Process for incorporating rosmarinic acid into a type i collagen solution

Abstract

The invention relates to a process for preparing a formulation of rosmarinic acid in a type I collagen hydrogel of bovine origin for topical application in the treatment of pressure ulcers, also known as decubitus ulcers or pressure sores, in domestic animals and human beings, with the aim of accelerating the healing of the ulcerous wounds, reducing the area affected by the wound and improving wound contraction, thereby providing an effective and promising option for the treatment of pressure ulcers.

Description

[0001] DESCRIPTIVE REPORT

[0002] PROCESS FOR INCORPORATING ROSMARINIC ACID INTO A TYPE I COLLAGEN SOLUTION

[0003] Brief description

[0004]

[0001] The present invention describes a process for incorporating rosmarfonic acid into a type I collagen solution in order to form a formulation for topical application in the treatment of pressure ulcers. Intended for the sector of materials for non-surgical wound treatment in veterinary or human patients, this formulation aims to treat pressure ulcers, characterized by local damage to the skin and / or underlying tissues due to ischemia and reperfusion, along with increased levels of reactive oxygen species. The formulation contains rosmarfonic acid, recognized for its antioxidant efficacy, and type I collagen extracted from bovine digital flexor tendon, formulated as a hydrogel. Efficacy was confirmed by a comparative study including untreated control groups and groups treated with type I collagen from bovine digital flexor tendon in hydrogel form, without rosmarfonic acid.

[0005] Field of application

[0006]

[0002] The present invention falls within the field of medical and veterinary needs, especially in the sector of materials for non-surgical wound treatment in veterinary or human patients, focusing on the incorporation of rosmarinic acid into type I collagen extracted from bovine digital flexor tendon, formulating a hydrogel for topical use. This development is intended for the treatment of pressure ulcers, also known as decubitus ulcers or bedsores, characterized by local damage to the skin and / or underlying tissues due to ischemia and reperfusion. Fundamentals of the technique

[0007]

[0003] Pressure ulcers, also known as bedsores or decubitus ulcers, are chronic lesions in the skin and underlying tissues caused by restriction of blood flow (ischemia) followed by restoration of that flow (reperfusion) (KWEK et al., 2023. Targeting Cx43 to reduce the severity of pressure ulcer progression). They develop due to persistent pressure and friction, mainly over bony prominences (GASPAR et al., 2019. Effectiveness on hospital-acquired pressure ulcers prevention: a systematic review). Ischemia occurs when there is a significant reduction or interruption of the blood supply to a specific tissue, depriving it of oxygen and essential nutrients. Reperfusion, in turn, occurs when blood flow is restored after a period of ischemia, triggering inflammation, oxidative stress, increased oxidative phosphorylation, and alterations in cell recruitment (SALCIDO; POPESCU; AHN, 2007).Animal models in pressure ulcer research).

[0008]

[0004] Pressure ulcers are characterized by an intense inflammatory response and reduced antioxidant capacity (ZHAO et al., 2016. Inflammation in chronic wounds). Both the development of ulcers and the associated inflammation involve at least two pathophysiological mechanisms: ischemia-reperfusion induced injury and increased levels of reactive oxygen species (SALCIDO; POPESCU; AHN, 2007. Animal models in pressure ulcer research). Previous studies have demonstrated the interconnection of these mechanisms, showing that cycles of ischemia and reperfusion raise reactive oxygen species levels to cytotoxic levels, resulting in tissue damage that prolongs tissue repair time (DUNNILL et al., 2017. Reactive oxygen species (ROS) and wound healing: the functional role of ROS and emerging ROS-modulating technologies for augmentation of the healing process).

[0009]

[0005] In the context of pressure ulcers, the superoxide anion is the main free radical involved in the formation of reactive oxygen species, produced by neutrophils through the xanthine oxidase pathway (KUMAR et al., 2021. Reactive oxygen species and pressure ulcer formation after traumatic injury to spinal cord and brain). In addition, more stable byproducts, such as hydrogen peroxide, can also cause tissue damage when not properly metabolized (BORTOLOTTI et al., 2021. Xanthine oxidoreductase: one enzyme for multiple physiological tasks).

[0010]

[0006] Various factors negatively influence the repair of pressure ulcers, resulting in a prolonged time required for tissue repair. Interruptions in the phases of the repair process represent one of the main obstacles, frequently preventing the progression of the repair phases (PROTZMAN et al., 2023. Placental-derived biomaterials and their application to wound healing: a review). The repair process can be divided into four overlapping phases: hemostasis, inflammation, proliferative, and remodeling (WALLACE et al., 2023. Wound healing phases). Previous studies have highlighted that the accumulation of reactive oxygen species interferes with the transition from the inflammatory phase to the proliferative phase (DENG et al., 2019. Ml macrophage mediated increased reactive oxygen species (ROS) influence wound healing via the MAPK signaling in vitro and in vivo) (MALONE-POVOLNY; MALONEY; SCHOENFISCH, 2019. Nitric oxide therapy for diabetic wound healing).

[0011]

[0007] In the context of human health, pressure ulcers, also known as bedsores, are a growing public health problem, especially for health systems, due to the high costs associated with their treatment (JARBRINK et al., 2016. Prevalence and incidence of chronic wounds and related complications: a protocol for a systematic review). The older population is the most susceptible to this type of wound, with the average age of patients ranging between 70 and 80 years (GOULD et al., 2015. Chronic wound repair and healing in older adults: current status and future research). The aging of the global population is expected to intensify the impact of pressure ulcers on health services, making the development of new therapies to alleviate the financial and social burden that this condition represents urgent.The global market for advanced wound care for pressure ulcers and other chronic wounds is estimated to reach £16.5 billion in 2024, not including the costs of other associated therapies (SEM, 2019. Human wounds and its burden: an updated compendium of estimates).

[0012]

[0008] Among humans, predisposing factors for pressure ulcers include reduced mobility, poor nutritional status, impaired sensory perception, advanced age, cognitive impairment, neurological disorders, cardiovascular diseases, prolonged anesthesia, dehydration, malnutrition, hypotension, surgical interventions, and comorbidities such as diabetes mellitus, which interfere with tissue repair. Immobility for just 2 hours in bedridden patients or those undergoing surgical procedures is sufficient to trigger the development of pressure ulcers (ZAl Dl; SHARMA, 2024. Pressure ulcer).

[0013]

[0009] In the context of animal health, debilitated animals recovering from serious injuries or illnesses, who have difficulty or reluctance to change position, are subject to the development of pressure ulcers. In these cases, many factors contribute to the appearance of ulcers, such as reduction of the cushioning between the skin and bone, atrophy or loss of adipose tissue, loss of tissue elasticity, malnutrition, maceration of the skin, contusion of soft tissues, friction and abrasion, or rubbing and stretching of the skin, and skin irritation due to exposure to urine and feces (PARISH; WITKOWSKI, 1987. The decubitus ulcer: reflections of a decade of concern).

