Anti-inflammatory composition for companion animals comprising epsilon-viniferin as active ingredient and uses thereof

Abstract

The present invention relates to an anti-inflammatory composition for companion animals comprising epsilon-viniferin as an active ingredient, and uses thereof. The anti-inflammatory composition comprising epsilon-viniferin as an active ingredient according to an embodiment of the present invention exhibits effects of suppressing inflammatory response pathways in canine macrophages and inhibiting expression of genes and secretion of proteins involved in inflammatory responses and thus can be advantageously applied to the development of cosmetic composition formulations for suppressing inflammation of companion animals.

Description

Anti-inflammatory composition for companion animals containing epsilon-viniferin as an active ingredient and uses thereof

[0001] The present invention relates to an anti-inflammatory composition for companion animals comprising epsilon-viniferin as an active ingredient and the use thereof.

[0002] The pet market is growing rapidly worldwide, and demand for medical and welfare services is on the rise, particularly as a culture of considering pets as family members spreads. Along with the expansion of the pet market, the incidence of inflammatory diseases is increasing due to the aging of pets and changes in their living environments, with chronic inflammatory conditions such as arthritis, dermatitis, and digestive inflammation emerging as major issues.

[0003] The inflammatory response is the body's defense mechanism against cell damage or infection, a process that heals tissue damage and protects the body from external pathogens. Inflammatory responses include fever, redness, swelling, and pain, and are achieved through the complex interaction of various factors at the cellular and molecular levels. The immune system activates the phagocytosis of foreign factors and promotes the chemotaxis of immune cells, which amplifies the inflammatory response, through the activation of pattern recognition receptors (PRRs) capable of recognizing pathogen-associated molecular patterns (PAMPs). This activation leads to the activation of various inflammatory signaling networks, including nuclear factor-κB (NFκB) and mitogen-activated protein kinase (MAPK). Key factors involved in the inflammatory response include cytokines, chemokines, prostaglandins, and histamine, which induce immune cells to the damaged site to aid in cell repair and contribute to the elimination of pathogens.

[0004] Currently, the primary methods used to treat inflammatory diseases in companion animals are steroids and non-steroidal anti-inflammatory drugs (NSAIDs). While steroids offer powerful anti-inflammatory effects, long-term use can lead to side effects such as immunosuppression and impaired liver and kidney function. NSAIDs are relatively safe anti-inflammatory agents, but they can cause gastrointestinal side effects, including gastrointestinal disorders and bleeding. Furthermore, these drugs focus on symptom relief and often provide only temporary effects rather than long-term inflammation suppression. Due to the limitations of these existing treatments, there is a need for new alternatives that can be used long-term with minimal side effects. Additionally, given that side effects can be more pronounced in companion animals due to physiological differences from humans, there is a pressing need for the development of safe anti-inflammatory treatments tailored specifically for pets.

[0005] In order to solve the above-mentioned problems, the inventors completed the present invention by treating dog macrophages with epsilon-viniferin (ε-viniferin) and confirming the inhibitory effect of epsilon-viniferin on the inflammatory response pathway and the inhibitory effect on gene expression and protein secretion involved in the inflammatory response in dog macrophages.

[0006] Therefore, the objective of the present invention is to provide an anti-inflammatory composition for companion animals comprising epsilon-viniferin (ε-viniferin) as an active ingredient.

[0007] Another objective of the present invention is to provide a cosmetic composition for anti-inflammatory use in pets comprising epsilon-viniferin (ε-viniferin) as an active ingredient.

[0008] The terms used in this specification are for descriptive purposes only and should not be interpreted as being limiting. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this specification, terms such as "comprising" or "having" are intended to indicate the existence of the features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, and should be understood as not precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof.

[0009] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by those skilled in the art to which the embodiments pertain. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and should not be interpreted in an ideal or overly formal sense unless explicitly defined in this application.

[0010] The present invention will be described in detail below.

[0011] To achieve the above objective, one aspect of the present invention provides an anti-inflammatory composition for companion animals comprising epsilon-viniferin (ε-viniferin) as an active ingredient.

[0012] The term "viniferin" used in this invention refers to a natural compound of the stilbenoid family extracted from the grapevine (Vitis vinifera), which is mainly found in grape stems or roots. Viniferin is in the form of an oligomer combined with the monomer resveratrol, and there are various types such as alpha-viniferin (α-viniferin), beta-viniferin (β-viniferin), delta-viniferin (δ-viniferin), and epsilon-viniferin (ε-viniferin). In one embodiment of the present invention, for epsilon-viniferin (ε-viniferin) (IUPAC: 5-{(2R,3R)-6-Hydroxy-2-(4-hydroxyphenyl)-4-[(E)-2-(4-hydroxyphenyl)ethen-1-yl]-2,3-dihydro-1-benzofuran-3-yl}benzene-1,3-diol) represented by the following chemical formula, the inhibitory effect on the inflammatory response pathway and the inhibitory effect on gene expression and protein secretion involved in the inflammatory response were confirmed in canine macrophages.

