Dermatologic composition for external application to prevent or treat diabetic foot ulcers comprising pinitol as active ingredient

Abstract

The present invention relates to a dermatologic composition for external application to prevent or treat diabetic foot ulcers, comprising pinitol as an active ingredient. By confirming the effects of pinitol on: wound healing of diabetic foot ulcers; promoting collagen synthesis in diabetic foot ulcers; anti-inflammation and antioxidation in fibroblasts; restoration of mitochondrial membrane potential in fibroblasts; enhancing ATP production; increasing AMPK expression in fibroblasts; promoting angiogenesis in diabetic foot ulcers; promoting angiogenesis; reducing the expression of inflammatory factors in vascular endothelial cells; wound healing; inhibiting reactive oxygen species; mitochondrial membrane potential restoration; and reducing MAPK phosphorylation, a dermatologic composition for external application to ameliorate diabetic foot ulcers, comprising pinitol as an active ingredient, of the present invention, can be prepared.

Description

Composition for external skin application for prevention or treatment of diabetic foot ulcers containing pinitol as an active ingredient

[0001] The purpose of the present invention is to provide a composition for external application of skin for the prevention or treatment of diabetic foot ulcers, which contains pinitol as an active ingredient.

[0002] Diabetic foot disease, also known as "diabetic foot," refers to any problem that occurs in the feet of people with diabetes. The most common problem that can occur in diabetic patients is foot ulcers, which develop when the skin or mucous membranes of the feet become worn away. Diabetic foot disease can be narrowly defined as foot ulcers, as they are the most common lesion of diabetic foot disease.

[0003] Diabetes causes blood vessels to gradually narrow or become blocked. When blood vessels to the lower extremities narrow or become blocked, the feet cannot receive adequate blood flow. This disrupts the supply of nutrients and oxygen, and hinders the movement and excretion of waste products, making ulcers more likely to form. Furthermore, diabetic neuropathy can cause numbness in the feet, making them more susceptible to injury and ulcers. Furthermore, nerve abnormalities can cause foot deformities, and changes in weight-bearing during walking can increase pressure on certain parts of the foot, all of which contribute to the development of ulcers. Furthermore, diabetes itself delays wound healing. Therefore, traumatic lesions on the feet can slow their healing process, making them prone to ulcers and delaying healing.

[0004] Diabetic foot disease accounts for approximately 47% of all foot diseases, and approximately 15% of diabetic patients will experience a foot ulcer at least once in their lifetime, and approximately 1-3% of these patients will undergo surgery to amputate part of their leg. Recurrence of diabetic foot ulcers is common once a patient has suffered one, with approximately 30% of patients experiencing a recurrence within one year, and more than half of those who have undergone surgery for a diabetic foot ulcer will undergo surgery on the other side within four years. In particular, diabetic foot disease is said to account for approximately 40% of hospitalizations in diabetic patients. Furthermore, diabetic foot ulcers are known to be the cause of more than half of leg amputations, excluding cases due to trauma. Methods for treating diabetic foot ulcers include local treatment (surgical removal of necrotic tissue (debridement), dressing, negative pressure occlusion therapy), treatment of blood flow disorders (drug therapy such as vasodilators, endovascular treatment, blood flow reconstruction surgery such as arterial bypass surgery), treatment of inflammation (administration of antibiotics), and systemic treatment (improvement of edema through blood sugar control). However, as the disease progresses, the quality of life is significantly reduced, so early detection or prevention is important.

[0005] Furthermore, diabetic wounds are caused by hyperglycemia, and when angiogenesis occurs, dermal endothelial cells are severely damaged, leading to peripheral ischemia and difficulties in wound healing. The wound healing process is complex and consists of four phases: inflammation, proliferative phase, re-epithelialization, and maturation phase. Among these, the granulation phase is an angiogenic process in which vascular cells form new blood vessels from existing ones. Once new blood vessels are formed, the cells enhance the activity of existing blood vessels, improving blood circulation and ensuring that oxygen and nutrients are effectively supplied to the destination.

[0006] Therefore, angiogenesis and blood flow improvement are complementary concepts, and their functions are essential for wound healing and restoration to normal tissue.

[0007] Previous studies have shown that human dermal microvascular endothelial cells (HDMECs) derived from diabetic foot ulcers (DFUs) exhibit impaired angiogenesis, consistent with changes observed in high-glucose-stimulated HDMECs. In vitro bulk-seq and scRNA-seq analyses of HDMECs isolated from DFU patient foot skin samples confirmed that these cells exhibited significantly impaired angiogenic capacity. Therefore, diabetic wound healing is closely linked to the proliferation and apoptosis of vascular endothelial cells. Therefore, providing a microenvironment that promotes cell migration, proliferation, and prevention of apoptosis may be a therapeutic strategy for repairing wounds derived from diabetic foot ulcers. Furthermore, this approach may also be applicable to renal abnormalities, such as renal failure leading to dialysis, and ocular diseases, such as diabetic retinopathy, which can lead to blindness, caused by microvascular problems in chronic diabetes.

[0008] Meanwhile, pinitol (D-Pinitol, 3-O-methyl-D-chiro-inositol) is a natural blood sugar regulating component found in beans and pine needles. It is a substance in which a methyl group is attached to the 3rd carbon of chiro-inositol, a structural isomer of myo-inositol, by an ether bond. Pinitol exists in various plants including legumes in nature, and in particular, it has been reported to exist in large quantities in soybeans, which are in the spotlight as folk remedies, as well as black-eyed beans, Acanthopanax senticosus, silkworm powder, and the Korean walnut tree. Since pinitol has been revealed to be an effective component in various plants used as folk remedies for diabetics around the world, we sought to confirm its correlation with diabetic wound healing.

[0009] Accordingly, the inventor of the present invention confirmed the diabetic foot ulcer wound healing effect, collagen synthesis promotion effect, and anti-inflammatory activity of pinitol, and confirmed the angiogenesis promotion effect in vascular endothelial cells, wound healing effect under hyperglycemic conditions, and inflammatory factor expression reduction effect, thereby completing the present invention.

[0010] The purpose of the present invention is to provide a composition for external application of skin for the prevention or treatment of diabetic foot ulcers, which contains pinitol (D-Pinitol, 3-O-methyl-D-chiro-inositol) as an active ingredient.

[0011] In addition, the present invention aims to provide a pharmaceutical composition for preventing or treating diabetic foot ulcers, which comprises pinitol as an active ingredient.

[0012] In addition, the present invention aims to provide an external composition for wound treatment in diabetic foot ulcers, which contains pinitol as an active ingredient.

[0013] In addition, the present invention aims to provide a composition for promoting collagen synthesis for diabetic foot ulcers, which contains pinitol as an active ingredient.

[0014] In addition, the present invention aims to provide an anti-inflammatory composition for diabetic foot ulcers containing pinitol as an active ingredient.

[0015] In addition, the present invention aims to provide an antioxidant composition for diabetic foot ulcers containing pinitol as an active ingredient.

[0016] In addition, the present invention aims to provide a composition for enhancing ATP production for diabetic foot ulcers, which contains pinitol as an active ingredient.

[0017] In addition, the present invention aims to provide a composition for promoting angiogenesis containing pinitol as an active ingredient.

[0018] The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating angiogenesis-dependent diseases, which comprises pinitol as an active ingredient.

[0019] In addition, the present invention aims to provide a pharmaceutical composition for preventing or improving angiogenesis-dependent diseases, which comprises pinitol as an active ingredient.

[0020] In addition, the present invention aims to provide a composition for wound healing of vascular endothelial cells, which contains pinitol as an active ingredient.

[0021] In addition, the present invention aims to provide a method for preventing or treating diabetic foot ulcers, which comprises a step of administering pinitol to a subject.

[0022] In addition, the present invention aims to provide a method for preventing or treating angiogenesis-dependent disease, which comprises a step of administering pinitol to a subject.

[0023] To achieve the above purpose, the present invention provides a composition for external application of skin for preventing or treating diabetic foot ulcers, which contains pinitol (D-Pinitol, 3-O-methyl-D-chiro-inositol) as an active ingredient.

[0024] In addition, the present invention provides a pharmaceutical composition for preventing or treating diabetic foot ulcers, which comprises pinitol as an active ingredient.

[0025] In addition, the present invention provides an external composition for wound treatment in diabetic foot ulcers, which contains pinitol as an active ingredient.