[0014]

[0010] Certain dog breeds are predisposed to developing pressure ulcers due to specific neurological conditions associated with them. Among these conditions, cervical vertebral instability / malformation syndrome stands out, observed in breeds such as Dobermans, Pinschers, and Great Danes, which can result in secondary pressure ulcers due to temporary tetraplegia (SWAIM; HANSON; COATES, 1996. Pressure wounds in animals). - In addition, in dogs, pressure ulcers may be related to orthopedic abnormalities. In this context, neurological orthopedic pathology resulting from spinal trauma plays a secondary role in the development of pressure ulcers, along with paraplegia or tetraplegia associated with the abnormality (SWAIM; ANGARANO, 1990. Chronic problem wounds of dog limbs).Another relevant condition that can lead to the development of pressure ulcers is elbow hygroma, characterized by a fluid-filled cavity surrounded by a dense fibrous wall. This condition is frequently observed in young, large dogs such as German Shepherds, Great Danes, Bullmastiffs, and Irish Wolfhounds, due to repeated exposure to pressure or trauma in the olecranon region (SWAIM; HANSON; COATES, 1996. Pressure wounds in animals).

[0015]

[0011] In large animal veterinary medicine, horses that remain recumbent for long periods, especially those affected by post-anesthetic myopathies, neurological diseases, limb fractures, or laminitis, are susceptible to the development of pressure ulcers (LAUNOIS et al., 2021. Use of negative pressure wound therapy in the treatment of limb wounds: a case series of 42 horses). Pressure ulcers in horses generally result from prolonged pressure on a relatively small area of ​​the body, leading to tissue ischemia followed by necrosis. This condition is particularly concerning when it occurs near a joint, as it can result in infection of the synovial spaces (VAJS; NEKOUEI; BIERMANN, 2022. Descriptive study of the clinical presentation, management, and outcome of horses with acute soft tissue trauma of the tarsus and the association with synovial involvement).

[0016]

[0012] The treatment of pressure ulcers requires an interdisciplinary approach. In the surgical context, techniques such as debridement stand out, while in non-surgical therapies, wound dressings or related treatments play a fundamental role (GUSHIKEN et al., 2021. Cutaneous wound healing: an update from physiopathology to current therapies). Dressings or related treatments should keep the pressure ulcer moist, free from infection, devitalized or sloughed tissue, and toxic chemicals.There are three main categories of dressings: passive dressings, which act as protective barriers, isolating the ulcer from the external environment and controlling exudate; interactive dressings, which stimulate repair through mechanisms such as maintaining optimal hydration, autolytic debridement, and antimicrobial action; and active dressings, which accelerate repair by releasing growth factors or bioactive compounds, activating cellular mechanisms and controlling inflammation (NICE CLINICAL GUIDELINES).

[0017] 2014. The prevention and management of pressure ulcers in primary and secondary care).

[0018]

[0013] Technological advances in the area of ​​wound dressings or related products have made available to the market a wide variety of options, including films, foams, hydrocolloids, and hydrogels (SHI et al., 2020. Selection of appropriate wound dressing for various wounds). Among wound dressings, hydrogels stand out due to their ability to maintain a favorable environment for tissue repair (ASWATHY; NARENDRAKUMAR; MANJUBALA, 2020. Commercial hydrogels for biomedical applications). They contain a large amount of water, keeping ulcers hydrated and preventing dryness. Hydrogels can form a semi-closed protective film on the injured area, providing a breathable environment for repair. Furthermore, hydrogels can carry bioactive substances that are slowly released into the ulcerative wound bed, accelerating the repair process (SOLANKI; VINCHHI; PATEL, 2023).Design considerations, formulation approaches, and strategic advances of hydrogel dressings for chronic wound management. This capacity to carry bioactive substances adds an additional therapeutic dimension to hydrogel dressings, making them even more effective in treating pressure ulcers.

[0019]

[0014] Hydrogels are materials with a highly hydrophilic three-dimensional structure, allowing them to absorb and retain large volumes of water without dissolving (JIN et al., 2023. Progress of hydrogel dressings with wound monitoring and treatment functions). The synthesis of hydrogels involves two main methods: chemical and physical bonding. In chemical bonding, permanent covalent bonds are formed between the polymer chains, resulting in robust and stable structures. In physical bonding, the interactions between the chains are non-covalent in nature, such as ionic, hydrogen, or hydrophobic bonds (VARAPRASAD et al., 2017. A mini review on hydrogels classification and recent developments in miscellaneous applications). This approach allows the formation of soft and adaptable hydrogels, with properties that can be easily adjusted. The choice of polymer for the construction of the hydrogel is crucial in determining its final properties.Synthetic polymers, such as polyacrylic acid, polymethacrylic acid, and N-isopropyl acrylamide, provide greater control over the physical and chemical characteristics of the material, while polymers of natural origin, such as chitosan, cellulose, sodium alginate, gelatin, and collagen, offer improved biocompatibility and biodegradability (JIN et al., 2023. Progress of hydrogel dressings with wound monitoring and treatment functions).

[0020]

[0015] Collagen is an abundant extracellular protein in animal connective tissue. With a wide range of functions, collagen not only provides structural support to the body's tissues and organs, but also influences biological processes such as cell adhesion, growth, migration, metabolism, and signaling. To date, 28 distinct types of collagen have been identified and described in the scientific literature, each with specific characteristics. Among these, collagens I, III, and V stand out in the composition of the skin, giving it firmness, elasticity, and tensile strength (SILVIPRIYA et al., 2015. Collagen: animal sources and biomedical application).

[0021]

[0016] Type I collagen is widely explored in the biomedical field due to its potential in the production of hydrogels, biomaterials, and implants. Its structure presents four levels of molecular interaction. In the primary structure, there is a tripeptide sequence -(Gly-XY)-, with glycine (Gly) corresponding to about 30% of the amino acids, while X is usually a proline and Y is a hydroxyproline. The secondary structure consists of repetitions of triple chains of amino acids. In the third level of organization, a triple helix is ​​formed, with each chain containing approximately one thousand amino acids. Collagen fibers represent the quaternary structure and are composed of self-organized fibrils (SORUSHANOVA et al., 2019. The collagen suprafamily: from biosynthesis to advanced biomaterial development).

[0022]

[0017] Type I collagen, derived from bovine sources such as bones, skin, and tendons, is an important raw material in the manufacture of hydrogels (SILVI PRIYA et al., 2015. Collagen: animal sources and biomedical application). Studies have shown that type I collagen peptides have several beneficial properties. Among them, the regulation of the immune system, the promotion of tissue repair, antitumor and antibacterial effects, and anti-inflammatory and antioxidant properties stand out (LU et al., 2024. Antioxidant and anti-aging potential of collagen peptide conjugated with ionic liquid). In the treatment of pressure ulcers, the anti-inflammatory and antioxidant properties of type I collagen may be particularly useful in controlling inflammation and oxidative stress.

