KR101820306B1 - Gallic Acid-Chitosan Complexes and Composition for Wound Healing Comprising Them - Google Patents

Abstract

The present invention relates to a chitosan complex which has excellent adhesion and hemostasis effects, has antibacterial efficacy and is effective in healing wounds due to high biocompatibility, and to a composition for healing wounds comprising the same and, more specifically, to a gallic acid-chitosan complex in which gallic acid is bound to chitosan by an ester bond or an ester bond and an amine bond, and a composition for healing wounds comprising the same.

Description

[0001] The present invention relates to gallic acid-chitosan complexes, and compositions for treating wounds using the same. [0002] Gallic Acid-Chitosan Complexes and Composition for Wound Healing Comprising Them,

The present invention relates to a composition for wound healing which is excellent in adhesive force and hemostatic effect, has antibacterial effect and high biocompatibility and is effective for wound healing.

The wounds are in a state where the continuity of the tissue is destroyed by external pressure or a defect is formed in some areas. Wounds of hardness, such as simple trauma, can be regenerated by self-repair function, but it is difficult to restore intractable wounds such as severe burns, multiple wound, pressure sores, and surgical wounds. Because intractable wounds or extensive wounds can leave defects in the functioning of the tissue, treatment is needed to quickly treat the wound area and minimize secondary side effects.

It is known that the wound healing rate is about twice as fast when the wounds are maintained in a dry state. Conventionally, a method of dressing with a solution containing a physiological solution or a disinfecting agent in a wet state has been used to maintain the wet state. However, since the replacement cycle of the dressing should be frequent, management is troublesome, and not only the wound area but also the peripheral area is wetted by the physiological solution or the disinfectant, thereby causing skin rash. In recent years, a method has been widely used in which a wound covering material such as hydrogel or hydrocolloid is used to protect the affected part, to prevent water leakage from the body, and to absorb the exudate to maintain the wet environment.

The wounds usually involve bleeding, and the wider the wounds are, the more severe the bleeding is. In order to be able to effectively treat wounds effectively at the initial stage, a hemostatic effect on the bleeding associated with the wound is required, and then it is necessary to prevent tissue necrosis by inhibiting infection of the wound tissue caused by bacterial contamination. It is also effective to reduce the frequency of treatment of the wound healing agent and make the wound healing agent itself sticky so as to obtain a long-term effect by a single treatment. In addition, biocompatibility should be excellent because it is likely to be absorbed into the body by direct contact with the wound area, and biodegradability should be possible in addition to biocompatibility when applied to deep wound.

Chitosan is a type of polysaccharide present in nature and is obtained by deacetylation of chitin contained in the shell of crustaceans such as crabs, shrimp, and microbes such as bones, fungi and bacteria of squid. Chitin is not only resistant to chemicals but also soluble in water and most solvents, while chitosan is readily soluble in low concentrations of inorganic or organic acids and forms hydrogels. Chitosan has been mainly used in industrial fields such as wastewater treatment and agrochemicals, but its application range of foods, health hygiene and pharmaceuticals is expanding as various physiological activities are known. Chitosan, which is composed of glucosamine bonds, is very promising as a medical material because its molecular structure is very similar to that of human tissues and does not cause an immune response and is highly biocompatible and biodegradable by enzymes such as chitosanase and lysozyme.

Since the chitin powder pulverized in German Patent No. 1,906,159 has been shown to exhibit an excellent effect on wound healing, gauze dressing materials using chitin or chitosan have been actively developed. In recent years, it has been known that chitosan can rapidly clot blood, and has been approved as a hemostatic agent in the United States and Europe. Chitosan hemostatic agents reduce blood loss and improve patient survival compared to gauze dressings, have less irritation, have antibacterial properties, and have a role in reducing pain by blocking nerve endings. Patent Nos. 1700107 and 1429455 disclose hydrogel patches for hemostatic dressings and wounds using the above characteristics of chitosan. However, the conventional gauze type dressing material has a problem that the dressing material adheres to the wound surface, which is not easy to replace, and causes damage to the new tissue at the time of replacement.

In recent years, complexes in which chitosan is conjugated with glucan, hyaluronic acid and catechol have been developed in order to further improve the properties of chitosan as a composition for treating wounds. Patent No. 1301276 is for use as a tissue adhesive by combining a catechol-chitosan conjugate with a flakoser, and Patent No. 1320960 discloses a mixture of a chitosan-glucan conjugate and a disinfectant by a wetting wound healing and a bandage As an anti-adhesion agent. Patent No. 1686343 discloses a medical implantable material with enhanced strength using a bioabsorbable polymer composite comprising a chitosan-hyaluronic acid complex.