[0026] In addition, the present invention provides a composition for promoting collagen synthesis for diabetic foot ulcers, which contains pinitol as an active ingredient.

[0027] In addition, the present invention provides an anti-inflammatory composition for diabetic foot ulcers, which comprises pinitol as an active ingredient.

[0028] In addition, the present invention provides an antioxidant composition for diabetic foot ulcers, which contains pinitol as an active ingredient.

[0029] In addition, the present invention provides a composition for enhancing ATP production for diabetic foot ulcers, which contains pinitol as an active ingredient.

[0030] In addition, the present invention provides a composition for promoting angiogenesis, which contains pinitol as an active ingredient.

[0031] The present invention provides a pharmaceutical composition for preventing or treating angiogenesis-dependent diseases, comprising pinitol as an active ingredient.

[0032] In addition, the present invention provides a pharmaceutical composition for preventing or improving angiogenesis-dependent diseases, which comprises pinitol as an active ingredient.

[0033] In addition, the present invention provides a composition for wound healing of vascular endothelial cells, which contains pinitol as an active ingredient.

[0034] Additionally, the present invention provides a method for preventing or treating diabetic foot ulcers, comprising administering pinitol to a subject.

[0035] Additionally, the present invention provides a method for preventing or treating angiogenesis-dependent disease, comprising administering pinitol to a subject.

[0036] The present invention relates to a composition for external application of skin for preventing or treating diabetic foot ulcers, which contains pinitol as an active ingredient, and has confirmed the following effects: wound healing effect of pinitol for diabetic foot ulcers, collagen synthesis promotion effect in diabetic foot ulcers, anti-inflammation and antioxidant effect in fibroblasts, membrane potential restoration of mitochondria in fibroblasts, ATP production promotion effect, AMPK expression promotion effect in fibroblasts, angiogenesis promotion effect in diabetic foot ulcers, angiogenesis promotion effect of pinitol, reduction of inflammatory factor expression in vascular endothelial cells, wound healing, inhibition of reactive oxygen species, restoration of mitochondrial membrane potential, and MAPK phosphorylation reduction effect. Therefore, the present invention can produce an external application for skin for improving diabetic foot ulcers, which contains pinitol, and can be usefully used in businesses related thereto.

[0037] Figure 1 is a diagram showing changes in the size of foot wounds in streptozotocin (STZ)-induced diabetic rats.

[0038] Figures 2 and 3 are diagrams showing the increase and decrease in collagen expression in the feet of streptozotocin (STZ)-induced diabetic rats.

[0039] Figure 4 is a diagram showing the antigen-antibody reaction and DAPI staining results of NF-κB, an inflammatory biomarker, in LPS-induced infected fibroblasts.

[0040] Figure 5 is a diagram showing the movement of NF-κB into the nucleus in LPS-induced infected fibroblasts.

[0041] Figure 6 is a diagram showing the results of evaluating the cytotoxicity of LPS, pinitol, and metformin in fibroblasts.

[0042] Figures 7 and 8 are diagrams showing the results of evaluating the wound healing efficacy according to pinitol and metformin treatment in LPS-induced infected fibroblasts.

[0043] Figures 9 and 10 are diagrams showing the results of evaluating the inhibition effect of reactive oxygen species (ROS) according to pinitol and metformin treatment in LPS-induced infected fibroblasts.

[0044] Figures 11 and 12 are drawings showing the expression of p-Nrf2 observed in LPS-induced infected fibroblasts using a fluorescence microscope.

[0045] Figures 13 to 16 are diagrams showing the results of evaluating the expression level of antioxidant proteins (Nrf2, HO-1, Keap1) according to pinitol and metformin treatment in LPS-induced infected fibroblasts.

[0046] Figures 17 to 19 are diagrams showing the results of evaluating the expression level of antioxidant proteins (SOD1, SOD2, Catalase) according to pinitol and metformin treatment in LPS-induced infected fibroblasts.

[0047] Figures 20 to 22 are diagrams showing the results of evaluating the expression level of inflammatory proteins (NF-κB, IκBα) according to pinitol and metformin treatment in LPS-induced infected fibroblasts.

[0048] Figures 23 to 25 are diagrams showing the results of evaluating the expression level of inflammatory proteins (IL-6, IL-8, IL-1β) according to pinitol and metformin treatment in LPS-induced infected fibroblasts.

[0049] Figures 26 and 27 are diagrams showing the results of evaluating the efficacy of mitochondrial membrane potential recovery according to pinitol and metformin treatment in LPS-induced infected fibroblasts.

[0050] Figures 28 and 29 are diagrams showing the results of evaluating the level of ATP expression according to pinitol and metformin treatment in LPS-induced infected fibroblasts.

[0051] Figures 30 and 31 are diagrams showing the results of evaluating the level of AMPK expression according to pinitol and metformin treatment in LPS-induced infected fibroblasts.

[0052] Figure 32 is a diagram showing the results of measuring blood sugar and weight in streptozotocin (STZ)-induced diabetic rats.

[0053] Figures 33 to 36 are photographs of streptozotocin (STZ)-induced diabetic rat paw tissues showing the results of evaluating the expression levels of collagen, Ki67, and TUNEL.

[0054] Figure 37 is a diagram showing the results of evaluating the expression levels of MDA and GSH in streptozotocin (STZ)-induced diabetic rat paw tissue.

[0055] Figures 38 and 39 are photographs of streptozotocin (STZ)-induced diabetic rat paw tissues, showing blood vessel formation through CD31.

[0056] Figures 40 and 41 are diagrams showing the results of evaluating the expression level of proteins (VEGFA, HIF-1α, CXCL12, CXCR4) involved in angiogenesis in streptozotocin (STZ)-induced diabetic rat paw tissue.

[0057] Figure 42 is a diagram showing the angiogenesis-promoting effect of pinitol confirmed by applying Matrigel to vascular endothelial cells.

[0058] Figures 43 and 44 are diagrams showing the effect of pinitol on reducing the expression of NF-κB, a key biomarker related to inflammation.

[0059] Figure 45 is a diagram showing the wound healing effect of pinitol, confirmed through the establishment of a hyperglycemic in vitro model in vascular endothelial cells.

[0060] Figures 46 and 47 are diagrams showing the results of evaluating the inhibitory effect of reactive oxygen species (ROS) on LPS-induced infected vascular endothelial cells by pinitol and metformin treatment.

[0061] Figures 48 and 49 are diagrams showing the results of evaluating the efficacy of mitochondrial membrane potential recovery according to pinitol and metformin treatment in LPS-induced infected vascular endothelial cells.

[0062] Figures 50 to 53 are diagrams showing the results of evaluating the inhibitory effect of pinitol on the expression of MAPKs, ERK, JNK, and p38 proteins in vascular endothelial cells.

[0063] Hereinafter, the present invention will be described in detail with reference to the attached drawings, using exemplary embodiments of the present invention. However, the following exemplary embodiments are provided as illustrative examples of the present invention. If a detailed description of a technology or configuration well known to those skilled in the art is judged to unnecessarily obscure the gist of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the following claims and equivalents interpreted therefrom.

[0064] In addition, the terminology used in this specification is a term used to appropriately express preferred embodiments of the present invention, and this may vary depending on the intention of the user or operator, or the customs of the field to which the present invention belongs. Therefore, the definition of these terms should be determined based on the contents throughout this specification. Throughout the specification, when a part is said to "include" a certain component, this does not mean that other components are excluded, but rather that other components may be included, unless specifically stated otherwise.

[0065] The present invention provides a composition for external application of skin for the prevention or treatment of diabetic foot ulcers, comprising pinitol (D-Pinitol, 3-O-methyl-D-chiro-inositol) as an active ingredient.

[0066] In the present invention, pinitol (D-Pinitol, 3-O-methyl-D-chiro-inositol) is represented by the following chemical formula 1, and may include a pharmaceutically acceptable salt of pinitol.