[0023]

[0018] Compared to other types of hydrogels, collagen hydrogels have the advantage of possessing a high water content, greater than 99%, giving them a flexibility similar to that of body tissues, such as skin (CHEEMA, 2013. Three-dimensional collagen biomatrix development and control). In addition, they are a sustainable option, since they are constructed with raw materials of animal origin that would otherwise be discarded, thus minimizing environmental impacts. Type I collagen hydrogels can carry bioactive compounds, such as antibiotics, metallic nanoparticles, plant extracts, growth factors, and natural exogenous antioxidants (SOLANKI; VINCHHI; PATEL, 2023. Design considerations, formulation approaches, and strategic advances of hydrogel dressings for chronic wound management) (ZHANG et al., 2023. Application of collagen-based hydrogel in skin wound healing).

[0024]

[0019] Natural exogenous antioxidants are chemical compounds with the ability to inhibit the production of reactive oxygen species (SCHILRREFF; ALEXIEV. 2022. Chronic inflammation in non-healing skin wounds and promising natural bioactive compounds treatment). In addition, some of these antioxidants are capable of catalyzing complex biochemical reactions, converting reactive oxygen species into more stable molecules, such as water and oxygen. They also have the ability to stimulate endogenous antioxidant enzyme systems and increase the production of non-enzymatic antioxidants in vivo. As a result, these compounds can maintain non-toxic levels of reactive oxygen species in the wound and promote a significant acceleration of the tissue repair process (HE et al., 2017. Antioxidants maintain cellular redox homeostasis by elimination of reactive oxygen species) (COMINO-SANZ et al., 2021. The role of antioxidants on wound healing: a review of the current evidence).

[0025]

[0020] Among natural antioxidants, rosmarinic acid (C18H16O8) stands out. It is a commercially available phenolic substance belonging to the hydroxycinnamic acid family, being an ester of caffeic acid and 3,4-dihydroxyphenyl lactic acid (SILVEIRA FACHEL et al., 2019. Glioprotective effect of chitosan-coated rosmarinic acid nanoemulsions against lipopolysaccharide-induced inflammation and oxidative stress in rat astrocyte primary cultures). Found in more than 30 plant families, rosmarinic acid exhibits a variety of biological and pharmacological activities, notably its antioxidant potential, superior to that of vitamin E (AMOAH et al., 2016. Rosmarinic acid - pharmaceutical and clinical aspects).The in vitro antioxidant capacity of rosmarinic acid has been demonstrated by methods such as 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), 2,2-diphenyl-1-picrylhydrazyl, reducing antioxidant power of ferric acids, hypochlorite and deoxyribose, showing superior results in comparison with other hydroxycinnamic acids, such as caffeic acid, chlorogenic acid, ferulic acid and p-coumaric acid (AMOAH et al., 2016. Rosmarinic acid - pharmaceutical and clinical aspects). Furthermore, rosmarinic acid is able to increase the expression of the antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase and reduce the levels of thiobarbituric acid reactive substances, related to the upregulation of nuclear factor erythroid 2-related factor 2 (VERAS et al., 2022. Technological strategies applied for rosmarinic acid delivery through different routes - a review).

[0026]

[0021] The antioxidant property of rosmarinic acid, attributed to the catechol groups present in its structure (AMOAH et al., 2016. Rosmarinic acid pharmaceutical and clinical aspects), is associated with a variety of beneficial activities widely documented in the technical-scientific literature. Among these, the anti-inflammatory, photoprotective and wound-healing properties stand out (FADEL; KI AT; MORANDAT, 2011. The natural antioxidant rosmarinic acid spontaneously penetrates membranes to inhibit lipid peroxidation in situ) (CUTRIM; CORTEZ, 2018. A review on polyphenols: classification, beneficial effects and their application in dairy products) (WAN I; RAZA; KHAN, 2019. Rosmarinic acid loaded chitosan nanoparticles for wound healing in rats).Furthermore, rosmarinic acid demonstrates the ability to reduce the formation of reactive oxygen species and the secretion of interleukin-6 by immune system cells, aiding in protection against cellular damage (VOST LOV; ZDARILOVA; SVOBODOVA, 2010. Prunella vulgaris extract and rosmarinic acid prevent UVB-induced DNA damage and oxidative stress in HaCaT keratinocytes).

[0027]

[0022] Rosmarinic acid is used in the treatment of peptic ulcers, cataracts, rheumatoid arthritis, herpes simplex, arteriosclerosis, and bronchial asthma (KANTAR et al., 2022. Deciphering the therapeutical potentials of rosmarinic acid). Although it can be administered via various routes, such as aqueous solutions, including intravenous, dermal / transdermal, ocular, and nasal, most administrations are oral (VERAS et al., 2022. Technological strategies applied for rosmarinic acid delivery through different routes ■■■ a review). Administering rosmarinic acid in powder form is not always convenient, since, despite being one of the simplest pharmaceutical forms, it presents problems related to its physicochemical and microbiological instabilities.Thus, other strategies for administering rosmarinic acid have emerged, such as emulsions, nanoemulsions, nanoparticles, and liposomes; in addition to complexation techniques with polymers to form different materials, including hydrogels, with special interest in topical applications on the skin and in the treatment of wounds (KÜBA et al., 2020. Comparison of local rosmarinic acid and topical dexpanthenol applications on wound healing in a rat experimental wound model) (CHHABRA et al., 2020. Augmented healing of full thickness chronic excision wound by rosmarinic acid loaded chitosan encapsulated graphene nanopockets) (HUERTA-MADRŌNAL et al., 2021. Chitosan – Rosmarinic acid conjugates with antioxidant, anti-inflammatory and photoprotective properties) (ZHONG et al., 2023. Fabrication and characterization of PVA@PLA electrospinning nanofibers embedded with Bletilla striata polysaccharide and Rosmarinic acid to promote wound healing).

[0028]

[0023] When applied topically to the skin, rosmarinic acid demonstrates good penetration capacity and promotes the survival of keratinocytes exposed to ultraviolet radiation, reducing the generation of intracellular reactive oxygen species and DNA damage (OSAKABE et al., 2004. Rosmarinic acid, a polyphenolic compound from rosemary, protects against UVA-induced skin damage in hairless mice). At concentrations of 2 μg / mL or 4 μg / mL, rosmarinic acid activates the sodium-hydrogen exchanger, an important factor in the acidification of the stratum corneum of the skin. This is significant because the pH of healthy skin is slightly acidic, with a range of 4.8 to 5.7 (JUNG et al., 2022. Rosmarinic acid, as an NHE1 activator, decreases skin surface pH and improves the skin barrier function).In chronic wounds, the skin's pH becomes alkaline, and studies show that acidifying this environment can increase oxygen supply, induce fibroblast proliferation, and facilitate tissue repair (MILLER et al., 2017. The pH of wounds during healing and infection: a descriptive literature review) (WALLACE; GWYNNE; JENKINS, 2019. Challenges and opportunities of pH in chronic wounds).

