Wound healing materials containing amorphous chitin

Abstract

To provide wound healing materials with better performance than existing wound healing materials, more specifically, wound healing materials that are highly safe, can be mass-produced at a lower price, and act more effectively.SOLUTION: Provided is a wound healing material containing amorphous chitin as an active ingredient. More specifically, it is a wound healing material containing as an active ingredient amorphous chitin that has been deacetylated to the extent that it maintains a fluid state.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a wound-healing material containing amorphous chitin as an active ingredient. [Background technology]

[0002] Chitin is widely found in nature as a biomass second only to cellulose, particularly in the shells of crustaceans, the cells of insects and fungi, the backbone of squid, etc. Chitin has excellent biocompatibility, and it has been dissolved and processed into fibers like nonwoven fabrics to be used for artificial skin, etc.

[0003] For example, a wound healing material using crystalline chitin (product name: Beschitin F) is already on the market. Its principle is that sponge-like chitin absorbs exudate and creates an appropriate moist environment at the wound site, aiding in tissue regeneration and promoting healing. However, the chitin used in the wound healing material is crystalline, and due to its strong crystal structure, it does not dissolve in organic solvents or water, and has low reactivity in chemical treatments, so there are limitations to its use as a material for the development of a wide range of applications. In industry, chitin is often deacetylated and converted into chitosan for use.

[0004] Chitosan is defined as a deacetylated product of chitin, and generally has a degree of deacetylation of 70 to 80% or more. Chitosan is insoluble in water, but has the characteristic of being soluble in dilute acid solutions in which chitin does not dissolve. For example, a hemostatic agent (product name: Hemcondot) in which chitosan is impregnated with acetic acid is already on the market. When the hemostatic agent is applied to a puncture site by injection or the like, negatively charged red blood cells and platelets are attracted to the positively charged chitosan, causing blood to clot, thereby achieving a hemostatic effect.

[0005] In order to make chitin itself widely usable, it is considered important to change its strong crystalline structure into an amorphous state. Various methods have been tried to produce amorphous chitin, such as a method in which the viscosity of an alkaline chitin dope is set to 400 cps or less, and the chitin is partially deacetylated so that the deacetylation rate is 35 to 65%, and then neutralized with an acid or dealkalized with alcohols, ion exchange resins, etc. (Patent Document 1), a method in which a high-concentration solvent is added to pulverized chitin, the chitin is dissolved while stirring to make it uniform, the chitin is swelled, and then ice of approximately the same volume as the high-concentration solvent and a neutralizing agent of approximately the same volume as the high-concentration solvent but with the opposite chemical properties are added (Patent Document 2). In addition, the relationship between deacetylation and gelation is also discussed in Non-Patent Document 1 ("Deacetylation and Gelation of Amorphous Chitin Cake" Abstracts of the 2019 Chitin Chitosan Society Research Presentation (Vol. 25, No. 2, pp. 136-137)), and this content has been filed as Patent Application No. 2019-131912.

[0006] As uses of amorphous chitin, there are disclosures of a sponge-like hemostatic agent mainly composed of an amorphous partially deacetylated chitin salt (Patent Document 3) and amorphous chitin for treating joint diseases in cattle (Patent Document 4).

[0007] However, there have been no reports on wound healing materials containing amorphous chitin as an active ingredient, and Patent Documents 3 and 4 do not disclose or suggest anything about them. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Patent No. 2990248 [Patent Document 2] Patent Publication No. 2018-053067 [Patent Document 3] Patent No. 5588465 [Patent Document 4] Patent Publication No. 2004-256402 [Non-patent literature]

[0009] [Non-Patent Document 1] Abstracts of the 2019 Chitin Chitosan Society Research Presentation, Vol. 25, No. 2, pp136-137 Summary of the Invention [Problem to be solved by the invention]

[0010] The present invention aims to provide a wound-healing material that has better performance than existing wound-healing materials, more specifically, to provide a wound-healing material that is highly safe, can be mass-produced at low cost, and acts more effectively. [Means for solving the problem]

[0011] As a result of extensive research aimed at solving the above-mentioned problems, the inventors discovered that the above-mentioned problems could be achieved by using a wound-healing material containing as an active ingredient amorphous chitin that has been deacetylated to an extent that allows it to maintain a fluid state, and thus completed the present invention.