[0067] [Chemical Formula 1]

[0068]

[0069] The term pharmaceutically acceptable salt means any organic or inorganic addition salt of the basic compound of formula 1 at a concentration that is relatively non-toxic and harmless to the patient and has an effective effect, and the side effects caused by this salt do not reduce the beneficial efficacy of the basic compound of formula 1. These salts may use inorganic acids and organic acids as free acids, and inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, perchloric acid, phosphoric acid, etc. may be used, and organic acids such as citric acid, acetic acid, lactic acid, maleic acid, fumaric acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, tartaric acid, galacturonic acid, embonic acid, glutamic acid, aspartic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, ethanesulfonic acid, 4-toluenesulfonic acid, salicylic acid, citric acid, benzoic acid, or malonic acid may be used. Additionally, these salts include alkali metal salts (sodium salts, potassium salts, etc.) and alkaline earth metal salts (calcium salts, magnesium salts, etc.). For example, acid addition salts include acetate, aspartate, benzate, besylate, bicarbonate / carbonate, bisulfate / sulfate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulfate, naphthylate, 2-naphthylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogen phosphate / dihydrogen phosphate, saccharate, stearate, succinate, May include, but are not limited to, tartrate, tosylate, trifluoroacetate, aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, zinc salts, etc.

[0070] The term "prevention" as used herein refers to any action that reduces the frequency or severity of a pathological phenomenon. Prevention may be complete or partial. In this case, it may refer to a reduction in the symptoms of diabetic foot ulcers in a subject compared to when the composition was not used.

[0071] The term "treatment" above refers to any clinical intervention intended to alter the natural processes of the target or cells to be treated, and may be performed during or to prevent the progression of a clinical pathological condition. The desired therapeutic effect may include preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the progression of the disease, alleviating or temporarily alleviating the disease state, or improving the prognosis.

[0072] In one embodiment of the present invention, the pinitol may be included in a proportion of 0.001 to 1.0 wt% relative to the total weight of the composition, preferably in a proportion of 0.001 to 0.75 wt%, but is not limited thereto.

[0073] In one embodiment of the present invention, the composition comprises an extract selected from the group consisting of a carob bean extract, a soybean extract, a black bean extract, a pine needle extract, a schisandra berry extract, and a walnut extract, and the extract may be characterized in that it contains pinitol, but is not limited thereto.

[0074] The extract according to the present invention can be obtained by extracting and separating from nature using extraction and separation methods known in the art, and the “extract” defined in the present invention is extracted using an appropriate solvent, and includes, for example, a crude extract, a polar solvent-soluble extract, or a non-polar solvent-soluble extract. Any pharmaceutically acceptable organic solvent may be used as a suitable solvent for extracting the above extract, and water or an organic solvent may be used, and is not limited thereto, for example, purified water, alcohols having 1 to 4 carbon atoms including methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane, and cyclohexane, and various solvents may be used alone or in combination. As an extraction method, any one of hot water extraction, cold immersion extraction, reflux cooling extraction, solvent extraction, steam distillation, ultrasonic extraction, dissolution, and pressing may be selected and used. Additionally, the desired extract may be subjected to additional conventional fractionation processes and purified using conventional purification methods.

[0075] There is no limitation on the method for preparing the extract of the present invention, and any known method can be used. For example, the extract included in the composition of the present invention can be prepared in a powder form by additional processes such as reduced pressure distillation and freeze drying or spray drying of the primary extract extracted by the above-mentioned hot water extraction or solvent extraction method. In addition, the primary extract can be further purified to obtain a fraction using various chromatography methods such as silica gel column chromatography, thin layer chromatography, high performance liquid chromatography, etc. Therefore, in the present invention, the extract is a concept that includes all extracts, fractions, and purified products obtained at each stage of extraction, fractionation, or purification, as well as their dilutions, concentrates, or dried products.

[0076] In one embodiment of the present invention, the extract may be included in an amount of 0.03 to 25 wt% relative to the total weight of the composition, but is not limited thereto.

[0077] In one embodiment of the present invention, the composition may be characterized by promoting collagen synthesis, but is not limited thereto.

[0078] In one embodiment of the present invention, the composition may be characterized by having anti-inflammatory activity, but is not limited thereto.

[0079] In one embodiment of the present invention, the anti-inflammatory activity is to inhibit the movement of NF-κB into the nucleus, to inhibit the expression of inflammatory proteins, and to inhibit the secretion of cytokines, wherein the inflammatory proteins are NF-κB or IκBα, and the cytokines may be IL-8, IL-1β, or IL-6, but are not limited thereto.

[0080] In one embodiment of the present invention, the pinitol may enhance the wound healing activity of fibroblasts, but is not limited thereto.

[0081] In one embodiment of the present invention, the wound healing activity may be, but is not limited to, increasing collagen area and cell growth compared to the control group and inhibiting cell death.

[0082] In one embodiment of the present invention, the increase in cell growth may be due to, but is not limited to, promotion of ki67+ (Antigen identified by monoclonal antibody Ki-6)+ expression.

[0083] In one embodiment of the present invention, the cell death may be due to an increase in TUNEL (TdT-mediated dUTP Nick-End Labeling) positive cells, but is not limited thereto.

[0084] In one embodiment of the present invention, the pinitol enhances antioxidant activity in reactive oxygen fibroblasts, and the enhancement of antioxidant activity is due to enhancement of expression of antioxidant proteins, and the antioxidant proteins may be one or more selected from the group consisting of Nrf2 (Nuclear factor erythroid 2-related factor 2), HO-1 (Heme oxygenase-1), SOD1 (Superoxide dismutase 1), SOD2 (Superoxide dismutase 2), and catalase, but are not limited thereto.

[0085] In one embodiment of the present invention, the pinitol may have an activity of restoring mitochondrial membrane potential, but is not limited thereto.

[0086] In one embodiment of the present invention, the pinitol may increase mitochondrial ATP production capacity, but is not limited thereto.

[0087] In one embodiment of the present invention, the pinitol may increase AMPK (AMP-activated protein kinase) expression, but is not limited thereto.

[0088] In one embodiment of the present invention, the pinitol activates angiogenesis in diabetic foot ulcers, and the angiogenesis activation is by promoting the expression of angiogenesis-related proteins, and the angiogenesis-related proteins may be, but are not limited to, VEGFA (Vascular Endothelial Growth Factor A), HIF-1α (Hypoxia-Inducible Factor 1 Alpha), CXCL12 (CXC Motif Chemokine Ligand 12), or CXCR4 (CXC Chemokine Receptor 4).

[0089]

[0090] In addition, the present invention provides a pharmaceutical composition for preventing or treating diabetic foot ulcers, which comprises pinitol as an active ingredient.

[0091] The pharmaceutical composition of the present invention may further include an adjuvant in addition to the active ingredient. Any adjuvant known in the art may be used without limitation. However, for example, Freund's complete or incomplete adjuvant may be further included to increase immunogenicity.

[0092] The pharmaceutical composition according to the present invention can be prepared in a form in which the active ingredient is mixed with a pharmaceutically acceptable carrier. Here, the pharmaceutically acceptable carrier includes carriers, excipients, and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0093] The pharmaceutical composition of the present invention can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injection solutions, each according to a conventional method.

[0094] When formulated, it can be prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants that are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and such solid preparations can be prepared by mixing the active ingredient with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups, and in addition to commonly used diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives can be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, Tween 61, cocoa butter, laurin, and glycerogelatin.

[0095] The pharmaceutical composition according to the present invention can be administered to a subject via various routes. All modes of administration are contemplated, including oral, intravenous, intramuscular, subcutaneous, and intraperitoneal injection.

[0096] The dosage of the pharmaceutical composition according to the present invention is selected taking into consideration the age, weight, sex, physical condition, etc. of the subject.

[0097] The above pharmaceutical composition can be formulated into various oral or parenteral dosage forms.

[0098] Oral dosage forms include, for example, tablets, pills, hard and soft capsules, solutions, suspensions, emulsions, syrups, and granules. These dosage forms may further contain, in addition to the active ingredient, diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and / or glycine), lubricants (e.g., silica, talc, stearic acid and its magnesium or calcium salts, and / or polyethylene glycol). In addition, the tablets may contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidine, and, if desired, disintegrants or effervescent mixtures such as starch, agar, alginic acid or its sodium salt, and / or absorbents, coloring agents, flavoring agents, and sweetening agents. The above formulation can be prepared by conventional mixing, granulating or coating methods.

[0099] In addition, representative parenteral administration formulations include injectable preparations, and solvents for injectable preparations include water, Ringer's solution, isotonic saline solution, or suspensions. Sterile fixed oils for the injectable preparations can be used as solvents or suspension media, and any non-irritating fixed oil, including mono- and di-glycerides, can be used for this purpose.

[0100] Additionally, the above injectable formulation may use a fatty acid such as oleic acid.