[0029]

[0024] Regarding the therapeutic potential of rosmarinic acid in wound treatment, recent studies have presented promising results. In one study, a gelatin / dextran hydrogel containing rosmarinic acid was found to be effective in promoting tissue repair (YIN et al., 2023. Rosmarinic acid-grafted dextran / gelatin hydrogel as a wound dressing with improved properties: strong tissue adhesion, antibacterial, antioxidant, and anti-inflammatory). This effect is mainly attributed to the antioxidant and anti-inflammatory properties of rosmarinic acid, which were evidenced by the regulation of inflammatory cytokine levels and oxidative stress. Another approach involved the development of a nanofiber material using polylactic acid, a natural plant polysaccharide, Bletillo striata polysaccharide, and rosmarinic acid to facilitate the repair of cutaneous wounds (ZHONG et al., 2023).Fabrication and characterization of PVA@PLA electrospinning nanofibers embedded with Bletilla striata polysaccharide and rosmarinic acid to promote wound healing. This material induced macrophage proliferation and favored skin repair. Another study evaluated the effect of a formulation of encapsulated chitosan and graphene nanoparticles containing rosmarinic acid (CHHABRA et al., 2020. Augmented healing of full thickness chronic excision wound by rosmarinic acid loaded chitosan encapsulated graphene nanopockets). This formulation demonstrated significant wound repair and stimulated angiogenesis.

[0030] Background of the invention

[0031]

[0025] In the current state of the art, some prior art is presented that describes the effects of collagen products of bovine origin or other sources, with or without the addition of natural exogenous antioxidants other than rosmarinic acid, on skin quality or skin wound repair. In parallel, some prior art is presented that explores the effects of rosmannic acid in various concentrations, formulations, and routes of administration on skin quality or skin wound repair. However, no reference or document presented addresses the specific treatment of pressure ulcers. Furthermore, there are no records of formulations, products, or materials that combine collagen of animal origin and rosmannic acid powder for therapeutic use in skin wounds.

[0032]

[0026] Document CN108014366B, entitled “Marine biological material composite hydrogel dressing and preparation method thereof”, describes a hydrogel dressing produced with marine biological material. This hydrogel is composed of two solutions: the first contains sodium alginate, fish collagen, polyvinyl alcohol, sodium carboxymethylcellulose, pectin, glycerol, propylene glycol, and distilled water; while the second contains chitosan, a crosslinking agent, glacial acetic acid, and distilled water. This hydrogel dressing demonstrates good mechanical properties, water retention capacity, and excellent biocompatibility. It can accelerate wound healing and reduce inflammation. Furthermore, it is easily removable and does not stick to the wound bed.

[0033]

[0027] Document CN107549812B, entitled “A skin caring and antiaging food prepared from acerola cherry extract, Haematococcus pluvialis, and fish collagen peptide, and its preparation method”, describes an invention related to an anti-aging product, for oral administration, which uses acerola cherry extract, Haematococcus pluvialis, and fish collagen peptide as raw materials. The product is composed of the following ingredients in parts by weight: 20-30 parts acerola cherry extract, 20-30 parts Haematococcus pluvialis powder, and 40-60 parts fish collagen peptide. It exhibits excellent anti-aging and antioxidant effects, promoting skin cell regeneration, maintaining hydration, reducing pigment formation, and providing elasticity and wrinkle resistance.

[0028] Document EP3419684B1, entitled “Biomateriaux composites a liberation controlee de principe actif, procede de preparation et utilisations”, describes an innovation related to a composite biomaterial that combines collagen, at least one hydrophobic organic polymer, and at least one active ingredient. This biomaterial finds application in the treatment of chronic wounds, and is biocompatible, biodegradable, and possesses mechanical properties comparable to or improved upon conventional collagen hydrogels. Furthermore, it stands out for its ability to release the active ingredients in a controlled and prolonged manner, providing an effective therapeutic action.

[0034]

[0029] A study describes the formulation of chitosan / polyvinyl alcohol / collagen hydrogels containing jackfruit extract (Artocarpus heterophyllus Lam) for application to wounds. The hydrogels are prepared from mixed solutions of 1% w / v chitosan, 1% w / v polyvinyl acid, and collagen in various proportions, including 5 / 4 / 1, 5 / 3 / 2, 4 / 5 / 1, and 4 / 4 / 2 by weight of the solution. The antioxidant activity of the jackfruit extract was evaluated by the 1,1-diphenyl-2-picrylhydrazyl assay. All hydrogels demonstrate an antioxidant activity of approximately 46-51% and have potential for use as wound dressings (WITTICHAROENMOGKOL et al., 2022. Chitosan / poly(vinyl alcohol) / collagen hydrogel composites containing jackfruit axis extract for wound dressing application).

[0035]

[0030] A study describes the preparation of an injectable, antioxidant hydrogel based on collagen and hyaluronic acid, mediated with gallic acid and dopamine. This hydrogel demonstrates the ability to neutralize free radicals and adapt to the wound site, accelerating tissue repair. Tests show that the hydrogel promotes cell proliferation and migration. In vitro assays demonstrate the antioxidant property of the hydrogel and its ability to neutralize excess reactive oxygen species. In vivo assays demonstrate that the hydrogel can promote angiogenesis, inhibit inflammation, and promote collagen fiber deposition, accelerating wound repair (YANG et al., 2023. An injectable, self-healing, and antioxidant collagen- and hyaluronic acid-based hydrogel mediated with gallic acid and dopamine for wound repair).

[0036]

[0031] The effects of rosmarinic acid and dexpanthenol are compared in an experimental wound model in rats. After creating 2 cm thick skin defects, the wounds were washed with a sterile 0.9% sodium chloride (NaCl) solution. Before excision, the skin was evaluated macroscopically; after excision, microscopic analyses of epithelialization, inflammation, fibrosis, and granulation parameters were performed. The results show that the group treated with rosmarinic acid presents a greater reduction in wound size and may be an effective therapeutic option as a topical cream (KUBA et al., 2020. Comparison of local rosmarinic acid and topical dexpanthenol applications on wound healing in a rat experimental wound model).

[0037]

[0032] A study describes the fabrication and evaluation of a formulation of graphene nanoparticles encapsulated in chitosan loaded with rosmarinic acid to promote wound repair in Sprague Dawley rats. Through histopathological evaluations, it was demonstrated that the nanoparticles promote wound repair, epithelial migration, and collagen synthesis (CH HABRA et al., 2020. Augmented healing of full thickness chronic excision wound by rosmarinic acid loaded chitosan encapsulated graphene nanopockets).

[0038]

[0033] A study describes the synthesis of water-soluble chitosan and rosmarinic acid conjugates, potentially useful for dermatological applications. Chitosan was functionalized with different concentrations of rosmarinic acid, confirmed by techniques such as Fourier transform infrared spectroscopy, proton nuclear magnetic resonance, and ultraviolet spectroscopy. These conjugates demonstrate a radical scavenging capacity three times greater compared to free rosmarinic acid, in addition to reducing cellular damage caused by ultraviolet radiation and the formation of reactive oxygen species (HUERTA-MADRONAL et al., 2021. Chitosan – Rosmarinic acid conjugates with antioxidant, anti-inflammatory and photoprotective properties).

[0039]

[0034] A study describes the fabrication of a nanofibrillar material composed of polylactic acid, Bletilla striata polysaccharide, and rosmarinic acid with the aim of promoting wound repair. This material is biomimetic and demonstrates the ability to induce wound repair, as evidenced by animal models in dorsal skin of rats and detailed histological analyses. Histological analyses reveal that the material facilitates the proliferation and differentiation of macrophages in the initial phases of the repair process. Furthermore, it induces downregulation of myeloperoxidase expression in the wound area, contributing to efficient tissue repair (ZHONG et al., 2023. Fabrication and characterization of PVA@PLA electrospinning nanofibers embedded with Bletilla striata polysaccharide and rosmarinic acid to promote wound healing).

