days the largest increase was in the rice oil-treated group) and decreased values. measured at day 4 (major decrease in the group treated with AGE). Statistically, however, it can be said that in none of the groups and on any day the animals presented different amounts of leptin systemically.
  • IL-4 had its level increased systemically in the rice oil group compared to the control group on days 2 and 10 and decreased on day 4.
  • the AGE-treated group had its systemic values decreased throughout the treatment.
  • This cytokine has its function in early healing phase by depositing tenascin on the injured tissue; It is a cytokine little expressed in the adult, being reexpressed in the cicatricial processes (Makhluf, 1996). Keeping this in mind, the result presented by rice oil is the best among the groups, as it was the one that presented the largest amount of this on day 2 of healing. This result was different from that found by Sierra, where oryzanol decreased the amount of IL-4 (Sierra, 2005). According to the analysis of variance table, however, it can be said that in none of the groups and on any day the animals showed statistically different blood amounts of IL-4.
  • IL-6 has its lowest values in the rice oil treated group throughout the healing process both locally and systemically.
  • the group treated with AGE presents systemic fall on days 2 and 10 compared to the control group and there is no difference on day 4.
  • the analysis of variance table regarding the days for Elisa tests we can say that the animals presented same amount of IL-6 in the blood every day they were evaluated.
  • the 3 groups showed differences. First, the group with the highest amount of IL-6 was group 1, then group 2, and finally the group with the lowest amount of IL-6 was group 3.
  • IL-12 rice oil showed a decrease in systemic expression of this cytokine on days 2 and 4 compared to the control group. This decrease in IL-12 is desirable also in other inflammatory diseases such as psoriasis, for example. In the AGE group systemic decrease was noted throughout the treatment. According to the analysis of variance table, it can be observed by Elisa's technique that the interaction between groups and days was not significant, indicating that the day does not interfere with the results of the groups and vice versa. Thus, each factor was evaluated separately and in this case the two factors were significant, that is, there are differences between the groups and between the days on which the animals were evaluated. The Scott-Knott test indicated that groups 1 and 3 are statistically equal and the amounts of IL-12 found are greater than the amounts. found in the blood of group 2 animals. Only animals that were analyzed on day 2 had higher IL-12 levels than the other days. The animals on days 4 and 10 showed statistically equal and lower amounts of IL-12 than the animals on day 2.
  • TNF- ⁇ had its systemic value increased throughout the treatment with rice oil in relation to the control group. Since this factor is responsible for initiating the healing event cascade and also acts on healing tension, it should be expressed throughout healing (ast, 1996), which can be seen systemically with rice oil. The increase found in this paper is consistent with that found by Sierra and colleagues in their study on the immunological effect of rice oil (Sierra, 2005). According to the ANAVA table of treatment day breakdown, it can be observed that the amounts of TNF- ⁇ , in relation to the groups, were significant only on days 4 and 10. On the second day, the groups did not behave differently. , that is, on this day the amounts of TNF- ⁇ were not affected in the groups.
  • the amounts of TNF-a in the groups had different behaviors, that is, that day the amounts of TNF- ⁇ in group 3 were higher than in the other groups.
  • the action of TNF- ⁇ may vary according to the amount administered at the injury site, where a low amount speeds up the process and a high amount slows down the process (Mast, 1996). In this sense, late systemic elevation may be related to the reduction of this stimulus during the remodeling period.
  • IGF-1 is elevated in the early phase (day 2) of the healing process, which can be treated with rice oil or AGE, a result observed in both the Elisa test and the real time PCR. From day 4, rice oil has decreased the amount of IGF-1 until day 10, both in PCR and Elisa, and the AGE continues to increase on day 4 and falls on day 10 in both tests.
  • the analysis of variance table for the Elisa test it can be said that in none of the groups and in none of the days did the animals present different amounts of IGF-1.
  • IFN- ⁇ decreased throughout the treatment in the rice oil and AGE treated groups.
  • the Scott-Knott test indicated that only group 1 had higher IFN- ⁇ levels than the other groups.
  • the amount of IFN- ⁇ found in the blood of animals from groups 3 and 2 can be considered statistically equal and lower than the amounts found in group 1.
  • Only animals that were analyzed on day 4 had a higher amount of IFN- ⁇ than the other days.
  • Systemic reduction of IFN- ⁇ is also desirable in certain inflammatory diseases such as psoriasis, for example. As both have anti-scarring action, it is important that their value be decreased, especially in the early stages of healing, so that collagen deposition can occur (Tredget, 2000), which occurred in the rice oil treated group and slightly less in the treated group. with AGE.
  • IFNgamma testicular interferon-gamma
  • Tredget EE Wang R, Shen Q, Scott PG, Ghahary A. Transforming growth factor-beta mRNA and protein in hypertrophic scar tissues and fibroblasts: antagonism by IFN-alpha and IFN-gamma in vitro and in vivo. J Interferon Cytokine Res. 2000; 20 (2): 143-51.

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Abstract

La présente invention concerne une composition et sa formulation à base d'huile de riz, à usage topique et à haute capacité de cicatrisation cutanée. Cette composition est capable de maintenir la zone cicatricielle à un niveau d'humidité idéal et les zones périphériques sèches et protégées, outre le fait de présenter une action systémique, facteurs qui accélèrent le processus cicactriciel.
PCT/BR2013/000457 2012-11-05 2013-11-01 Composition et formulation à base d'huile de riz, et utilisations WO2014066968A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3988436A (en) * 1974-02-11 1976-10-26 Carnation Company Sunscreening method using rice bran oil
DE3938284A1 (de) * 1989-11-17 1990-03-22 Josef Dipl Chem Dr Rer N Klosa Hautschutzmittel gegen vorzeitig alternde haut und falten

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3988436A (en) * 1974-02-11 1976-10-26 Carnation Company Sunscreening method using rice bran oil
DE3938284A1 (de) * 1989-11-17 1990-03-22 Josef Dipl Chem Dr Rer N Klosa Hautschutzmittel gegen vorzeitig alternde haut und falten

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BERNARDI, D.S.: "Desenvolvimento de nanoemulsao de oleo de arroz como adjuvante no tratamento de dermatite atopica e psoriase.", DISSERTAÇAO DE MESTRADO EM MEDICAMENTOS E COSMETICOS., 2011, RIBEIRAO PRETO: FACULDADE DE CIENCIAS FARMACEUTICAS DE RIBEIRAO PRETO, UNIVERSIDADE DE SAO PAULO, Retrieved from the Internet <URL:http://www.teses.usp.brlteses/disponiveis/60/60137/tde-11p42011-,140431> *
DE PAEPE, K. ET AL.: "Effect of rice starch as a bath additive on the barrier function . of healthy but, SLS-damaged skin and skin of atopic patients.", ACTA DERM. VENEREOL, vol. 82, 2002, pages 184 - 186 *

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