[0012] The present invention comprises the following. 1. A wound healing material containing amorphous chitin as an active ingredient. 2. A wound-healing material as described in the preceding paragraph 1, in which the amorphous chitin is in a state capable of maintaining a fluidity. 3. The wound-healing material according to item 1 or 2 above, wherein the amorphous chitin has a deacetylation degree of 0.1 to 50%. 4. The wound-healing material according to item 3 above, wherein the amorphous chitin has a deacetylation degree of 0.1 to 10%. 5. The wound-healing material according to the preceding paragraph 4, wherein the amorphous chitin has a deacetylation degree of 0.1 to less than 5%. 6. A wound-healing material according to any one of items 1 and 3 to 5 above, wherein the amorphous chitin contained in the wound-healing material is in a gel state or in a dried state of gel-like amorphous chitin. Effect of the Invention

[0013] The wound-healing material of the present invention containing the amorphous chitin as an active ingredient can provide an excellent wound-healing material that secretes various cytokines and cell growth factors at the wound site, attracts neutrophils, macrophages and lymphocytes, promotes the inflammatory, cell proliferation and remodeling processes necessary for the wound healing process, and reduces the area of ​​the wound site. [Brief description of the drawings]

[0014] [Figure 1] FIG. 3 is a graph showing the results of comparing wound areas over time when an amorphous chitin solution or distilled water as a control was applied to the wound of a skin ulcer model mouse, with the wound area at the time of wound creation set at 1 (Example 3). [Diagram 2] This is a photograph showing the results of histopathological observation of the area of ​​granulation tissue at the wound in a skin ulcer model mouse, which was performed 7 days after the initial application of an amorphous chitin solution or distilled water as a control to the wound in the model mouse, and the skin tissue of the model mouse was fixed with formalin to prepare tissue sections (Example 5). [Diagram 3] This is a diagram showing the results of histopathological observation of the expression of CD31, a marker for neovascularization, in tissue sections prepared in the same manner as in Fig. 2. This is a diagram showing the results of promoting the formation of neovascularization when a chitin solution is applied. (Example 5) [Figure 4] This is a diagram showing the results of observing neutrophil infiltration into skin tissue by Ly6G staining for tissue sections prepared in the same manner as in FIG. 2. It was confirmed that when the chitin solution was applied, the level of neutrophil infiltration was high, and the wound had progressed to the inflammatory stage of the wound healing process (Example 5). [Diagram 5] This is a diagram showing the results of observing macrophage infiltration into skin tissue by F4 / 80 staining for tissue sections prepared in the same manner as in FIG. 2. When the chitin solution was applied, the level of macrophage infiltration was somewhat higher, and it was confirmed that the wound had progressed to the inflammatory stage of the wound healing process. (Example 5) [Figure 6]FIG. 6 shows the results of measuring the expression level of VEGF (vascular endothelial growth factor) by real-time PCR when THP-1 cells, which are human monocytic cells derived from acute monocytic leukemia, were cultured for 6, 12, and 24 hours in a culture medium containing various concentrations of amorphous chitin solution or distilled water (Example 6). [Figure 7] FIG. 6 is a diagram showing the results of measuring the expression level of TNF (tumor necrosis factor)-α by real-time PCR when THP-1 cells were cultured for 6, 12, and 24 hours in a culture solution containing various concentrations of amorphous chitin or distilled water (Example 6). [Figure 8] 6 shows the results of measuring the expression level of IL (interleukin)-1 by real-time PCR when THP-1 cells were cultured for 6, 12 and 24 hours in a culture solution containing various concentrations of amorphous chitin or distilled water (Example 6). [Figure 9] This is a diagram showing the results of a cell proliferation test when THP-1 cells were cultured in a medium or a culture solution containing amorphous chitin at various concentrations. As a result, no particular difference in the number of live cells was observed between the culture system using only a medium and the culture system containing amorphous chitin at various concentrations, and no cytotoxicity due to amorphous chitin was confirmed. (Example 7) [Figure 10] FIG. 8 is a diagram showing the results of observing granulation formation during wound healing when amorphous chitin or Beschitin (registered trademark) as a control was applied to the wound of a skin ulcer model mouse (Example 8). [Figure 11] FIG. 9 is a graph showing the results of measuring the area of ​​granulation tissue during wound healing when amorphous chitin or Beschitin (registered trademark) as a control was applied to the wound of a skin ulcer model mouse (Example 9). [Figure 12] Example 9 shows the results of applying amorphous chitin or Beschitin (registered trademark) as a control to the wound of a skin ulcer model mouse, fixing the skin tissue with formalin, preparing tissue sections, and observing the infiltration of neutrophils into the skin tissue by Ly6G staining. [Figure 13]Example 9: Amorphous chitin or Beschitin (registered trademark) was applied to the wound of a skin ulcer model mouse, and the skin tissue was fixed with formalin to prepare tissue sections, and the expression of CD31, a marker for neovascularization, was observed histopathologically. [Figure 14] Example 9: Amorphous chitin or Beschitin (registered trademark) was applied to the wound of a skin ulcer model mouse, and the skin tissue was fixed with formalin to prepare tissue sections, and macrophage infiltration into the skin tissue was observed by F4 / 80 staining. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The present invention relates to a wound healing material containing amorphous chitin as an active ingredient. The "amorphous chitin" used as the active ingredient of the wound healing material of the present invention may be amorphous chitin, and is not particularly limited in its production method or properties, but is preferably amorphous chitin that has been deacetylated to a degree that allows it to maintain a fluid state, and may be amorphous chitin that has been post-treated by adding a solvent such as water or various additives after production, or by freeze-drying. As such amorphous chitin, amorphous chitin with a deacetylation degree of 0.1 to 95.0%, preferably 0.1 to 50%, more preferably 0.1 to less than 10%, and even more preferably 0.1 to 5% is suitable. The chitin obtained in this manner maintains a gel state.