[0040]

[0035] A study describes the fabrication of a hydrogel, ODex-AG-RA, developed by incorporating rosmarinic acid into dextran / gelatin matrices. The hydrogel demonstrates favorable characteristics, including rapid gelation time, high adhesive strength, and enhanced mechanical properties. In vitro biocompatibility tests involving hemolysis assessment and co-culture with L929 cells reveal excellent compatibility of the ODex-AG-RA hydrogels. In vivo assays using a rat model with skin defects demonstrate significant improvement in wound repair. The underlying mechanism of the wound repair properties of ODex-AG-RA is attributed to its anti-inflammatory effects, as evidenced by the modulation of inflammatory cytokines and reduction in oxidative stress markers (YIN et al., 2023).Rosmarinic acid-grafted dextran / gelatin hydrogel as a wound dressing with improved properties: strong tissue adhesion, antibacterial, antioxidant and anti-inflammatory). Purpose of the invention.

[0041]

[0036] The present invention aims to provide a process for obtaining a rosmannian acid formulation in bovine type I collagen hydrogel for topical application in the treatment of pressure ulcers, also known as decubitus ulcers or bedsores, in domestic animals and humans. The choice of this formulation is based on the antioxidant and anti-inflammatory properties of rosmannian acid, a phenolic substance frequently used in photoprotectors, characterized by its excellent skin penetration capacity and its aptitude to be complexed or associated with different materials, including polymers, for therapeutic purposes.

[0042]

[0037] Furthermore, the invention is based on the properties of type I collagen, a polymer that exhibits high skin biocompatibility, favorable cost-benefit ratio, and sustainability, since its extraction source comes from slaughterhouse waste. Additionally, the choice of a hydrogel formulation is justified by its hydrophilicity, which prevents wound drying, and its ability to incorporate bioactive substances. Specifically, collagen hydrogels possess excellent flexibility, making them ideal for skin application.

[0043] Brief description of the invention

[0044]

[0038] The present invention relates to a process for obtaining a formulation containing rosmarinic acid in bovine type I collagen hydrogel for the topical treatment of pressure ulcers. The preparation process begins with obtaining bovine flexor tendons, which are washed, cut, and treated with a sodium hypochlorite solution to remove impurities. Then, the collagen is extracted in an acidic solution, precipitated, and dialyzed to obtain the collagen gel. The gel is diluted in an aqueous solution of acetic acid and then combined with rosmarinic acid at concentrations of 2 pg / mL or 4 pg / mL, dialyzed, and subjected to gelation to form the hydrogel. Rosmarinic acid is widely recognized for its antioxidant and anti-inflammatory properties, and is used in pharmaceutical and cosmetic formulations.However, its solubility and stability are critical aspects, especially in formulations that require optimized interaction between the bioactive and a structural matrix. In conventional methods of incorporating bioactive compounds, the use of neutral or alkaline solutions is common, as these environments favor the solubility of rosmarfonic acid and protect the compound from degradation that could compromise its biological functions. In contrast, the use of acidic solutions has been avoided due to the instability of rosmarfonic acid under such conditions, in which it tends to degrade, losing its functionality. The technical innovation of this invention lies precisely in the incorporation of rosmarfonic acid into an acidic solution of type I collagen, followed by neutralization by dialysis. The final product is sterilized and stored under refrigeration.Infrared spectroscopy analyses confirm the preservation of the characteristic collagen structure, while swelling tests demonstrate the hydrogel's ability to absorb fluid. Macroscopic evaluation of wounds treated with the formulation containing rosmarinic acid at concentrations of 2 pg / mL or 4 pg / mL shows a significant reduction in wound size compared to control groups. Histopathological analyses reveal greater re-epithelialization and collagen density in the groups treated with the formulation. Furthermore, the formulation is able to reduce levels of reactive oxygen species at the wound sites.

[0045] Description of the figure

[0046]

[0039] The invention is described in a preferred embodiment, so, for better understanding, reference will be made to the figures:

[0040] FIG. 1. Macroscopic aspect of the products obtained. In A, formulation of rosmarfonic acid, at a concentration of 2 pg / mL, in bovine type I collagen hydrogel. In B, formulation of rosmarfonic acid, at a concentration of 4 pg / mL, in bovine type I collagen hydrogel.

[0047]

[0041] FIG. 2. Fourier transform infrared absorption spectra of rosmarinic acid formulations, at concentrations of 2 pg / mL (A) and 4 pg / mL (B), incorporated into type I bovine collagen hydrogel.

[0048]

[0042] FIG. 3. Swelling profile of rosmarinic acid formulations, at concentrations of 2 μg / mL (HC + AR 2 μg / mL) and 4 μg / mL (HC + AR 4 μg / mL), incorporated into type I bovine collagen hydrogel, before (initial weight - IP - baseline) and after 1h, 2h, 3h and 4h of immersion in phosphate-buffered saline.

[0049]

[0043] FIG. 4. In A, graph representing the average wound sizes, in cm2, at different evaluation times of the untreated, HC + AR 2 pg / mL (treatment with rosmarfonic acid formulation 2 pg / mL in type I collagen hydrogel), HC + AR 4 pg / mL (rosmarfonic acid formulation 4 pg / mL in type I collagen hydrogel) and HC (treatment with type I collagen hydrogel) groups. In B, graph showing the percentage of residual wound in the evaluation groups after 14 days of pressure ulcer induction. *, P < 0.05; **, P < 0.01; ***, P = 0.001; ****, p < 0.0001.

[0050]

[0044] FIG. 5. Graphical representation of the percentages of wound counteraction at different evaluation times of the untreated, HC + AR 2 pg / mL (treatment with rosmarfonic acid formulation 2 pg / mL in type I collagen hydrogel), HC + AR 4 pg / mL (rosmarfonic acid formulation 4 pg / mL in type I collagen hydrogel) and HC (treatment with type I collagen hydrogel) groups. *, P < 0.05.

[0051]

[0045] FIG. 6. Graphs showing re-epithelialized areas (A) and collagen density (B) for the untreated, HC + AR 2 pg / mL (treatment with rosmannian acid formulation 2 pg / mL in type I collagen hydrogel), HC + AR 4 pg / mL (rosmannian acid formulation 4 pg / mL in type I collagen hydrogel) and HC (treatment with type I collagen hydrogel) groups, after 6 and 14 days of pressure ulcer induction. Optical density (OD) was expressed in arbitrary units ****, p < 0.0001.

[0052]

[0046] FIG. 7. Graph representing the levels of reactive oxygen species in the wounds of the following groups: untreated, HC + AR 2 pg / mL (treatment with rosmarinic acid formulation 2 pg / mL in type I collagen hydrogel), HC + AR 4 pg / mL (rosmarinic acid formulation 4 pg / mL in type I collagen hydrogel) and HC (treatment with type I collagen hydrogel), after 6 and 14 days of pressure ulcer induction. Optical density (OD) was expressed in arbitrary units. ***, P = 0.001; ****, p < 0.0001.