[0101]

[0102] In addition, the present invention provides an external composition for wound treatment in diabetic foot ulcers, which contains pinitol as an active ingredient.

[0103]

[0104] In addition, the present invention provides a composition for promoting collagen synthesis for diabetic foot ulcers, which contains pinitol as an active ingredient.

[0105]

[0106] In addition, the present invention provides an anti-inflammatory composition for diabetic foot ulcers, which comprises pinitol as an active ingredient.

[0107]

[0108] In addition, the present invention provides an antioxidant composition for diabetic foot ulcers, which contains pinitol as an active ingredient.

[0109]

[0110] In addition, the present invention provides a composition for enhancing ATP production for diabetic foot ulcers, which contains pinitol as an active ingredient.

[0111]

[0112] In addition, the present invention provides a composition for promoting angiogenesis, which contains pinitol as an active ingredient.

[0113]

[0114] In addition, the present invention provides a pharmaceutical composition for preventing or treating angiogenesis-dependent diseases, which comprises pinitol as an active ingredient.

[0115] In one embodiment of the present invention, the angiogenesis-dependent disease may be selected from the group consisting of diabetic ulcer, diabetic nephropathy, diabetic retinopathy, diabetic nephropathy, and diabetic neuropathy, but is not limited thereto.

[0116] In one embodiment of the present invention, the pinitol may be characterized by having an angiogenesis-promoting effect of vascular endothelial cells, but is not limited thereto.

[0117] In one embodiment of the present invention, the pinitol may be characterized by reducing the expression of NF-κB, an inflammatory factor of vascular endothelial cells, but is not limited thereto.

[0118] In one embodiment of the present invention, the pinitol may have a wound healing effect on vascular endothelial cells, but is not limited thereto.

[0119] In one embodiment of the present invention, the pharmaceutical composition may be characterized by having an effect of inhibiting active oxygen in vascular endothelial cells, but is not limited thereto.

[0120] In one embodiment of the present invention, the pharmaceutical composition may have a membrane potential restoration effect of mitochondria in vascular endothelial cells, but is not limited thereto.

[0121] In one embodiment of the present invention, the pinitol reduces the expression of Mitogen-Activated Protein Kinases (MAPKs) in vascular endothelial cells, and the MAPKs in the vascular endothelial cells may be ERK (Extracellular signal-Regulated Kinase), JNK (c-Jun N-terminal Kinase), or p38 (p38 mitogen-activated protein kinases), but is not limited thereto.

[0122]

[0123] In addition, the present invention provides a pharmaceutical composition for preventing or improving angiogenesis-dependent diseases, which comprises pinitol as an active ingredient.

[0124]

[0125] In addition, the present invention provides a composition for wound healing of vascular endothelial cells, which contains pinitol as an active ingredient.

[0126]

[0127] Additionally, the present invention provides a method for preventing or treating diabetic foot ulcers, comprising administering pinitol to a subject.

[0128] The term "subject" as used in the present invention refers to a subject requiring a method for preventing, controlling, or treating a disease, and may be used without limitation as a human, dog, monkey, cat, rodent, such as a mouse, genetically modified mouse, etc. More specifically, it refers to a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, cow, etc.

[0129]

[0130] Additionally, the present invention provides a method for preventing or treating angiogenesis-dependent disease, comprising administering pinitol to a subject.

[0131] Hereinafter, the present invention will be described in more detail through examples. These examples are intended to more specifically illustrate the present invention, and the scope of the present invention is not limited to these examples.

[0132]

[0133] <Experimental Example 1> Evaluation of the wound healing ability of pinitol

[0134] 1-1. Establishment of an animal model of diabetic foot ulcers

[0135] Four-week-old Specific Pathogen-Free male Sprague-Dawley dogs weighing 180-200 g were purchased, and after a one-week acclimatization period, food and water were provided ad libitum. The temperature was maintained at 22±2℃, humidity at 50±10%, and light / dark cycle was controlled at 12 hours (09:00-21:00). After the adaptation period, rats were divided into a control group, a streptozotocin-treated group (Streptozotocin, STZ, 65 mg / mL), and a pinitol-treated group (Streptozotocin (STZ, 65 mg / mL) + Pinitol (100 mg / kg)), and each group consisted of 4 rats. In the streptozotocin-treated group and the pinitol-treated group, blood sugar levels were checked 3 days after intraperitoneal administration of streptozotocin (65 mg / mL), and 10 days later, blood sugar levels were checked in a fasting state. Rats with blood sugar levels of 16.7 mmol / L or higher were included in the experimental group and analyzed.

[0136] 1-2. Evaluation of the ability of pinitol to heal diabetic foot ulcers

[0137] To induce diabetic foot ulcers, rats were anesthetized with ether (10 to 20 ml) and a 5 × 5 mm punch was used to induce a wound on the paw. A ring-shaped silicone splint was fixed around the wound using tissue adhesive (VetBond 1469c, 3M, Saint Paul, MN), and a waterproof and bacterial barrier transparent occlusive dressing (Tegaderm Film 1622 W, 3M) was applied over the wound. A topical skin preparation containing pinitol was applied to the wound once daily for 8 days, while in the control group, only a base skin preparation without pinitol was applied. Photographs of the foot ulcers were taken on days 0, 2, 4, 6, and 8 after wound induction to determine the size of the foot ulcers. Figure 1 shows the change in the size of the foot ulcer wounds over time.

[0138] Looking at Figure 1, in the control group, the wound size of the foot ulcer was found to decrease over time, while in the streptozotocin-treated group, the wound size did not decrease significantly. However, in the pinitol-treated group, as in the control group, the wound size of the foot ulcer was found to decrease significantly over time.

[0139] Through the above results, it was confirmed that pinitol has a wound healing effect on diabetic foot ulcers.

[0140]

[0141] <Experimental Example 2> Analysis of changes in collagen and cell count in diabetic foot ulcers following pinitol treatment

[0142] To determine the effects of pinitol on collagen and cell numbers in diabetic foot ulcers, wound tissues were collected from each group of rats on the 8th day after wound induction, and collagen angiogenesis within the tissues was confirmed through H&E and Masson's Trichrome staining. In addition, changes in collagen in the wounds were confirmed through numerical comparison using Image J. Figures 2 and 3 show collagen changes according to H&E and Masson's Trichrome staining.

[0143] Looking at Figures 2 and 3, it can be confirmed that the collagen content in the streptozotocin treatment group decreases compared to the control group. However, the collagen content in the pinitol treatment group increased compared to the streptozotocin treatment group. Numerically, the collagen ratio in the control group was approximately 20%, but in the streptozotocin treatment group, it was less than 10%, and in the pinitol treatment group, it was found to have a collagen ratio of more than 30%.

[0144] Through the above results, the collagen production promoting effect of pinitol was confirmed.

[0145]

[0146] <Experimental Example 3> Evaluation of wound healing ability and inflammatory response in fibroblasts induced by wound infection with LPS.

[0147] 3-1. Culture conditions for human dermal fibroblasts

[0148] Normal human dermal fibroblasts (NHDFs) were purchased from Promo cell (Cat. No. C-12302, Heidelberg Germany). The human dermal fibroblasts were cultured in 75 cm 2 Subculture was performed in T-flasks and cultured in a CO2 incubator at 37°C under 5% CO2 conditions. Cell subculture was typically performed in the second or third passage.

[0149] In addition, to confirm the effect of each material on human dermal fibroblasts damaged by LPS, after treatment with LPS (10 μg / mL), pinitol at a concentration of 100 μM and metformin at a concentration of 1 mM were treated in 10% FBS medium and cultured for 24 hours.

[0150]

[0151] 3-2. Evaluation of the effect of materials on dermal fibroblasts through comparison of immunofluorescence images

[0152] Subculture of dermal fibroblasts was performed in confocal dishes, and permeabilization was performed using Triton X-100. Subsequently, the cells were blocked with 3% BSA and washed with PBS. Subsequently, antibody treatment was performed to detect antigen-antibody reactions, and fluorescence images were captured using a fluorescence microscope. Figure 4 shows the fluorescence images, and Figure 5 shows the nuclear translocation of NF-κB.

[0153] Looking at Figures 4 and 5, the number of nuclear translocations of NF-κB was found to increase in the LPS-treated group compared to the control group. However, the number of nuclear translocations in the pinitol-treated group was found to decrease compared to the LPS-treated group.

[0154] Through the above results, it was confirmed that pinitol inhibits the movement of NF-κB into the nucleus, thereby inhibiting the activation of NF-κB, and thus exhibiting anti-inflammatory activity.