[0013] [Chemical Formula]

[0014]

[0015]

[0016] The term "companion animal" as used in the present invention refers to an animal that lives as a companion to a human. In the present invention, a companion animal may be one or more selected from the group consisting of canids, felines, rodents, rabbits, and birds, and preferably may be a canine, but is not limited thereto.

[0017] The term "anti-inflammatory" in this invention refers to the action of suppressing inflammation. It is known that the regulation of inflammatory responses is highly complex, and this is intended to enhance the body's repair system and reduce damage. However, if the inflammatory response persists due to repeated tissue damage or regeneration, ROS and RNS are overproduced in inflammation-related cells, resulting in permanent genetic mutations. As such, ROS and RNS are deeply involved in inflammatory responses that regulate the functions of various cells in the body. During the inflammatory process, large amounts of pro-inflammatory cytokines, nitric oxide (NO), and prostaglandin E2 (PGE2) are produced by inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Inflammation is a cause of various inflammatory diseases, and the composition containing epsilon-viniferin of the present invention as an active ingredient can have preventive and improving effects on various inflammatory diseases through anti-inflammatory action.

[0018] The term "anti-inflammatory composition" as used in the present invention refers to a composition that acts to eliminate inflammation by removing inflammatory agents and reducing biological responses and symptoms.

[0019]

[0020] In addition, another aspect of the present invention provides a cosmetic composition for anti-inflammatory use in pets comprising epsilon-viniferin (ε-viniferin) as an active ingredient.

[0021] The anti-inflammatory cosmetic composition of the present invention comprising epsilon-viniferin as an active ingredient may include a cosmetically acceptable carrier in addition to the epsilon-viniferin. Examples include, but are not limited to, fatty substances, organic solvents, solvents, thickeners, gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, emulsifiers, fillers, metal ion chelating agents, chelating agents, preservatives, vitamins, blockers, humectants, oils, dyes, pigments, hydrophilic or lipophilic activators, lipid vesicles, etc., and may include all known substances applicable to cosmetic compositions.

[0022] In addition, the anti-inflammatory cosmetic composition comprising epsilon-viniferin of the present invention as an active ingredient may be formed into one or more formulations selected from the group consisting of softening lotion, nourishing lotion, astringent lotion, skin toner, lotion, essence, cream, massage cream, pack, makeup base, BB cream, foundation, powder, cleansing foam, cleansing cream, and cleansing water, but is not limited thereto, and may be formed into any known formulation.

[0023] Meanwhile, when an anti-inflammatory cosmetic composition containing epsilon-viniferin of the present embodiment having the above-described formulation is formed in a paste, cream, or gel state, it may further include animal oil, vegetable oil, wax, paraffin, starch, tragacanth gum, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc.

[0024] When the anti-inflammatory cosmetic composition containing epsilon-viniferin of the present invention as an active ingredient is formed in the form of a solution or an emulsion, it may further include a solvent, a solubilizing agent, an emulsifying agent, etc. The solvent, solubilizing agent, and emulsifying agent may include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, fatty acid ester of sorbitan, etc.

[0025] When the anti-inflammatory cosmetic composition containing epsilon-viniferin of the present invention as an active ingredient is formed in the form of a suspension, it may further include a liquid diluent such as water, ethanol, and propylene glycol; a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester; microcrystalline cellulose, aluminum metahydroxyoxide, bentonite, agar, tragacanth gum, etc.

[0026] When the anti-inflammatory cosmetic composition containing epsilon-viniferin of the present invention as an active ingredient is a cleansing foam, cleansing cream, or cleansing water, it may further include an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, isethionate, an imidazolinium derivative, methyl taurate, a sarcosinate-based compound, a fatty acid amide ether sulfate, an alkylamidobetaine, an aliphatic alcohol, a fatty acid glyceride, a fatty acid diethanolamide, a vegetable oil, a lanolin derivative, an ethoxylated glycerol fatty acid ester, etc.

[0027] An anti-inflammatory composition containing epsilon-viniferin as an active ingredient according to one embodiment of the present invention has an effect of inhibiting inflammatory response pathways, an effect of inhibiting pro-inflammatory gene expression and protein secretion, and an effect of anti-inflammatory gene expression and protein activation in canine macrophages, and can be usefully utilized in the development of cosmetic composition formulations for suppressing inflammation in companion animals.

[0028] The effects of the present invention are not limited to those described above, and unmentioned effects will be clearly understood by those skilled in the art from the present specification and the accompanying drawings.