[0053] Detailed description of the invention

[0054]

[0047] The process for obtaining the formulation of rosmannic acid in bovine type I collagen hydrogel for topical application in the treatment of pressure ulcers, the subject of this invention patent, begins with the use of bovine digital flexor tendons obtained from a commercial slaughterhouse as sources for the extraction of type I collagen.

[0055]

[0048] The first step of the process consists of washing and cutting the digital flexor tendons into 5 cm x 5 cm fragments, excluding the tendon ends, which have a significant content of non-collagenous molecules due to their insertion into bony and muscular ends. Next, each tendon fragment is immersed in 50 mL of 1% sodium hypochlorite under refrigeration at 9°C for 1 h. Subsequently, deep longitudinal incisions are made in each fragment, respecting the direction of the collagen bundle crimps.

[0056]

[0049] In the second stage, each tendon fragment is immersed in 50 mL of 4 molar sodium hydroxide for 4 hours at a controlled temperature of 25°C, followed by three washes in 200 mL of distilled water each, lasting 1 hour each.

[0050] In the third stage, each tendon fragment is placed in 100 mL of aqueous solution of 0.5 molar acetic acid containing 1% 1 molar hydrochloric acid, where it remains for seven days at 9°C.

[0057]

[0051] In the fourth step, the acidic solution containing the tendon fragment is sieved through a 0.59 mm mesh filter and the filtration is followed by the addition of 200 mL of 10% aqueous sodium chloride solution to promote the precipitation of collagen fibrils.

[0058]

[0052] In the fifth stage, the precipitated collagen fibrils are collected, placed in dialysis bags with a diameter of 50 mm and a molecular weight cutoff between 12000 and 14000, and dialyzed against 1000 mL distilled water for 48 h, under refrigeration, at a controlled temperature of 9 °C, with water changes every 12 h, to obtain a collagen gel.

[0059]

[0053] In the sixth step, the collagen gel is weighed and dissolved in a 0.5 molar aqueous solution of acetic acid, in a ratio of 1 gram for every 1 mL of solution. Then, commercial rosmannian acid (C18H16O8) powder (purity specifications in catalog number R4033, Sigma-Aldrich, Brazil) is added to the resulting product, at concentrations of 2 pg / mL (solute / solvent ratio 2:1) or 4 pg / mL (solute / solvent ratio 4:1).

[0060]

[0054] Although it is a known compound, rosmarinic acid presents challenges related to its solubility and stability in formulations, especially in acidic environments, where it tends to degrade. In this process, however, it was decided to dissolve it directly in the acidic collagen solution. This choice aims not only to facilitate its initial solubilization, but also to take advantage of the acidic environment to promote an efficient interaction with the collagen matrix, before the stability of the compound is compromised. Subsequently, the product is centrifuged at 400 revolutions per minute for 10 min and placed in dialysis bags with a diameter of 50 mm and a molecular weight cutoff between 12000 and 14000, being dialyzed against distilled water for 24 h, with water changes every 12 h.

[0055] In the seventh step, after opening the dialysis bag, the product is transferred to a sterile container.The pH of the collagen solution is adjusted to 7.0 using 2 molar sodium hydroxide and 2 molar acetic acid, with continuous monitoring and adjustments as needed to ensure the desired pH. The product is then stored in a refrigerator at a controlled temperature of 4°C to induce hydrogel formation by gelation at a reduced temperature. The time required for gelation is 12 hours.

[0061]

[0056] For sterilization, the material can be subjected to radiosterilization.

[0062] The final product is semi-transparent and flexible, as shown in figure 1 (FIG.1), odorless and must be stored under refrigeration at a temperature between 8°C and 12°C, avoiding contamination during handling.

[0063]

[0057] The product can be handled at room temperature without compromising its stability or the characteristics of the hydrogel.

[0064] Tests performed

[0065]

[0058] The tests performed for the proposed formulation are described below.

[0066]

[0059] Fourier Transform Infrared Spectroscopy: In this invention, Fourier transform infrared spectroscopy was used, using a PerkinElmer Spectrum RXI spectrometer, to determine whether rosmarfonic acid or the hydrogel preparation protocol affects the structure of type I collagen. Samples were scanned in the wavenumber range of 400 cm⁻¹ to 4000 cm⁻¹, encompassing typical functional groups of biological materials. Each sample was scanned 32 times to improve the signal-to-noise ratio and ensure data accuracy. The spectral resolution was set to 2 cm⁻¹, providing details on the chemical functionalities present. The spectra were analyzed using specialized software for spectral data processing. The characteristic peaks of each component were identified and compared with the literature to confirm the presence of specific functional groups (LOHRASBI et al., 2019).Collagen / cellulose nanofiber hydrogel scaffold: physical, mechanical and cell biocompatibility properties).

[0067]

[0060] Swelling test: In this invention, a gravimetric method was used to investigate the swelling, i.e., the ability to absorb liquids, of the formulations. The hydrogel samples were weighed on an electronic analytical balance (Mettler Toledo Products & Solutions, Brazil) before immersion and after 1, 2, 3 and 4 h of immersion in phosphate-buffered saline, pH 7.4.

[0068] The test was performed in triplicate, under a controlled temperature of 25 °C. The degree of swelling, expressed as mass gain, was calculated using the formula = (final weight - initial weight) / final weight x 100 (LI et al., 2015. In vitro and in vivo evaluation of a novel collagen / cellulose nanocrystals scaffold for achieving the sustained release of basic fibroblast growth factor).

[0069]

[0061] In vivo assay: In this invention, the in vivo assay protocols were implemented only after approval by the Institutional Ethics Committee on the Use of Animals. Forty-four male Swiss mice, in systemic health conditions, aged between five and seven weeks, and weighing between 30 and 35 grams, were used. During the experiment, the mice were kept in the Institutional Animal Facility in cage systems with forced ventilation (60 air changes per hour), lined with wood shavings, under controlled conditions of temperature (25±1°C), relative humidity (60±5%) and a 12-hour light / dark cycle (8 am-20 pm), with unrestricted access to potable water and commercial feed suitable for the species.

[0070]

[0062] Mice were randomly assigned to four groups, each consisting of eleven animals. All groups underwent an experimental protocol for the induction of pressure ulcers. In the untreated group, the ulcers received no therapeutic intervention. In contrast, the ulcers in the groups designated HC + AR 2 pg / mL, HC + AR 4 pg / mL and HC were treated, respectively, with a rosmannian acid formulation at a concentration of 2 pg / mL in collagen hydrogel, a rosmannian acid formulation at a concentration of 4 pg / mL in collagen hydrogel and collagen hydrogel.