[0016] The amorphous chitin used in the present invention can be obtained by a method known per se, but for example, chitin contained in the exoskeleton of crustaceans such as crabs and shrimps, or chitin contained in squids, insects, shellfish, mushrooms, etc. can be used as a raw material, crushed, dissolved uniformly, and regenerated and recovered. For example, it can be prepared as follows. A high-concentration solvent is added to the crushed chitin, and the chitin is dissolved and swelled while stirring to make it uniform. Then, ice of approximately the same volume as the high-concentration solvent and / or a neutralizing agent having approximately the same amount as the high-concentration solvent and opposite chemical properties is added to neutralize the solution so that the pH is within the range of 6 to 9. The heat of neutralization generated at this time is lowered using ice or the like, and the dispersion of precipitated chitin is then washed as necessary and desalted, and the amorphous chitin can be filtered out and regenerated and recovered.

[0017] The "pulverized chitin" used in the production process of amorphous chitin may be a commercially available chitin powder, more specifically, α-chitin powder, etc., which has been pre-pulverized, or may be chitin contained in the exoskeleton of crustaceans such as crabs and shrimps, or chitin contained in squids, insects, shellfish, mushrooms, etc., which is appropriately pulverized in the production process. The method of pulverizing chitin is not particularly limited, and a method known per se can be applied. For example, a method known per se can be applied, such as mechanically pulverizing chitin in a crystalline state without using chemical agents such as an alkaline aqueous solution or concentrated hydrochloric acid. The method of pulverizing the chitin raw material is preferably a method that can obtain a strong shear force, such as a high-pressure homogenizer, a grinder (stone mill), or a media stirring mill such as a bead mill. When pulverizing the chitin raw material using the above-mentioned high-pressure homogenizer, the degree of fineness and homogenization can depend on the pressure at which the raw chitin is pumped into the ultra-high pressure chamber of the high-pressure homogenizer, the number of times it is passed through the ultra-high pressure chamber (number of treatments), and the chitin concentration in the aqueous dispersion.

[0018] In this specification, the "high concentration solvent" can be an alkaline solution or an acidic solution. Since a high concentration alkaline solution or a high concentration acidic solution is used as the solvent, chitin can be efficiently dissolved and swollen in a short time. When the high concentration solvent is an alkaline solution, an acidic solution can be used as a neutralizing agent, and when the high concentration solvent is an acidic solution, an alkaline solution can be used as a neutralizing agent. In this case, a sodium hydroxide solution can be used as the alkaline solution. If the concentration of the sodium hydroxide solution is 30% or less, sufficient dissolution and swelling are difficult to achieve, so 30 to 48% is preferable. As the acidic solution, a hydrochloric acid solution, concentrated sulfuric acid, formic acid, etc. can be used. If the concentration of the acidic solution is less than 6N, sufficient dissolution and swelling are also difficult to achieve, so an acidic solution of 6 to 12N is more preferable (N=normality. equivalent x molar concentration (mol / L)). High concentration alkaline solutions and high concentration acidic solutions are easy to obtain and inexpensive, so they are suitable for mass production.