[0029] Figure 1 is a figure showing the cytotoxicity of epsilon-viniferin (ε-viniferin) at different concentrations according to one embodiment of the present invention using an MTT assay.

[0030] Figure 2 is a figure showing the inhibitory effect of inflammation-related signaling factors according to the concentration of epsilon-viniferin according to one embodiment of the present invention using Western blot analysis.

[0031] Figure 3 is a figure showing the inhibitory efficacy of epsilon-viniferin on inflammation-related cytokines according to concentrations according to one embodiment of the present invention using qRT-PCR.

[0032] Figure 4 is a figure showing the inhibitory effect of epsilon-viniferin on the expression of inflammation-related proteins according to an embodiment of the present invention using ELISA.

[0033] The present invention will be explained in more detail below through examples. The following examples are intended solely to explain the present invention more specifically, and it will be obvious to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention.

[0034]

[0035] Example 1. Cytotoxicity Evaluation

[0036] 1-1. Experiment Preparation

[0037] Cell viability was evaluated using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT). After treating DH82 canine macrophages with epsilon-viniferin at different concentrations (0.2 to 4 μM), MTT solution (1 mg / mL) was added to each well, and the plates were incubated at 37°C for 1 hour. Subsequently, the optical density of each well was measured at 540 nm using a microplate reader.

[0038]

[0039] 1-2. Confirmation of Cytotoxicity of Epsilon Viniferin at Different Concentrations

[0040] To confirm the cytotoxicity of epsilon-viniferin, DH82 cells were treated with epsilon-viniferin at a concentration of 0.2 to 4 μM for 24 hours as in Example 1-1, and cell viability was evaluated using an MTT assay, and the results are shown in Figure 1.

[0041] As shown in Figure 1, when epsilon-viniferin was treated to a concentration of 0.2 to 1 μM, no cytotoxicity was observed in DH82 cells; however, when treated to a concentration of 2 μM or higher, it was confirmed that cell viability decreased significantly. Therefore, subsequent experiments were conducted using epsilon-viniferin at a concentration of 0.2 to 1 μM.

[0042]

[0043] Example 2. Evaluation of efficacy in inhibiting inflammation-related signaling pathways

[0044] 2-1. Experiment Preparation

[0045] DH82 cells were treated with epsilon-viniferin at a concentration of 0.2 to 1 μM for 24 hours, then the supernatant was removed and washed with cold DPBS. Subsequently, cells were lysed by treating them with RIPA lysis buffer (ATTO, Tokyo, Japan), and total protein was extracted. 30 μg of protein was loaded onto 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to separate proteins by size, and then transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was then blocked with 5% skim milk (BD, Difco, Franklin Lakes, NJ, USA) at room temperature for 1 hour, followed by overnight incubation with the primary antibody at 4°C at specific concentrations. The overnight-incubated membrane was then incubated with the peroxide-conjugate secondary antibody for 1 hour at room temperature. Protein bands on the membrane were visualized using chemiluminescence solution (Thermo Fisher Scientific, Waltham, MA, USA) and Azure Biosystems instruments (Azure Detection was performed using Biosystems (Dublin, CA, USA). Quantitative analysis of specific protein bands was performed using ImageJ software version 1.8 (National Institutes of Health, Bethesda, MD, USA), and GAPDH was used as an internal loading control.

[0046]

[0047] 2-2. Confirmation of the Inhibitory Efficacy of Epsilon-Viniferin on Inflammation-Related Signaling Pathways

[0048] To confirm the inhibitory effect of epsilon-viniferin on inflammation-related signaling pathways, DH82 cells were treated with epsilon-viniferin at a concentration of 0.2 to 1 μM for 24 hours as in Example 2-1, and the expression of inflammation-related signaling factors was evaluated using Western blot analysis, and the results are shown in Figure 2.

[0049] As shown in Figure 2, when treated with Lipopolysaccharide (LPS), it was confirmed that the expression ratio of inflammation-related signaling factors p-ERK, p-JNK, p-IkB, and p-NFkB relative to GAPDH increased compared to the untreated control group. However, when treated with epsilon-viniferin at 0.2 to 1 μM, it was confirmed that the expression ratio of p-ERK, p-JNK, p-IkB, and p-NFkB relative to GAPDH decreased in a concentration-dependent manner.

[0050]

[0051] Example 3. Evaluation of Inhibitory Efficacy of Inflammation-Related Cytokines

[0052] 3-1. Experiment Preparation

[0053] qRT-PCR was performed to evaluate the expression levels of inflammation-related cytokine genes. DH82 cells were treated with epsilon-viniferin at concentrations of 0.2 to 1 μM for 24 hours, after which total RNA was extracted from DH82 cells using the RNeasy extraction kit (QIAGEN) according to the manufacturer's instructions. cDNA synthesis from the extracted total RNA was performed using the iScript™ cDNA synthesis kit. Real-time PCR was performed using the CFX Connect™ Real-Time System (Bio-Rad Laboratories) with iScript™ Green Supermix, cDNA, and custom-designed primers. Data analysis was performed using CFX Maester™ Analysis Software 3.1 (Bio-Rad Laboratories).