[0071]

[0063] All protocols were performed by the same team of examiners, who were unaware of the research groups. To induce pressure ulcers, three ischemia-reperfusion cycles were performed with mice anesthetized with ketamine (100 mg / kg, Cristalia, Itapira, Brazil) and xylazine (15 mg / kg, Cristalia) via intraperitoneal injection. During each ischemia-reperfusion cycle, the skin on the back of each mouse was shaved, pinched, and held between a pair of sterile magnets with 300 g of magnetic force and 5 mm in diameter. The ischemia period was 16 h per cycle, while the reperfusion period was 8 h. At the end of the third ischemia-reperfusion cycle, each mouse presented two ulcers, with a distance of approximately 1 cm between them (PEIRCE; SKALAK; RODEHEAVER, 2000. Ischemia-reperfusion injury in chronic pressure ulcer formation: a skin model in the rat) (TONG et al., 2011.Heparan sulfate glycosaminoglycan mimetic, OTR4120, reduces skin ulceration and improves ulcer healing in a rat model of ischemia-reperfusion injury.

[0072]

[0064] Protocols for the treatment of pressure ulcers: In this invention, the management or treatment protocols began on the first day after the third cycle of ischemia and reperfusion, considered day zero (DO). In the untreated group, the ulcers were cleaned every two days for fourteen days using a sterile solution of 0.9% sodium chloride (NaCl). In the HC + AR 2 ug / mL, HC + AR 4 ug / mL and HC groups, the ulcers were cleaned in the same way as in the untreated group. However, after each cleaning, a rosmannian acid formulation was applied as a primary dressing, at concentrations of 2 ug / mL and 4 ug / mL, in collagen hydrogel, and collagen hydrogel alone, respectively.

[0073]

[0065] Macroscopic analysis of ulcerative wounds: In this invention, wounds were evaluated on day zero (DO) and 2, 6, 10, and 14 days after the start of management and treatment, using a digital caliper (Thermo Scientific, Franklin, Mass.). This was positioned on the edges of the wounds to measure their lengths and widths. The wound sizes were then calculated based on the area of ​​an ellipse, using the mathematical formula: radius of length x radius of width x π.

[0074]

[0066] In addition, to assess ulcer counterpain, a sheet of transparent paper was placed over the wound and the edges were outlined. The resulting images were digitized and the area of ​​each ulcer was measured in mm2, using ImageJ® software (National Institutes of Health, Bethesda, MD) (ROMANA-SOUZA; DOS SANTOS; MONTE-ALTO-COSTA, 2018. Caffeic acid phenethyl ester promotes wound healing of mice pressure ulcers affecting NF-κB, NOS2 and NRF2 expression).

[0075]

[0067] Collection and processing of material: On days six and fourteen after the start of wound management and treatment, five and six mice from each group, respectively, were euthanized, selected randomly. Euthanasia was performed using a lethal dose of injectable barbiturate anesthetic (sodium thiopental, Cristalia), administered intraperitoneally (150 mg / kg). After euthanasia, the two ulcerated areas of each mouse were collected. Each ulcerated area was divided into two halves, one of which was fixed in buffered neutral formalin (Labsynth, Diadema, Brazil) for histopathological analysis, while the other was frozen in liquid nitrogen and stored at -80°C for quantification of reactive oxygen species levels.

[0076]

[0068] Histopathological analysis: In this invention, formalin-fixed samples were subjected to a dehydration process in progressively increasing ethanol solutions, followed by xylene clarification and routine paraffin embedding. Subsequently, sections with a thickness of 3 µm were obtained, which were stained using the hematoxylin and eosin technique or Masson's trichrome method. For histopathological analysis, three non-sequential sections from each ulcer were examined using a conventional Carl Zeiss light microscope equipped with UPL Neofluar objectives and a digital video-image analysis system. In each section, the re-epithelialized area was evaluated, that is, the area covered by neoepidermis, determined by the sum of the lengths of the migratory epithelial tongues (ROMANA-SOUZA; PIRES; MONTE-ALTO-COSTA, 2015).Mate tea-mediated reduction in catecholamine synthesis improves cutaneous wound healing of chronically stressed mice) (ROMANA-SOUZA; DOS SANTOS; MONTE-ALTO-COSTA, 2018. Caffeic acid phenethyl ester promotes wound healing of mice pressure ulcers affecting NF-κB, NOS2, and NRF2 expression). Furthermore, the amount of collagen in the dermis was evaluated using ImageJ® software calibrated in optical density mode (RIEPPO et al., 2019. Histochemical quantification of collagen content in articular cartilage).

[0077]

[0069] Analysis of reactive oxygen species levels: Ulcerative wounds, previously stored at -80°C, were thawed and macerated in 800 microliters of lysis buffer containing 20 millimolar Tris-hydroxymethylaminomethanehydrochloride (pH 7.5), 138 millimolar sodium chloride, 10% glycerol, 1% Triton X-100, and a protease inhibitor cocktail (Sigma-Aldrich). The lysate was centrifuged at 4°C for 30 min at 6149 times the force of gravity. The resulting supernatant was collected and used for the assay of reactive oxygen species (ROMANA-SOUZA; DOS SANTOS; MONTE-ALTO-COSTA, 2018. Caffeic acid phenethyl ester promotes wound healing of mice pressure ulcers affecting NF-κB, NOS2 and NRF2 expression).

[0078]

[0070] To determine the total amount of reactive oxygen species, the colorimetric method was used, which is based on the reaction of nitrotetrazol chloride blue (NBT) with these species, resulting in the formation of formazan crystals. In the procedure, 100 microliters of 0.1% NBT were mixed with 100 microliters of the sample and incubated at 37°C for 1 h. Then, the mixture was centrifuged at 983.84 times the force of gravity for 10 min to remove the supernatant. The resulting pellet was washed with phosphate-buffered saline (pH 7.4) and resuspended in a 2 molar potassium hydroxide solution in dimethylisothiazolinone. This solution served as a blank control. The reaction reading was performed at 630 nanometers in an enzyme immunoassay reader. The results were expressed as optical density of formazan crystals per milligram per milliliter of total protein (CHOI et al., 2006).A quantitative nitroblue tetrazolium assay for determining intracellular superoxide anion production in phagocytic cells).

[0079]

[0071] Data analysis: Statistics were conducted using two-way ANOVA or a mixed-effects model to investigate variations in measurements over time among the different groups evaluated. Sidak's adjustment method was used to control for type I error in multiple comparisons. Differences were considered significant when P < 0.05.

[0080] Fourier transform infrared spectroscopy

[0081]

[0072] Analysis of rosmannian acid formulations, at concentrations of 2 pg / mL and 4 pg / mL, in bovine type I collagen hydrogels, revealed the presence of functional groups characteristic of collagen. Figure 2 (FIG. 2) shows that peaks were observed around 3408 cm-1, attributed to NH stretching vibrations of amine groups, and at 2932 cm-1, attributed to the asymmetric stretching of CH2. The peaks at 1655 cm-1 and 1238 cm-1 correspond, respectively, to the O=O stretching for amide I and to the CN stretching and NH bending vibrations of amide III bonds. Based on the objective of verifying whether rosmannian acid or the hydrogel preparation protocol affects the collagen structure, the results obtained by Fourier transform infrared spectroscopy indicate that bovine type I collagen maintains its characteristic structure, as evidenced by the presence of representative peaks.