[0019] A high-concentration solution of 7 to 12 times the weight of the crushed chitin, particularly preferably 10 times the weight, is added to the crushed chitin and allowed to penetrate sufficiently to dissolve and swell the crushed chitin. If the amount of the high-concentration solution is less than 7 times the weight of the crushed chitin, sufficient dissolution and swelling are difficult to achieve, and conversely, if it exceeds 12 times, the solvent that has not reacted with the chitin remains, which is uneconomical. In order to allow the high-concentration solution to penetrate sufficiently into the crushed chitin, it is preferable to treat it for a day and night, specifically for 10 to 24 hours, preferably for 12 to 16 hours. The reaction temperature is not particularly limited and may be room temperature, but can be, for example, 15 to 35°C, preferably 18 to 28°C.

[0020] Since the high-concentration solvent is an alkaline or acidic solution, approximately the same volume of water as the high-concentration solvent and / or approximately the same volume of a neutralizing agent having the opposite chemical properties as the high-concentration solvent is added to neutralize the high-concentration solvent that has penetrated into the crushed chitin so that the pH of the high-concentration solvent is within the range of 6 to 9, preferably 7.5 to 9, and most preferably 8.0 to 8.5. The temperature can be lowered by using ice or the like to reduce the heat of neutralization generated at this time. After that, the dispersion of precipitated chitin is washed as necessary after being left to stand, desalted, and the amorphous chitin can be filtered out and regenerated / recovered. If the residual salt concentration after desalting is 0.1% or less, preferably 0.05 or less, and most preferably 0.01 or less, washing can be considered to be complete.

[0021] The filtration can be carried out by a method known per se. The material of the filter cloth is not particularly limited, and any known material can be used. However, if the filtration is carried out using filter paper or filter cloth, the chitin that is the filtrate may adhere to and be mixed with the filter material, so that a synthetic fiber filter is preferred, and a polyester filter is particularly preferred. For example, the filtration can be carried out while repeatedly washing using a filter press. When a filter press is used, the filtrate can be repeatedly washed and filtered while removing salt, so that the operation can be carried out efficiently, and as a result, amorphous chitin can be mass-produced.

[0022] The chitin produced by the above method is amorphous, and is preferably amorphous chitin having a deacetylation degree of 0.1 to 95.0%, preferably 0.1 to 50%, more preferably 0.1 to less than 10%, and even more preferably 0.1 to 5%.

[0023] The amorphous chitin used in the present invention is preferably chitin that has been deacetylated to an extent that allows it to maintain a fluid state and has enhanced dispersibility. The amorphous chitin in this specification is chitin that has a fiber width of 1 to 20 nm, preferably 1 to 10 nm, and more preferably 5 to 10 nm in TEM observation. The amorphous chitin used in the present invention is preferably in a gel state, but may be in a state of being dried by freeze-drying or the like using gel-like amorphous chitin, or may be in a state of being held on a carrier or the like and then dried.

[0024] In the present invention, the term "wound" refers to wounds caused by various factors, such as trauma, bedsore, vascular (arterial, venous), and neuropathic (diabetic). In addition to general trauma, wound dehiscence after surgery is also included as a wound in the present specification. Ulcers, scars, keloids, and associated contractures resulting from various types of trauma are also included in the wounds of the present specification. For example, diabetic wounds include foot ulcers, diabetic neuropathy, and peripheral circulatory disorders. Ulcers occurring in the ankle joint are often venous. The "wound healing material" of the present invention is particularly effective in healing skin wounds, which are damage to the epidermis or dermis of the skin. Here, the severity and depth of the skin wound are not particularly limited. For example, in addition to general wounds such as incisions, lacerations, cracks, abrasions, crush wounds, contusions, puncture wounds, and bite wounds, bedsores, burns, burns, diabetic ulcers, lower limb ulcers, and lower limb aneurysms are included.

[0025] The wound healing process progresses through the inflammatory, cell proliferation, and remodeling phases. The first phase is the inflammatory phase, in which platelets aggregate when a wound is formed, sealing the wound and secreting various cytokines and cell growth factors, causing neutrophils, macrophages, lymphocytes, and other cells to infiltrate the wound. In the cell proliferation phase, epidermal cells, fibroblasts, vascular endothelial cells, and other cells proliferate, resulting in re-epithelialization and the formation of granulation tissue. This is followed by a relatively long-lasting process called the remodeling phase, in which the scar tissue that has formed is replaced by normal tissue structure.