[0054]

[0055] 3-2. Confirmation of Inhibitory Effilon-Viniferin’s Inflammatory Cytokines

[0056] To confirm the inhibitory effect of epsilon-viniferin on inflammation-related factors, DH82 cells were treated with epsilon-viniferin at a concentration of 0.2 to 1 μM for 24 hours as in Example 3-1, and the expression level of inflammation-related cytokine genes was evaluated using qRT-PCR, and the results are shown in Figure 3.

[0057] As shown in Figure 3, when LPS was treated, it was confirmed that compared to the untreated control group, the expression ratio of pro-inflammatory cytokine (TNF-α, IL-6) mRNA increased due to the influence of the inflammatory response, and the expression ratio of anti-inflammatory cytokine (IL-10, TGF-β1) mRNA increased due to the influence of the immune response activation and balance regulation mechanism.

[0058] However, when epsilon-viniferin was treated at 0.2 to 1 μM, it was confirmed that the expression of pro-inflammatory cytokine (TNF-α, IL-6) mRNA was reduced compared to when LPS was treated.

[0059]

[0060] Example 4. Evaluation of inflammation-related protein secretion

[0061] 4-1. Experiment Preparation

[0062]

[0063] The levels of secreted TNF-α and IL-10 were measured using an ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA). DH82 cells were cultured at 37°C for 24 hours. Then, the cells were treated with epsilon-viniferin at concentrations of 0.2 to 1 μM for 24 hours, followed by the use of LPS (0.1 μg / mL) for 6 hours to induce an inflammatory response. After culture, the culture supernatant was collected and centrifuged at 1,000 × g at 4°C for 10 minutes to remove debris before measuring cytokine levels.

[0064]

[0065] 4-2. Confirmation of the Inhibitory Efficacy of Epsilon-Viniferin on the Secretion of Inflammation-Related Proteins

[0066] To confirm the inhibitory effect of epsilon-viniferin on the secretion of inflammation-related proteins, DH82 cells were treated with epsilon-viniferin at a concentration of 0.2 to 1 μM for 24 hours as in Example 4-1, and the degree of secretion of inflammation-related cytokine proteins was evaluated using ELISA, and the results are shown in Figure 4.

[0067] As shown in Figure 4, when LPS was treated, it was confirmed that the secretion of pro-inflammatory cytokines (TNF-α, IL-6) increased due to the influence of the inflammatory response, and the secretion of anti-inflammatory cytokines (IL-10) increased due to the influence of the immune response activation and balance regulation mechanisms compared to the untreated control group.

[0068] However, when epsilon-viniferin was treated at 0.2 to 1 μM, it was confirmed that the secretion of pro-inflammatory cytokine (TNF-α, IL-6) proteins decreased compared to when LPS was treated.

[0069] An anti-inflammatory composition containing epsilon-viniferin as an active ingredient according to one embodiment of the present invention has an effect of inhibiting inflammatory response pathways in canine macrophages, an effect of inhibiting pro-inflammatory gene expression and protein secretion, and an effect of anti-inflammatory gene expression and protein activation, so it can be usefully utilized in the development of cosmetic composition formulations to suppress inflammation in companion animals, and thus has industrial applicability.

Claims

1. An anti-inflammatory composition for pets comprising epsilon-viniferin (ε-viniferin) represented by the following chemical formula as an active ingredient. [Chemical Formula] 2. In Paragraph 1, An anti-inflammatory composition characterized in that the concentration of epsilon-viniferin in the above composition is 0.2 μM to 1 μM.

3. In Paragraph 1, An anti-inflammatory composition in which the above-mentioned companion animal is one or more selected from the group consisting of canids, felines, rodents, rabbits, and birds.

4. An anti-inflammatory composition according to paragraph 3, wherein the companion animal is a canine.

5. A cosmetic composition for anti-inflammatory use in pets comprising epsilon-viniferin (ε-viniferin) represented by the following chemical formula as an active ingredient. [Chemical Formula] 6. In Paragraph 5, An anti-inflammatory cosmetic composition characterized in that the formulation of the above cosmetic composition is any one selected from the group consisting of softening lotion, nourishing lotion, astringent lotion, skin toner, lotion, essence, serum, cream, massage cream, pack, makeup base, BB cream, foundation, powder, cleansing foam, cleansing cream, and cleansing water.

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