[0155]

[0156] <Experimental Example 4> Analysis of toxicity and growth rate in fibroblasts

[0157] 4-1. Toxicity analysis of pinitol on human dermal fibroblasts

[0158] Cytotoxicity of pinitol, metformin, and LPS was evaluated in normal human dermal fibroblasts (NHDFs).

[0159] Human dermal fibroblasts (HDFs) are 75 cm 2 Subculture was performed in T-flasks and cultured in a CO2 incubator at 37°C under 5% CO2 conditions. Cell passage was usually performed in the second or third passage. Fibroblasts were cultured at 1.0X10 4Cells were dispensed into a 96-well plate at a concentration of 10 cells / well, and after 24 hours, pinitol (0, 2.5, 5, 10, 25, 50, 100 μM), metformin (0, 25, 50, 100, 250, 500, 1000 μM), and LPS (0, 0.001, 0.01, 0.1, 1, 10, 100 μg / mL) were dispensed and cultured for 24 hours. After culture, media suction was performed, and 110 μl of EZ-Cytox 10 μl and phenol red-free DMEM 100 μl were dispensed into each well, and the reaction was performed in an incubation for 1 hour, and the absorbance was measured at a wavelength of 450 nm. The absorbance measurement results are shown in Figure 6. As shown in Fig. 6, it can be confirmed that cell viability decreases depending on the concentration when treated with LPS, and it was confirmed that no toxicity was observed as the viability was over 90% at all concentrations of pinitol and metformin.

[0160] 4-2. Wound healing assay of human dermal fibroblasts with pinitol

[0161] Wound healing assays were performed in normal human dermal fibroblasts (NHDFs) treated with pinitol.

[0162] For wound healing analysis, human dermal fibroblasts were seeded in a confocal dish at a density of 4.0 × 10 4 The cells were dispensed at a concentration of 10 cells / well and cultured at 37 °C for 24 h. After creating a scratch with a 200 μL pipette tip, 1 μg / mL LPS, 1 mM metformin, and 100 μM pinitol were treated and cultured for 24 h. The change in scratch width between 0 h and 24 h was observed.

[0163] The results of the wound healing analysis are shown in Figures 7 and 8. As shown in Figures 7 and 8, the wound healing ability of the LPS-treated group was found to be reduced compared to the control group. However, the pinitol-treated group showed an increase in wound healing ability compared to the LPS-treated group.

[0164] Through the above results, it was confirmed that pinitol does not exhibit toxicity and increases wound healing ability.

[0165]

[0166] <Experimental Example 5> Analysis of the inhibitory effect of reactive oxygen species (ROS)

[0167] To confirm the ROS inhibitory effect in normal human dermal fibroblasts (NHDFs), DCF-DA analysis was performed according to pinitol treatment.

[0168] Normal human dermal fibroblasts (NHDFs) 4.0X10 4 Cells were cultured in a confocal dish at a concentration of 10 cells / mL and treated with LPS (1 μg / mL), pinitol (100 μM), and metformin (1 mM) for 24 h. After washing with PBS, cells were treated with DCFDA and incubated for 20 min. After washing with PBS, fluorescence images were captured using a fluorescence microscope.

[0169] The results of the ROS inhibition efficacy analysis are shown in Figs. 9 and 10. As shown in Figs. 9 and 10, when pinitol was treated, ROS fluorescence was found to decrease compared to the LPS-treated group, confirming that pinitol exhibits the efficacy of reducing ROS in human dermal fibroblasts.

[0170]

[0171] <Experimental Example 6> Analysis of antioxidant efficacy in fibroblasts induced by wound infection with LPS.

[0172] 6-1. Analysis of the antioxidant protein expression-enhancing activity of pinitol in dermal fibroblasts through immunofluorescence image comparison.

[0173] Normal human dermal fibroblasts (NHFFs) were subcultured in a confocal dish and permeabilized using Triton X-100. Afterwards, they were blocked with 3% BSA and washed with PBS. Afterwards, antigen-antibody reaction was performed by antibody treatment, and fluorescence images were captured using a fluorescence microscope. Figures 11 and 12 show graphs that quantify the fluorescence images and the expression level of Nrf2. As shown in Figures 11 and 12, the expression level of Nrf2, an antioxidant protein, also increased as the pinitol treatment concentration increased. The results confirmed that pinitol enhances the expression of antioxidant proteins and exhibits an antioxidant effect.

[0174] 6-2. Analysis of the antioxidant protein expression-enhancing activity of pinitol in dermal fibroblasts using Western blot.

[0175] To confirm the activation of antioxidant proteins (Nrf2, HO-1, Keap1) in normal human dermal fibroblasts (NHDFs) damaged by LPS, the following was performed. Human dermal fibroblasts (HDFs) were cultured at 50X10 4After culturing cells in 100 mm dishes at a density of cells / mL, LPS (1 μg / mL), pinitol (100 μM), and metformin (1 mM) were treated for 24 h. Nuclear and cytoplasmic proteins were separated using the Cell Fractionation Kit Standard (Abcam, UK), and protein lysates (30 mg protein) were loaded and separated by electrophoresis on a 10% SDS polyamide gel. The membranes were then blotted onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% skim milk for 2 h at room temperature and washed three times for 5 min each with 1X buffer. Afterwards, the membrane was incubated with primary antibodies for 1 hour at room temperature with a 5% BSA solution containing Nrf2 (1:1000, Novus Biologicals, CO, USA), HO-1 (1:1000, Cell Signaling Technology, Beverly, MA, USA), and Keap1 (1:1000, Cell Signaling Technology, Beverly, MA, USA), and then washed three times with 1X buffer for 5 minutes each. After that, the membrane was incubated with secondary antibodies for 1 hour at room temperature and then washed three times with 1X buffer for 5 minutes each. β-Actin expression was used as an internal standard, and the immobilized protein was measured using an enhanced chemiluminescence (ECL) detection system. Figures 13 to 16 show the results of measuring antioxidant protein expression. As shown in Figures 13 to 16, when pinitol was treated, the expression of Keap1 decreased, and the expression of Nrf2 and HO-1 increased. The above results confirmed that pinitol increases the expression of antioxidant proteins and thereby reduces ROS.

[0176] 6-3. Analysis of the antioxidant protein expression-enhancing activity of pinitol in dermal fibroblasts using quantitative real-time RT-qPCR.

[0177] 50X10 human dermal fibroblasts (NHDFs) 4 After culturing in a 100 mm dish at 10 cells / mL, LPS (1 μg / mL), pinitol (100 μM), and metformin (1 mM) were treated for 24 hours. After washing the cells twice with DPBS, RNA was extracted with TRIzol (Thermo Fisher Scientific, Waltham, MA, USA) reagent, and cDNA was synthesized using the RevertAid First Strand cDNA synthesis kit using 2 μg of RNA. Quantitative Real-time RT-PCR was performed using TaqMan Universal Master Mix II (containing UNG). The results of Quantitative Real-time RT-PCR analysis are shown in Figures 17 to 19. As shown in Figures 17 to 19, the expression levels of SOD1, SOD2, and Catalase also increased as the concentration of pinitol increased. These results confirmed that pinitol increases the expression of antioxidant proteins, thereby reducing ROS.

[0178] When the results of the above experimental example 6 are summarized, it can be confirmed that pinitol increases the movement of Nrf2 into the nucleus, increases antioxidant proteins, and exhibits the activity of enhancing antioxidant activity.

[0179]

[0180] <Experimental Example 7> Analysis of anti-inflammatory activity of pinitol on wound-infected fibroblasts

[0181] 7-1. Analysis of pinitol's inhibitory activity on inflammatory protein expression in normal human dermal fibroblasts (NHDFs) using Western blot

[0182] To confirm the inhibition of inflammatory protein (NF-κB, IκBα) activation in human dermal fibroblasts damaged by LPS, the following was performed.