[0026] During the inflammatory phase, immune cells such as neutrophils, macrophages, and T cells infiltrate the wound, exerting local immune and inflammatory effects such as the removal of bacteria and foreign bodies, and releasing cytokines and cell growth factors. Neutrophils activate fibroblasts and epidermal cells by producing and releasing cytokines such as TNF-α and IL-1. Macrophages stimulate the synthesis of extracellular matrix from fibroblasts, promote angiogenesis and granulation tissue formation, and induce re-epithelialization by epidermal cells by producing and releasing various cell growth factors and cytokines such as VEGF, PDGF (platelet-derived growth factor), TGF (transforming growth factor)-β, and bFGF (basic fibroblast growth factor). Furthermore, T lymphocytes also play an important role in the wound healing process by producing growth factors. It has also been revealed in recent years that B cells also produce cell growth factors and are involved in the wound healing process. In this way, the wound healing process of the skin progresses through close cooperation between cells that are originally present in the skin and various inflammatory cells that infiltrate after wounding.

[0027] The amorphous chitin as an active ingredient contained in the wound healing material of the present invention may be a pharma- ceutically acceptable salt. In this specification, the pharma- ceutically acceptable salt means a salt that does not have a harmful effect on the subject of administration and does not eliminate the pharmacological activity of the active ingredient in the pharmaceutical composition, and specifically includes acid addition salts with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, mandelic acid, tartaric acid, dibenzoyltartaric acid, ditoluoyltartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, aspartic acid, and glutamic acid, salts with inorganic bases such as sodium, potassium, magnesium, calcium, and aluminum, and organic bases such as methylamine, ethylamine, ethanolamine, lysine, and ornithine, salts with various amino acids and amino acid derivatives such as acetylleucine, and ammonium salts.

[0028] The pharmaceutical composition as the wound-healing material of the present invention can be prepared using pharma- ceutical acceptable carriers, excipients, etc. that are commonly used in the art. The wound-healing material of the present invention is particularly used as a composition for external application to the skin, and may be administered in any form, such as a transdermal solution, ointment, transdermal patch, transmucosal solution, or transmucosal patch.

[0029] As used herein, the pharma- ceutically acceptable carriers and excipients include various components, additives, bases, etc. that are commonly used in skin external compositions. The dosage form may be any of liquid, emulsion, cream, gel, paste, spray, etc. Examples of components that may be included in skin external compositions include oils and fats (olive oil, coconut oil, evening primrose oil, jojoba oil, castor oil, hardened castor oil, etc.), waxes (lanolin, beeswax, carnauba wax, etc.), hydrocarbons (liquid paraffin, squalene, squalane, petrolatum, etc.), fatty acids (lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, etc.), higher alcohols (myristyl alcohol, cetanol, cetostearyl alcohol, stearyl alcohol, behenyl alcohol, etc.), esters (isopropyl myristate, palmitic acid, stearic acid, behenic acid, etc.), and the like. Examples of the suitable ingredients include isopropyl myristate, cetyl octanoate, glycerin trioctanoate, octyldodecyl myristate, octyl stearate, stearyl stearate, etc., organic acids (citric acid, lactic acid, α-hydroxyacetic acid, pyrrolidone carboxylic acid, etc.), sugars (maltitol, sorbitol, xylobiose, N-acetyl-D-glucosamine, etc.), proteins and protein hydrolysates, amino acids and their salts, vitamins, plant and animal extracts, various surfactants, moisturizers, UV absorbers, antioxidants, stabilizers, preservatives, bactericides, fragrances, etc. The wound healing material of the present invention can be prepared by selecting and appropriately mixing the above-mentioned pharma- ceutically acceptable carriers and excipients according to their types and by following a method known in the art.

[0030] The pharmaceutical composition as a wound healing material of the present invention can be mixed with pharma- ceutical acceptable additives and formulated into various preparations in a dosage form suitable for application to the affected area. As pharmacologically and pharma-ceutical acceptable additives, a suitable base material for preparation, a carrier, an excipient, a diluent, a binder, a lubricant, a coating agent, a disintegrant or disintegrant aid, a stabilizer, a preservative, an antiseptic, a bulking agent, a dispersant, a wetting agent, a buffer, a solubilizer or solubilizer, an isotonicity agent, a pH regulator, a propellant, a colorant, a sweetener, a flavoring agent, a flavoring agent, a flavoring agent, and the like can be appropriately selected according to the dosage form and use, and prepared into various dosage forms that can be administered orally or parenterally, systemically or locally, by various known methods.