Patent Document 10-2015-0131951 confirms that excellent adhesion is increased by introducing a gallic acid group into chitosan by covalently bonding gallic acid with an amine group of chitosan and applying this to a support for biomaterials, tissue engineering, or a drug delivery carrier . However, as gallic acid is introduced into the amine group of chitosan, the solubility of chitosan decreases rapidly. In order to maintain the physical properties of the hydrogel, a small amount (less than 10%) of the gallic acid must be bonded to the free amine group. That is, if the introduction rate (binding rate) of gallic acid is low, the effect of introducing gallic acid is not sufficient, and if the introduction rate (binding rate) is increased to increase the effect of introducing gallic acid, the solubility is lowered. Also, as the free amine groups of chitosan are decreased by galactosylation, the hemostatic and antimicrobial effects of chitosan are decreased.

German Patent No. 1,906,159 Patent No. 1700107 Patent No. 1429455 Patent No. 1301276 Patent No. 1320960 Patent No. 1686343 Published Japanese Patent Application No. 10-2015-0131951

Disclosed is a composition for wound healing which is high in biocompatibility, excellent in adhesive force and hemostatic effect, exhibits an antimicrobial effect incidentally, and has a solubility suitable for formation of a hydrogel, so that it is effective for wound healing.

Another object of the present invention is to provide a composition for wound healing which is biodegradable and applicable not only to skin but also to internal tissues during surgery.

In order to accomplish the above object, the present invention relates to a gallic acid-chitosan complex characterized in that gallic acid is bound to chitosan by an ester bond or an ester bond and an amide bond.

Chitosan is made by making the N-acetyl group of chitin water-soluble by alkaline deacetylation. In the present invention, it is preferable to use chitosan having a deacetylation level of 70% or more so as to exhibit adequate solubility. However, it does not exclude the use of chitosan having a lower deacetylation level than 70%. The gallic acid-chitosan complex of the present invention is obtained by ester bonding of an alcohol group, particularly -OH group at position 6 of chitosan and gallic acid. In addition, in the gallic acid-chitosan complex of the present invention, gallic acid is bound to chitosan by ester bonding. In some of the glucosamid units in chitosan, gallic acid is also bonded by an amide bond. That is, the gallic acid may be bonded to chitosan by an ester bond and an amide bond. "Conjugate" in the present invention means that gallic acid is chemically bonded to chitosan.

The ester bond between gallic acid and chitosan can be achieved by an ester bond formation reaction by reaction of a carboxylic acid or its derivative with an alcohol. Also, as in the embodiment, ester groups may be protected before formation of an ester bond to form an ester bond, and when the protecting group is removed, the acid may be bound only to the acid by the ester bond. However, if the amine is reacted in a non-protected state, it is also possible to prepare a conjugate in which the bonding of the acid group in the conjugate is simultaneously performed by the ester bond and the amide bond.

The gallic acid-chitosan complex of the present invention preferably has a number average molecular weight of 4,000 to 1,000,000, more preferably 10,000 to 300,000. When the molecular weight was too small, the adhesion decreased. When the molecular weight was too large, the biodegradability and solubility in water were low.

The gallic acid group in the gallic acid-chitosan complex is preferably contained in an amount of 0.1 to 30% by weight based on the chitosan back-bone. As the ratio of gallic acid in the gallic acid - chitosan complex increased, the adhesion increased and the hemostatic effect also increased.

In the gallic acid-chitosan complex of the present invention, since gallic acid is bound to chitosan by ester bonding, the free amine group of chitosan remains. As a result, the solubility in water can be maintained even when the ratio of bound gallic acid increases to 30%, and the antimicrobial effect and hemostatic effect are also remarkable. According to the results of preliminary experiments, the gallic acid-chitosan conjugate in which gallic acid was bound to chitosan by amide bond greatly decreased the solubility of gallic acid when the content of gallic acid was 10 w% or more, and the hemostatic effect was greatly reduced. And the efficacy of wound healing was low.

The present invention also relates to a wound-healing composition comprising the galance-chitosan conjugate.

In the present invention, a wound does not only mean a wound on the skin, which is an external tissue, but also includes a wound of the internal body tissue. The cause of wound is not limited to trauma, burn, pressure ulcer, surgical operation, etc., and it is applicable to all parts requiring wound treatment. Chitosan is known to be useful as a hemostatic agent, but it is mainly used in gauze and pads because of its weak adhesion. On the other hand, the gallic acid-chitosan complex has a superior hemostatic effect due to the introduction of a gallic acid group, and the adhesion is increased. As a result, even when applied in the form of powder or granules on the wound area itself, And it was able to maintain the state of sticking to the wound area. That is, even if a separate support such as a gauze or a band is not used, it is possible to perform continuous action without being detached from the wound region, and it is possible to maintain a wet state as a hydrogel, thereby enabling rapid healing without causing pain due to drying have. In addition, it has high biocompatibility and biodegradability. Therefore, about 90% of the biodegradable biodegradable material can be biodegraded in about 4 weeks in the test tube test. Therefore, it is not necessary to remove biodegradable material even if it is used for internal tissues. low. Even when the gallic acid-chitosan complex is applied as a powder or a granule, it is converted into a hydrogel by absorption of a biotissue. However, it may be formulated into a hydrogel itself for more effective application. In addition, galance-chitosan conjugates may be applied to a support such as a gauze, band or patch, or incorporated into gauze, band, patch material, or used as a material to produce gauze, band or patch, It can also be used.