[0183] 50X10 human dermal fibroblasts (NHDFs) 4After culturing cells in a 100 mm dish at a density of 10 cells / mL, LPS (1 μg / mL), pinitol (100 μM), and metformin (1 mM) were treated for 24 h. Proteins were extracted using lysis buffer and phosphatase inhibitors, and the protein lysate (30 mg protein) was loaded and separated by electrophoresis on a 10% SDS polyamide gel. The membrane was then blotted onto a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% skim milk for 2 h at room temperature and washed three times for 5 min each with 1X buffer. Afterwards, the membrane was incubated with primary antibodies for 1 hour at room temperature with a 5% BSA solution containing p-NF-κB (1:1000, Cell Signaling Technology, Beverly, MA, USA), NF-κB (1:1000, Cell Signaling Technology, Beverly, MA, USA), IκBα (1:1000, Cell Signaling Technology, Beverly, MA, USA), and p-IκBα (1:1000, Novus Biologicals, CO, USA), and washed three times for 5 minutes each with 1X buffer. After that, the membrane was incubated with secondary antibodies for 1 hour at room temperature and washed three times for 5 minutes each with 1X buffer. β-Actin expression was used as an internal standard, and the immobilized proteins were measured using an enhanced chemiluminescence (ECL) detection system. The results of measuring the expression levels of inflammatory proteins (NF-κB, IκBα) are shown in Figures 20 to 22. Looking at Figures 20 to 22, it was shown that the expression levels of NF-κB and IκBα decreased as the pinitol treatment concentration increased. The above results confirmed that pinitol inhibits the expression of inflammatory proteins and suppresses the inflammatory response.

[0184] 7-2. Analysis of the cytokine expression inhibitory activity of pinitol in dermal fibroblasts using quantitative real-time RT-qPCR.

[0185] 50X10 human dermal fibroblasts (NHDFs) 4 After culturing in a 100 mm dish at 10 cells / mL, LPS (1 μg / mL), pinitol (100 μM), and metformin (1 mM) were treated for 24 hours. After washing the cells twice with DPBS, RNA was extracted with TRIzol (Thermo Fisher Scientific, Waltham, MA, USA) reagent, and cDNA was synthesized using the RevertAid First Strand cDNA synthesis kit using 2 μg of RNA. Quantitative Real-time RT-PCR was performed using TaqMan Universal Master Mix II (containing UNG). The results of quantitative Real-time RT-PCR analysis are shown in Figures 23 to 25. As shown in Figures 23 to 25, IL-8, IL-1β, and IL-6, which were increased by LPS, were reduced when pinitol was treated. Through the above results, it was confirmed that pinitol reduces the inflammatory response by inhibiting the expression of cytokines IL-8, IL-1β, and IL-6.

[0186]

[0187] <Experimental Example 8> Evaluation of the efficacy of restoring mitochondrial membrane potential

[0188] Fluorescence microscopy analysis was performed to determine the efficacy of mitochondrial membrane potential restoration in normal human dermal fibroblasts (NHDFs) damaged by LPS.

[0189] Normal human dermal fibroblasts (NHDFs) were subcultured in confocal dishes and treated with LPS (1 μg / mL), pinitol (100 μM), and metformin (1 mM) for 24 h. After washing with DPBS, the cells were treated with JC-1 (AAT bioquest, Sunnyvale, CA, USA) and incubated for 10 min. After washing with DPBS, fluorescence images were captured using a fluorescence microscope.

[0190] Figures 26 and 27 show graphs quantifying fluorescence images and fluorescence intensity values. Examining Figures 26 and 27, it was confirmed that the mitochondrial membrane potential in the LPS-treated group exhibited green fluorescence, whereas in the pinitol-treated group, the mitochondrial membrane potential exhibited red fluorescence similar to the control group. These results confirm that pinitol exhibits the activity of restoring membrane potential damaged by LPS.

[0191]

[0192] <Experimental Example 9> Analysis of mitochondrial ATP production capacity

[0193] To evaluate the ATP production capacity of mitochondria in LPS-injured human dermal fibroblasts (NHDFs), fluorescence analysis was performed using Mitolite™ and ATP Red™.

[0194] After subculture of human dermal fibroblasts in confocal dishes, the cells were treated with LPS (1 μg / mL), pinitol (100 μM), and metformin (1 mM) for 24 h. The cells were washed with DPBS and treated with Mitolite™ (Thermo Fisher Scientific, USA) and ATP Red™ (Sigma-Aldrich, USA), respectively, according to the manufacturers' protocols. After fluorescence staining at 37°C for 20 min, fluorescence images were captured under a microscope, and the amount of ATP produced was measured.

[0195] The results of ATP production analysis are shown in Figures 28 and 29. While the LPS-treated group exhibited a decrease in ATP production, the pinitol-treated group demonstrated a recovery in mitochondrial ATP production to the control level. These results confirm that pinitol increases the ATP production capacity of LPS-damaged mitochondria.

[0196]

[0197] <Experimental Example 10> Evaluation of AMPK expression activation by pinitol

[0198] To confirm AMPK activation in human dermal fibroblasts damaged by LPS, the following was performed.

[0199] 50X10 human dermal fibroblasts (NHDFs) 4After culturing cells in 100 mm dishes at a density of 10 cells / mL, LPS (1 μg / mL), pinitol (100 μM), and metformin (1 mM) were treated for 24 h. Proteins were extracted using lysis buffer and phosphatase inhibitors, and protein lysates (30 mg protein) were loaded and separated by electrophoresis on a 10% SDS polyamide gel. The samples were then blotted onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% skim milk for 2 h at room temperature and washed three times for 5 min each with 1X buffer. Afterwards, the membrane was incubated with primary antibodies for 1 hour at room temperature with 5% BSA solution containing p-AMPK (1:1000, Cell Signaling Technology, Beverly, MA, USA) and AMPK (1:1000, Cell Signaling Technology, Beverly, MA, USA), and washed three times for 5 minutes each with 1X buffer. After that, the membrane was incubated with secondary antibodies for 1 hour at room temperature and washed three times for 5 minutes each with 1X buffer. β-Actin expression was used as an internal standard, and the immobilized protein was measured using an enhanced chemiluminescence (ECL) detection system. The results of the enhanced chemiluminescence (ECL) analysis are shown in Figs. 30 and 31. As shown in Figs. 30 and 31, the AMPK expression level increased as the pinitol treatment concentration increased. Through the above results, it was confirmed that pinitol exhibits activity in suppressing inflammatory responses.

[0200]

[0201] <Experimental Example 11> Establishment of an animal model of diabetic foot ulcers

[0202] Four-week-old specific pathogen-free male Sprague-Dawley rats weighing 180-200 g were purchased, and allowed to acclimate for one week. Food and water were provided ad libitum. The temperature was maintained at 22±2℃, the humidity at 50±10%, and the light / dark cycle was controlled at 12 h (09:00-21:00). After the acclimation period, the rats were divided into the control group, streptozotocin treatment group (Streptozotocin, STZ, 65 mg / mL), pinitol treatment group (Streptozotocin (STZ, 65 mg / mL) + Pinitol (0.5 %)), and metformin treatment group (Streptozotocin (STZ, 65 mg / mL) + Metformin (0.5 %)), and each group consisted of 12 rats. The streptozotocin-treated and pinitol-treated groups were administered streptozotocin (65 mg / mL) intravenously, and their blood sugar levels were checked 48 hours later and 10 days later in a fasting state. The blood sugar analysis results are shown in Figure 32. As shown in Figure 32, when streptozotocin was treated, blood sugar levels were confirmed to be 300 mg / dL or higher for 2 hours. Therefore, rats with blood sugar levels 300 mg / dL or higher were included in the experimental group, and the following analyses were performed.

[0203]

[0204] <Experimental Example 12> Analysis of collagen, Ki67, and TUNEL expression levels in diabetic foot ulcers treated with pinitol

[0205] To confirm the therapeutic effect of pinitol on diabetic foot ulcers, changes in collagen, Ki67, and TUNEL expression levels were analyzed when pinitol was treated in mice of Experimental Example 11.

[0206] During the period of pinitol application, the wound size was measured and at a visually comparable time point (e.g., day 8), the corresponding wound tissues were collected from six animals in each group and collagen formation, cell growth, and cell death in the tissue were confirmed using Massin Trichrome and immunohistochemistry.

[0207] Changes in collagen area within tissues in Figs. 33 to 36, ki67 + The changes in expression and the percentage of TUNEL-positive cells were shown. Looking at Figures 33 to 36, the collagen area and ki67 were increased compared to the control group according to streptozotocin treatment. + The expression was decreased and the percentage of TUNEL-positive cells increased. However, when pinitol was treated, the collagen area and ki67 + Expression was found to recover to levels comparable to the control group, and the percentage of TUNEL-positive cells was lower than that of the control group. These results confirmed that pinitol improves diabetic foot ulcers by increasing collagen and cell growth and preventing cell death.