[0031] The active ingredient of the pharmaceutical composition as a wound healing material of the present invention is derived from a natural product, and therefore is very safe and has no side effects, so when used as a wound healing material, it can be administered to mammals such as humans, mice, rats, rabbits, dogs, cats, etc. The dosage of the wound healing material of the present invention is not particularly limited, and an effective amount can be used as appropriate. EXAMPLES

[0032] In order to aid in the understanding of the present invention, the present invention will be specifically described below with reference to examples and experimental examples. However, it goes without saying that the present invention is not limited to these.

[0033] (Example 1) Preparation of amorphous chitin In Example 1, an example of a method for producing amorphous chitin as an active ingredient of a wound healing material used in each of the following experimental examples is described, but the method is not limited to the described content. As the amorphous chitin as an active ingredient of a wound healing material, a wet cake-like dispersion containing nano-chitin at a high concentration was prepared as described below.

[0034] Crab shell-derived α-chitin powder (42 mesh sieved product, deacetylation degree = 1.0% to 2.0% ("Chitin L-PC", Koyo Chemical Co., Ltd.) was mixed with 10 times the weight of 48% sodium hydroxide solution, and the chitin powder was allowed to soak in the sodium hydroxide solution, then left to stand overnight at 20°C or below. Next, crushed ice was added until the sodium hydroxide concentration reached about 10 (w / v)%, and the mixture was stirred at -10°C or below until it became a uniform liquid, obtaining an alkaline chitin dope. Next, concentrated hydrochloric acid was added while adding ice to neutralize the solution until the pH of the solution reached about 8.0 to 8.5, and chitin was precipitated. The precipitated chitin dispersion was repeatedly washed with a large amount of water, and desalted until the salt content measured with a digital salinity meter ("ES-421", Atago Co., Ltd.) was 0.01% by weight or less. The desalted chitin dispersion was dehydrated using a filter press (manufactured by Yabuta Kikai Co., Ltd.) to obtain a wet cake-like dispersion, which was used below as amorphous chitin (gel-like).

[0035] (Example 2) Preparation of a mouse model of intractable skin ulcer Male mice with spontaneous diabetes (db / db mice) were brought in at 7 weeks of age and were pre-bred in cages until they reached 8 weeks of age. Under general anesthesia using an intraperitoneal injection of a mixture of three anesthetics, medetomidine hydrochloride, midazolam, and butorphanol tartrate, the backs of the mice were shaved and depilated to create circular full-thickness skin defects (ulcers) measuring 6 to 10 mm in diameter. In this and the following examples, the "ulcers" in the skin ulcer model mice are referred to as "wounds." To prevent the wounds from shrinking, donut-shaped silicone sheets were attached to the skin around the wounds using adhesive.

[0036] (Example 3) Effect on wound area 1 The wound of the model mouse created in Example 2 was applied with a chitin solution (100 μl) with a chitin concentration of 2.3 to 3% by dissolving the amorphous chitin (gel-like) created in Example 1 in a solvent (pure water) or distilled water (100 μl) as a control, and the wound was covered with a polyurethane dressing film. The chitin solution or distilled water was applied twice, on the first day (day 1) and the fourth day of the wound creation. Four, seven, and ten days after the wound creation, the mouse was euthanized by overdosing with an anesthetic, the wound was photographed with a digital camera, and the wound area was measured.

[0037] As a result, on the 4th and 7th days after wound creation, the wound area in the chitin solution application group had shrunk to 83% and 56%, respectively, compared to Day 0. On the other hand, in the control group, the wound area was 97% and 86%, respectively, demonstrating a wound reduction effect of approximately three times in the chitin solution application group (P=0.23, 0.12). However, after 10 days, the difference tended to decrease (Figure 1).

[0038] (Example 4) Preparation of intractable skin ulcer model mice 2 Male spontaneously diabetic mice (db / db mice) were brought in at 7 weeks of age and were kept in cages until they reached 8 weeks of age. Under general anesthesia using an intraperitoneal injection of a mixture of three anesthetics, medetomidine hydrochloride, midazolam, and butorphanol tartrate, the backs of the mice were shaved and depilated to create a circular full-thickness skin defect (ulcer) with a diameter of 6 mm. To prevent the wound from shrinking, a doughnut-shaped silicone sheet was attached to the skin surrounding the wound using adhesive.