[0082] Swelling test

[0083]

[0073] The swelling profile of rosmannic acid formulations, at concentrations of 2 pg / mL and 4 pg / mL, in bovine type I collagen hydrogels, showed similarities, with both hydrogels gaining mass in the first h of immersion in phosphate-buffered saline, pH 7.4, and maintaining this mass gain until the end of the experiment, after 4 h of immersion, as shown in Figure 3 (FIG. 3). Despite the similarity in the swelling profile, the fluid absorption capacity in the formulation containing rosmannic acid at a concentration of 4 pg / mL was approximately 44% lower compared to the 2 pg / mL formulation.

[0084] Macroscopic analysis of ulcerative wounds

[0085]

[0074] Figure 4 (FIG. 4) shows the evolution of wounds in the untreated, HC + AR 2 pg / mL, HC + AR 4 pg / mL and HC groups, from baseline (DO) to day 14 after the start of management and treatment, with assessments on days 2, 6, 10 and 14. In the untreated group, the average wound size decreased significantly compared to DO until the day 10 assessment (P < 0.05), subsequently stagnating (P > 0.05). The HC + AR 2 pg / mL and HC + AR 4 pg / mL groups showed continuous improvement, presenting a significant reduction (P < 0.05) in wound size from DO until day 14 (P < 0.05). Wound healing in the HC + AR 2 pg / mL and HC + AR 4 pg / mL groups was superior to that of the HC group, where there was a delay in repair, indicated by the absence of change in the wound area between days 6 and 10 (P > 0.05). On day 14, the percentage of residual wound in the HC + AR 2 pg / mL and HC + AR 4 pg / mL groups was significantly lower compared to the untreated or HC groups (P < 0.05).

[0086]

[0075] Biologically, the results regarding wound evolution show that HC + AR formulations (at concentrations of 2 pg / mL and 4 pg / mL) can promote more effective repair through improved re-epithelialization and granulation tissue formation. This process is crucial for rapid wound coverage and minimization of scar tissue, thus facilitating complete skin recovery.

[0076] Figure 5 (FIG. 5) shows the percentage of wound counter-reduction, i.e., the reduction in the ulcerated area, in the untreated, HC + AR 2 pg / mL, HC + AR 4 pg / mL and HC groups, evaluated at different intervals after the start of wound management or treatment (2, 6, 10 and 14 days). On days 2 and 6, no significant differences were observed in wound counteraction between the groups (P > 0.05). However, on day 10, the percentage of wound counteraction was significantly higher in the HC + AR 2 pg / mL, HC + AR 4 pg / mL and HC groups compared to the untreated group (P < 0.05).At this time, the percentages of wound counteraction were 71.23% for the HC + AR 2 pg / mL group, 73.95% for the HC + AR 4 pg / mL group, and 70.11% for the HC group, with no significant difference between them (P > 0.05). On day 14, the HC + AR 2 pg / mL and HC + AR 4 pg / mL groups showed a significant improvement (P < 0.05) in the percentage of wound counteraction, reaching values ​​of 90% and 92%, respectively, while wound counteraction in the HC group remained unchanged.

[0087]

[0077] Wound counteraction is a fundamental process in wound closure and occurs primarily through the counteraction of myofibroblastic cells around the wound. Analysis of wound counteraction shows that, over time, treatments with HC + AR (at concentrations of 2 pg / mL and 4 pg / mL) were more effective than treatment with HC alone or no treatment in promoting wound counteraction and, consequently, accelerating the repair process.

[0088] Histopathological analyses

[0089]

[0078] Figure 6 (FIG. 6) presents the results of the histopathological analyses of the untreated, HC + AR 2 pg / mL, HC + AR 4 pg / mL and HC groups, performed on days 6 and 14 after the start of wound management and treatment. It was observed that, at these times, the re-epithelialized areas were significantly larger (P < 0.05) in the HC + AR 2 pg / mL and HC + AR 4 pg / mL groups compared to the untreated group, but without significant differences compared to the HC group (P > 0.05). Furthermore, the collagen density in the dermis was significantly higher (P < 0.05) in the HC + AR 2 pg / mL and HC + AR 4 pg / mL groups compared to the other groups.

[0090] Analysis of reactive oxygen species levels

[0091]

[0079] Figure 7 (FIG. 7) shows the levels of reactive oxygen species, expressed as optical density of formazan crystals per milligram per milliliter of total protein, in the untreated, HC + AR 2 pg / mL, HC + AR 4 pg / mL and HC groups, on days 6 and 14 after the start of wound management and treatment. The results indicate that the rosmarfonic acid formulations, at concentrations of 2 pg / mL and 4 pg / mL, resulted in a significant reduction (P < 0.05) in the levels of reactive oxygen species at the wound sites compared to the controls; therefore, they had an antioxidant effect. No significant differences were observed in the levels of reactive oxygen species between the HC + AR 2 pg / mL and HC + AR 4 pg / mL groups (P > 0.05). Advantages of the invention

[0092]

[0080] The present invention has the following main advantages:

[0093]

[0081] To provide a described formulation of rosmarinic acid in bovine type I collagen hydrogel for topical application in the treatment of pressure ulcers, for the non-surgical treatment of pressure ulcers in human and veterinary patients;

[0094]

[0082] To provide a formulation of rosmarfonic acid in bovine-derived type I collagen hydrogel for topical application in the treatment of pressure ulcers, which incorporates an innovative method of solubilizing rosmarfonic acid in an acidic medium followed by neutralization by dialysis, ensuring its stability and functionality. This approach not only ensures the interaction between the bioactive and the collagen matrix, but also preserves its antioxidant properties, promoting effective skin repair and control of the wound environment;

[0083] To provide a formulation of rosmarfonic acid in bovine-derived type I collagen hydrogel for topical application in the treatment of pressure ulcers, which promotes effective repair of ulcerative wounds;

[0095]

[0084] To provide a formulation of rosmarinic acid in bovine type I collagen hydrogel for topical application in the treatment of pressure ulcers, which significantly reduces the levels of reactive oxygen species in wounds, controlling the wound environment and minimizing further damage to surrounding cells and tissues;

[0096]

[0085] To provide a formulation of rosmarinic acid in bovine type I collagen hydrogel for topical application in the treatment of pressure ulcers, made using bovine tendon-derived type I collagen as raw material, which offers high biocompatibility with human and animal skin, in addition to being a sustainable source from slaughterhouse waste, ensuring a favorable cost-benefit ratio and environmentally responsible practices.

Claims

CLAIMS 1) Process for incorporating rosmarfonic acid into a type I collagen solution characterized by comprising (i) dissolving 2 pg / mL to 4 pg / mL of rosmarfonic acid in 1 g of bovine type I collagen gel diluted in 1 mL of 0.5 molar aqueous acetic acid solution, followed by (ii) centrifuging the mixture at 400 revolutions per minute for 10 min and (iii) dialysis in dialysis bags with a molecular weight cutoff between 12000 and 14000 against distilled water for 24 h, with water change every 12 h. 2) Formulation obtained through the process of claim 1, characterized by comprising rosmarfonic acid incorporated into a type 2 collagen hydrogel.

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