[0208]

[0209] <Experimental Example 13> Evaluation of Pinitol's Oxidative Stress Reduction in Diabetic Foot Ulcers

[0210] On the last day of the application period, diabetic foot ulcer mice were sacrificed, 10 mg of tissue was collected, and oxidative stress concentration was measured using an MDA assay kit (Abcam, Cambridge, UK) and a GSH assay kit (Abcam, Cambridge, UK).

[0211] Figure 37 shows the results of measuring oxidative stress concentration. As shown in Figure 37, streptozotocin treatment increased lipid peroxidation content and decreased reduced glutathione concentration. However, pinitol treatment decreased lipid peroxidation content and increased reduced glutathione concentration, and showed a better effect than the positive control group, metfomin treatment group, confirming the effect of pinitol in reducing reactive oxygen species on diabetic foot ulcers.

[0212]

[0213] <Experimental Example 14> Analysis of the angiogenic ability of pinitol in diabetic foot ulcers.

[0214] To confirm the angiogenic ability of pinitol in diabetic foot ulcers, the following analyses were performed.

[0215] 14-1. Analysis of the angiogenic ability of pinitol in diabetic foot ulcers through comparison of immunofluorescence images.

[0216] During the application period, the wound size was measured, and at a point where it could be visually compared (e.g., on the 8th day), the corresponding wound tissues from six animals in each group were collected and the neovascularization was analyzed through CD31 expression in the tissue using immunofluorescence.

[0217] The results of angiogenesis analysis are shown in Figures 38 and 39. As seen in Figures 38 and 39, streptozotocin decreased blood vessel area, but pinitol treatment increased CD31 expression, resulting in a significant increase in blood vessel area. These results confirm that pinitol increases angiogenesis by increasing CD31 expression.

[0218] 14-2. Analysis of Pinitol's Enhanced Expression of Angiogenesis-Related Proteins in Diabetic Foot Ulcers Using Quantitative Real-Time RT-qPCR

[0219] To investigate the activity of pinitol to enhance the expression of angiogenic proteins, we confirmed the changes in the expression of angiogenic proteins VEGFA, HIF-1α, CXCL12, and CXCR4. Wound size was measured during the application period, and at a visually comparable time point (e.g., day 8), wound tissues from six mice in each group were collected, RNA was extracted with TRIzol (Thermo Fisher Scientific, Waltham, MA, USA) reagent, and cDNA was synthesized using the RevertAid First Strand cDNA synthesis kit using 2 μg of RNA. Quantitative real-time RT-PCR was performed using TaqMan Universal Master Mix II (containing UNG).

[0220] The results of Quantitative RealtimeRT-PCR are shown in Figures 40 and 41. Looking at Figures 40 and 41, it was confirmed that pinitol increases angiogenic capacity through increases in VEGFA, HIF-1α, CXCL12, and CXCR4 when pinitol was treated following streptozotocin treatment. Through the above results, it was confirmed that pinitol increases angiogenic-related proteins, thereby activating angiogenic capacity.

[0221]

[0222] <Experimental Example 15> Confirmation of the effect of pinitol on improving angiogenic capacity in vascular endothelial cells.

[0223] 50 μl of ECM gel solution was dispensed into a cooled 96-well cell culture dish and incubated at 37°C for 1 hour to form a gel. After that, 2 × 10 human-derived vascular endothelial cells were seeded into the 96-well cell culture dish. 5After culturing at 10 cells / ml, LPS (2 μg / ml), Metformin (1 mM), or D-pinitol (10, 50 μM) were treated, respectively. The LPS-treated group was set as the negative control group, the LPS and metformin-treated group was set as the positive control group, and the metformin and pinitol-treated group was set as the pinitol-treated group. After drug treatment, cell morphology and the degree of angiogenesis were confirmed using phase contrast microscopy every 0, 8, 24, and 48 hours. The confirmed cell morphology is shown in Figure 42. As shown in Figure 42, angiogenesis was inhibited in the LPS-treated group, and angiogenesis was increased in the pinitol-treated group compared to the LPS-treated group.

[0224] Through the above results, it was confirmed that pinitol has an effect of promoting blood vessel formation in vascular endothelial cells.

[0225]

[0226] <Experimental Example 16> Confirmation of the effect of pinitol on reducing NF-κB expression in vascular endothelial cells.

[0227] To confirm the effect of D-pinitol on improving angiogenic ability, the effect of reducing the expression of NF-κB, an inflammation-related biomarker, in vascular endothelial cells was confirmed through Western blot.

[0228] Human vascular endothelial cells were seeded at 5×10 in a 100 mm cell culture dish. 5 After culturing at 10 cells / ml, the cells were treated with Metformin (1 mM) and D-pinitol (10, 50, and 100 μM), as positive controls, for 24 hours. Afterwards, LPS (10 μg / ml) treatment was performed to induce reactive oxygen species.

[0229] Proteins were extracted using lysis buffer and phosphatase inhibitors, and the protein lysates (40 mg protein) were loaded and separated by electrophoresis on a 10% SDS polyamide gel. They were then blotted onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% skim milk for 2 h at room temperature and washed three times for 10 min each with 1X buffer. The membranes were then incubated overnight at 4°C with primary antibodies, including rabbit NF-κB (1:1000, Cell Signaling Technology, Beverly, MA, USA) and phosphorylated NF-κB (p-NF-κB) (1:1000, Cell Signaling Technology, Beverly, MA, USA). The membranes were then incubated with secondary antibodies for 2 h at room temperature. Expression of β-Actin or NF-kB was used as an internal standard, and immobilized proteins were measured using an enhanced chemiluminescence (ECL) detection system.

[0230] Figures 43 and 44 show the expression of NF-κB in vascular endothelial cells according to pinitol treatment. Looking at Figures 43 and 44, it was shown that the expression of NF-κB increased when LPS was treated, and that the expression of NF-κB decreased as the treatment concentration increased when D-pinitol was treated.

[0231] Through the above results, it was confirmed that pinitol has the effect of reducing the expression of NF-κB and has anti-inflammatory activity.

[0232]

[0233] <Experimental Example 17> Confirmation of the wound healing effect of pinitol in vascular endothelial cells.

[0234] To confirm the angiogenesis-improving effect of D-pinitol, the wound healing effect of D-pinitol was confirmed in vascular endothelial cells under hyperglycemic conditions.

[0235] Human dermal microvascular endothelial cells (3×10) were cultured in a 35 mm dish cell culture dish. 4 After culturing at a concentration of 100 cells / ml, the endothelial cells were wounded with the yellow tip. Afterwards, the cells were divided into high glucose conditions (HG: High Glucose, glucose 30 mM) and normal conditions (LG: Low Glucose, glucose 5 mM + mannitol 25 mM), and treated with LPS (10 μg / ml) or LPS (10 μg / ml) and D-pinitol (100 μM) for 24 hours, respectively. The LPS-treated group was set as the negative control group, and the group treated with LPS (10 μg / ml) and D-pinitol (100 μM) was set as the pinitol-treated group. The cell number and morphology of the vascular endothelial cells in the LPS-treated and pinitol-treated groups were confirmed by microscopy (phase contrast microscopy). The cell number and morphology of the vascular endothelial cells are shown in Figure 45.

[0236] Looking at Figure 45, under normal conditions, the wound width was significantly reduced in the pinitol-treated group compared to the LPS-treated group. On the other hand, under hyperglycemic conditions, the wound width was not significantly reduced in the pinitol-treated group compared to the LPS-treated group, but the number and density of vascular endothelial cells were found to increase.

[0237] Through the above results, it was confirmed that pinitol has a wound healing effect on vascular endothelial cells under normal and hyperglycemic conditions.

[0238]

[0239] <Experimental Example 18> Confirmation of the effect of pinitol on suppressing reactive oxygen species (ROS) in vascular endothelial cells.

[0240] To confirm the anti-oxidant effect of pinitol, DCF-DA analysis was performed according to pinitol treatment.

[0241] Human dermal microvascular endothelial cells (5×10) were cultured in a confocal dish. 4 After culturing at a concentration of 10 cells / ml, cells were treated with LPS (10 μg / ml) or LPS (10 μg / ml) and pinitol (100 μM) for 24 hours. The LPS-treated group was set as the negative control group, and the group treated with LPS (10 μg / ml) and pinitol (100 μM) was set as the pinitol-treated group. The levels of reactive oxygen species in the LPS-treated and pinitol-treated groups were confirmed under a microscope (Nikon Eclipse Ti2 fluorescence live-cell imaging microscope). Figures 46 and 47 show images showing that reactive oxygen species were suppressed by pinitol.