[0039] (Example 5) Histopathological examination of the wound 1 The wound of the model mouse prepared in Example 4 was applied with a chitin solution (100 μl) with a chitin concentration of 2.3 to 3% by dissolving the amorphous chitin (gel-like) prepared in Example 1 in a solvent (pure water) or distilled water (100 μl) as a control, and the wound was covered with a polyurethane dressing film. The chitin solution or distilled water was applied twice, on the first day (day 1) and the fourth day of wound preparation. Four and seven days after wound preparation, the mice were euthanized by overdosing on an anesthetic, and the wound skin tissue was collected. Histopathological examination was performed using the collected wound skin tissue. A part of the collected skin tissue was fixed in 10% formalin buffer, embedded in paraffin, and stained with hematoxylin and eosin. The area of ​​granulation tissue was measured by photographing under a microscope. The area of ​​granulation tissue in the chitin solution application group on the seventh day after wound preparation was significantly increased compared to the control group (P value = 0.003) (Figure 2).

[0040] The wound skin tissue after photographing with a digital camera in Example 5 was collected separately, degreased with cold acetone, embedded in OTC compound, frozen, and stained with CD31 (vascular endothelium), Ly6G (neutrophils), and F4 / 80 (macrophages). Photographs were taken under a microscope, and the number of positive cells per unit area was counted. It was thought that the application of chitin solution increased the number of new blood vessels and inflammatory cells such as neutrophils and macrophages. It was thought that the application of chitin solution induced inflammatory cells, contributing to the proliferation of granulation tissue (Figures 3, 4, and 5).

[0041] Example 6: Confirmation of inflammatory marker expression by real-time PCR THP-1 (DS PHARMA BIOMEDICAL) cells derived from acute monocytic leukemia and human monocytic cells were cultured in a culture medium prepared by mixing FBS (fetal bovine serum) and an antibiotic (1% diluted penicillin-streptomycin mixture (Nacalai Tesque)) in RPMI 1640 medium. THP-1 cells were cultured in a culture medium containing amorphous chitin at various concentrations (0.0043% (1000-fold dilution), 0.00043% (10000-fold dilution), 0.000043% (100000-fold dilution)) prepared in Example 1 or distilled water as a control. After 6, 12, and 24 hours of culture, THP-1 cells were harvested and gene expression of VEGF, TNF-α, and IL-1 was measured using real-time PCR.

[0042] With the addition of 0.0043% amorphous chitin, the most notable increase in gene expression of inflammatory cytokines, such as VEGF, an important factor in angiogenesis, and TNF-α and IL-1, which are important factors in early granulation formation at the wound site, was observed after 6 hours of culture (Figures 6, 7, and 8).

[0043] Example 7: Cell proliferation test A cell proliferation test (Cell Counting Kit-8: CCK-8, Dojin Chemical Laboratory) was performed when THP-1 cells were cultured. THP-1 cells were cultured in RPMI-1640 in a 10 cm diameter culture dish, and THP-1 cells in the logarithmic growth phase were seeded in each well of a 96-well microplate at 100 μl each, so that the number of cells was 5000 per well. After culturing for 24 hours in a CO2 incubator, five wells were cultured for 6, 12, and 24 hours using the culture solution or the culture solution containing amorphous chitin prepared in Example 1 at each concentration (0.0043% (1000-fold dilution), 0.00043% (10000-fold dilution), 0.000043% (100000-fold dilution)). After incubation, 10 μl of CCK-8 (WST-8: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium) solution was added to each well, and the color reaction was carried out in a CO2 incubator for 3 hours. The absorbance at 450 nm was then measured using a microplate reader (iMark Microplate Reader, BIO-RAD). As a result, no difference was observed between the medium and the chitin-added groups at different concentrations, suggesting that there is no cytotoxicity due to amorphous chitin (Figure 9).

[0044] (Example 8) Effect on wound area 2 The wound of a skin ulcer model mouse prepared by the same method as in Example 4 was applied with the amorphous chitin (gel-like) prepared in Example 1 dissolved in a solvent (pure water) at a chitin concentration of 2.3 to 3%, and as a control group, Beschitin (registered trademark) of a size adjusted to the wound area was applied. The wound was covered with a polyurethane dressing film. Amorphous chitin (chitin content: 0.06 mg / mm 2 ) was applied twice on the first and fourth days. 2 ) was applied only once on the first day.

[0045] Four and seven days after wound creation, the mice were euthanized by an overdose of anesthetic, and the wound was photographed with a digital camera and the wound area was measured. 2 ) and beschitin (chitin content: 0.2 mg / mm 2 ) showed little difference in the effect on wound area reduction (Figure 10).

[0046] (Example 9) Histopathological examination of the wound 12 In Experimental Example 8, the wound was photographed with a digital camera, and then skin tissue was collected for histological examination and gene expression measurement. A portion of the collected skin tissue was fixed in 10% formalin buffer, embedded in paraffin, and stained with hematoxylin and eosin. The tissue was photographed under a microscope, and the area of ​​granulation tissue was measured. Another portion was degreased with cold acetone, embedded in OTC compound, frozen, and stained with CD31 (vascular endothelium), Ly6G (neutrophils), and F4 / 80 (macrophage). The tissue was photographed under a microscope, and the number of positive cells per unit area was counted.

[0047] There was no significant difference in granulation formation during wound healing between amorphous chitin and Beschitin (registered trademark). Four and seven days after wound creation, the wound area in the amorphous chitin application group was reduced to (62% and 35%) compared to the wound on day 0, respectively, and there was no significant difference in the wound reduction effect compared to the control group (58% and 33%) (Figure 12). Histopathological examination showed that the granulation tissue area in the amorphous chitin application group on day 7 after wound creation was also not significantly different from the control group (Figure 11). Both amorphous chitin and Beschitin (registered trademark) increased neovascularization and infiltration of inflammatory cells such as neutrophils and macrophages, but it was thought that there was no significant difference in the effects (Figures 12, 13, and 14). These results revealed that amorphous chitin can achieve the same wound healing promotion effect at a lower concentration compared to the control Beschitin (registered trademark).

[0048] (Experimental Example 1) Measurement of the degree of deacetylation of amorphous chitin The degree of deacetylation of the amorphous chitin (gel-like) prepared in Example 1 and the raw chitin was measured by colloid titration.

[0049] The chitin concentration of amorphous chitin and raw chitin was calculated from the moisture content, and each was collected so that it was 2.5 g of chitin. 2.5 g of each chitin was added to 495 g of water (H2O) together with 2.5 g of acetic acid and stirred for 3 hours. 1 g of the solution was taken and added to 50 ml of H2O, toluidine blue indicator was added, and titration was performed with N / 400 potassium polyvinyl sulfate solution, and the degree of deacetylation was calculated for each using the formula.

[0050] The results of the measurement of the degree of deacetylation are as follows: [Table 1] [Industrial Applicability]

[0051] The wound healing material containing the amorphous chitin of the present invention as an active ingredient was subjected to in vivo and in vitro experiments. In the in vivo system, the group in which the chitin solution was applied showed a wound reduction effect compared to distilled water. Histopathological findings showed a significant increase in the proliferation of granulation tissue, and an increase in neovascularization and inflammatory cells such as neutrophils and macrophages. Furthermore, in comparison with the effect of crystalline chitin, it was confirmed that the same wound healing promotion effect could be obtained at a lower concentration. In the in vitro system, it was expected that various cytokines and cell growth factors would be secreted to induce neutrophil, macrophage and lymphocyte infiltration, and the inflammatory, cell proliferation and remodeling phases required for the wound healing process would be promoted. On the other hand, the amorphous chitin as an active ingredient is made from chitin derived from natural products, and has no cytotoxic effect. It can be said that it is industrially very excellent because it can exert an excellent wound healing effect at a low concentration of chitin.

Claims

1. A skin topical composition for wound healing comprising, as an active ingredient, amorphous chitin having a deacetylation degree of 0.1 to 5%.

2. The composition for external application to the skin according to claim 1, wherein the amorphous chitin contained as an active ingredient in the composition for external application to the skin is in a state capable of maintaining a fluid state.

3. The composition for external use on the skin according to claim 1 or 2, characterized in that in the amorphous chitin contained as an active ingredient in the composition for external use on the skin, the chitin concentration in an aqueous dispersion as a chitin solution is 2.3 to 3%.

4. The composition for external application to the skin for wound healing contains amorphous chitin as an active ingredient having a deacetylation degree of 0.1 to 5%, and the amorphous chitin is in a gel state or in a dried state of gel-like amorphous chitin.

5. A topical skin composition described in any of claims 1 to 4, characterized in that amorphous chitin contained as an active ingredient in the topical skin composition has the effect of enhancing factors important for angiogenesis and / or the effect of enhancing factors important for early granulation formation in a wound.

6. A topical skin composition as described in claim 5, wherein the factor important for angiogenesis is VEGF (vascular endothelial growth factor).

7. A topical skin composition as described in claim 5, wherein factors important for the early granulation formation in the wound are TNF (tumor necrosis factor) and / or IL-1.

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