[0242] Looking at Figures 46 and 47, the green fluorescence intensity was found to increase in the LPS treatment group compared to the control group, but the green fluorescence intensity was found to decrease in the pinitol treatment group compared to the LPS treatment group. Since the green fluorescence intensity is proportional to the amount of reactive oxygen species, the above results confirmed that pinitol has the effect of suppressing reactive oxygen species in vascular endothelial cells.

[0243]

[0244] <Experimental Example 19> Confirmation of the effect of pinitol on restoring mitochondrial membrane potential in vascular endothelial cells.

[0245] To confirm the mitochondrial membrane potential restoration effect of pinitol, the JC-1 assay of pinitol was performed in vascular endothelial cells.

[0246] Human dermal microvascular endothelial cells (5×10) were cultured in a confocal dish.4 After culturing at a concentration of 10 cells / ml, cells were treated with LPS (10 μg / ml) or LPS (10 μg / ml) and pinitol (100 μM) for 24 hours. The LPS-treated group was set as the negative control group, and the group treated with LPS (10 μg / ml) and pinitol (100 μM) was set as the pinitol-treated group. The degree of mitochondrial membrane potential recovery in the LPS-treated and pinitol-treated groups was confirmed under a microscope (Nikon Eclipse Ti2 fluorescence live-cell imaging microscope). Figures 48 and 49 show images showing the recovery of mitochondrial membrane potential by pinitol.

[0247] Looking at Figures 48 and 49, the green fluorescence intensity increased in the LPS-treated group compared to the control group. On the other hand, the red fluorescence intensity increased in the pinitol-treated group compared to the LPS-treated group. The higher the red (Aggregate) / green (Monomer) ratio, the higher the mitochondrial membrane potential, indicating a healthy cell state. The above results confirmed that pinitol has the effect of restoring the membrane potential of damaged mitochondria in vascular endothelial cells.

[0248]

[0249] <Experimental Example 20> Confirmation of the inhibitory effect of pinitol on MAPK expression in vascular endothelial cells.

[0250] To confirm the inhibitory effect of pinitol on MAPKs expression, protein expression analysis (Western blot assay) of D-pinitol was performed in vascular endothelial cells.

[0251] Human dermal microvascular endothelial cells (5×10) were seeded in a 100 mm cell culture dish. 4After culturing at 10 cells / ml, the cells were treated with pinitol (100 μM) and metformin (1 mM) for 24 hours each. Afterwards, LPS (10 μg / ml) was treated for 3 hours. The LPS-treated group was set as the negative control group, and the groups treated with LPS (10 μg / ml) and pinitol (10, 50, and 100 μM) were set as the pinitol-treated group. Figures 50 to 53 show the results of protein expression analysis of MAPKs, ERK, JNK, and p38.

[0252] Looking at Figures 50 to 53, the phosphorylation levels of ERK, JNK, and p38 were found to increase in the LPS-treated group compared to the control group, but the phosphorylation levels of ERK, JNK, and p38 were found to decrease in the pinitol-treated group. Through the above results, it was confirmed that pinitol has antioxidant and anti-inflammatory effects by reducing the phosphorylation levels of ERK, JNK, and p38 in vascular endothelial cells.

Claims

1. A composition for external skin application for the prevention or treatment of diabetic foot ulcers containing pinitol (D-Pinitol, 3-O-methyl-D-chiro-inositol) as an active ingredient.

2. In paragraph 1, A composition characterized in that the above pinitol promotes collagen synthesis.

3. In paragraph 1, A composition characterized in that the above pinitol has anti-inflammatory activity.

4. In paragraph 3, The above anti-inflammatory activity is to inhibit the movement of NF-κB into the nucleus, inhibit the expression of inflammatory proteins, and inhibit cytokine secretion. The above inflammatory protein is NF-κB or IκBα. A composition wherein the cytokine is IL-8, IL-1β or IL-6.

5. In paragraph 1, A composition wherein the above pinitol promotes wound healing activity of fibroblasts.

6. In paragraph 5, A composition having the above wound healing activity that increases collagen area and cell growth and inhibits cell death compared to the control group.

7. In paragraph 6, A composition wherein the above cell growth increase is due to promotion of ki67+ (antigen identified by monoclonal antibody Ki-6)+ expression.

8. In paragraph 6, A composition wherein the above cell death is caused by an increase in TUNEL (TdT-mediated dUTP Nick-End Labeling) positive cells.

9. In paragraph 1, The above pinitol enhances antioxidant activity in reactive oxygen fibroblasts. The above enhancement of antioxidant activity is due to the enhancement of antioxidant protein expression. A composition wherein the above antioxidant protein is at least one selected from the group consisting of Nrf2 (Nuclear factor erythroid 2-related factor 2), HO-1 (Heme oxygenase-1), SOD1 (Superoxide dismutase 1), SOD2 (Superoxide dismutase 2), and Catalase.

10. In paragraph 1, A composition wherein the above pinitol has the activity of restoring mitochondrial membrane potential.

11. In paragraph 1, A composition wherein the above pinitol increases mitochondrial ATP production capacity.

12. In paragraph 1, The above pinitol is a composition that increases the expression of AMPK (AMP-activated protein kinase).

13. In paragraph 1, The above pinitol activates angiogenesis in diabetic foot ulcers. The above angiogenesis activation is due to the promotion of angiogenesis-related protein expression. A composition wherein the angiogenesis-related protein is VEGFA (Vascular Endothelial Growth Factor A), HIF-1α (Hypoxia-Inducible Factor 1 Alpha), CXCL12 (CXC Motif Chemokine Ligand 12) or CXCR4 (CXC Chemokine Receptor 4).

14. A pharmaceutical composition for preventing or treating diabetic foot ulcers containing pinitol as an active ingredient.

15. A composition for external application for wound treatment of diabetic foot ulcers containing pinitol as an active ingredient.

16. A composition for promoting collagen synthesis for diabetic foot ulcers containing pinitol as an active ingredient.

17. An anti-inflammatory composition for diabetic foot ulcers containing pinitol as an active ingredient.

18. An antioxidant composition for diabetic foot ulcers containing pinitol as an active ingredient.

19. A composition for enhancing ATP production for diabetic foot ulcers containing pinitol as an active ingredient.

20. A composition for promoting angiogenesis containing pinitol as an active ingredient.

21. A pharmaceutical composition for the prevention or treatment of angiogenesis-dependent diseases containing pinitol as an active ingredient.

22. In paragraph 21, A pharmaceutical composition, wherein the angiogenesis-dependent disease is selected from the group consisting of diabetic ulcer, diabetic nephropathy, diabetic retinopathy, diabetic nephropathy and diabetic neuropathy.

23. In paragraph 21, A pharmaceutical composition characterized in that the above pinitol has an angiogenesis-promoting effect of vascular endothelial cells.

24. In paragraph 21, A pharmaceutical composition characterized in that the above pinitol reduces the expression of NF-κB, an inflammatory factor of vascular endothelial cells.

25. In paragraph 21, A pharmaceutical composition characterized in that the above pinitol has a wound healing effect on endothelial cells.

26. In paragraph 21, A pharmaceutical composition characterized in that the above pinitol has an effect of inhibiting active oxygen in vascular endothelial cells.

27. In paragraph 21, A pharmaceutical composition wherein the above pinitol has a membrane potential recovery effect of mitochondria in vascular endothelial cells.

28. In paragraph 21, The above pinitol reduces the expression of Mitogen-Activated Protein Kinases (MAPKs) in vascular endothelial cells. A pharmaceutical composition, wherein the MAPKs in the above vascular endothelial cells are ERK (Extracellular signal-regulated Kinase), JNK (c-Jun N-terminal Kinase) or p38 (p38 mitogen-activated protein kinases).

29. A pharmaceutical composition for preventing or improving angiogenesis-dependent diseases containing pinitol as an active ingredient.

30. A composition for wound healing of vascular endothelial cells containing pinitol as an active ingredient.

31. A method for preventing or treating diabetic foot ulcers, comprising the step of administering pinitol to a subject.

32. A method for preventing or treating angiogenesis-dependent disease, comprising the step of administering pinitol to a subject.

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