As the molecular weight of the chitosan complex increased, the glacial-chitosan complexes exhibited a delayed biodegradation rate. As the content of gallic acid increased, the adhesion increased and the hemostatic effect rapidly increased. Therefore, the amount of gallic acid added or the molecular weight of chitosan can be suitably selected in consideration of the cause of wound healing to which the wound healing composition of the present invention is applied, the wound area, and the range of wound healing. For example, when the range of the wound surface is narrow, it is possible to treat the healing more quickly than the case where the wound surface range is wide. Therefore, a composition containing a gallic acid-chitosan complex having a smaller molecular weight of chitosan can be selected so that biodegradation can proceed rapidly. Chronic acid-chitosan complexes with a high content of gallic acid can be used on the wounds of areas with a lot of movement, or where there is a lot of bleeding or concern about bleeding, which is highly sticky and has better haemostatic effect.

In addition, the composition for wound healing of the present invention can be used in combination with additives commonly used in the pharmaceutical field such as carriers and excipients. The composition of the present invention may be administered alone or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.

As described above, according to the gallic acid-chitosan complex of the present invention, antibiosis, stickiness, and hemostatic effect are further increased compared with chitosan while maintaining solubility in water even when the galactosyl group is introduced. When applied to the wound area without a separate support It quickly exhibits a hemostatic effect, absorbs the biotissue fluid, rapidly forms a hydrogel, and is maintained in a state of being adhered to the wound, so that it can be useful for wound healing.

In addition, the composition for wound healing including the gallic acid-chitosan complex of the present invention has high biocompatibility, is easily biodegradable, exhibits antimicrobial properties, and therefore does not require frequent replacement of dressing, Since it is decomposed and absorbed into a harmless substance in the body, it can be effectively used for the treatment of wound.

1 is a ¹ H-NMR spectrum of a gallic acid-chitosan complex.
FIG. 2 is a TGA curve of a gallic acid-chitosan complex. FIG.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these embodiments are merely examples for explaining the content and scope of the technical idea of the present invention, and thus the technical scope of the present invention is not limited or changed. It will be apparent to those skilled in the art that various changes and modifications can be made within the scope of the technical idea of the present invention based on these examples.

[Example]

Example: Preparation of gallic acid-chitosan ester conjugate

The gallic acid - chitosan complex in which gallic acid is bound to chitosan by ester bonding was prepared by esterification reaction from gallic acid derivative and chitosan.

5 g of chitosan HCl (KITO LIFE, Korea, number average molecular weight 4.7K Da, 12K Da, 300K Da or 1,000K Da) was dissolved in 50ml of distilled water, and then 0.928g of NaOH . 2.83 ml of p-anisaldehyde was added dropwise to the solution and reacted at 4 ° C for 8 hours. After 8 hours, the resulting white precipitate was filtered, washed sequentially with 100 ml of cold distilled water, ethanol, ethyl ether and dried to yield 4.8 g of a compound in which the free amine group was protected with a p-anisaldehyde group.

The compound in which 4.8 g of the amine group was protected was dissolved in 150 ml of DMF, and 1 g of 3,4,5-trihydroxybenzoyl chloride was added thereto, followed by reaction at room temperature for 48 hours. After the reaction was completed, 500 ml of distilled water was added to form a precipitate. This white precipitate solution was filtered, washed sequentially with 100 ml of cold distilled water, ethanol, and ethyl ether, and dried to synthesize 5.5 g of a halogen-substituted chemical.

The above 5.5 g of the compound was dissolved in 200 ml of acetone and refluxed. Then, 3.8 ml of 36% HCl was dissolved in 8 ml of distilled water, and the mixture was slowly added dropwise, followed by further refluxing for 20 minutes. The reaction solution was cooled and the insoluble precipitate of white was filtered, washed with 50 ml of ethyl ether, and then dried to convert some of the alcohol groups of the chitosan backbone into gallic groups. The amine groups were replaced with -NH ₂ .HCl, - chitosan ester conjugate.

Figure 1 is a gallic acid produced by this number-average molecular weight of the chitosan used to 4.7K - shows a ¹ H-NMR spectrum of the chitosan conjugate with ¹ H-NMR spectrum of gallic acid and of chitosan. In the gallic acid-chitosan complex, ¹ H derived from chitosan and ¹ H peak derived from gallic acid were all observed, confirming that a gallic acid group was bonded to chitosan. The gallic acid-chitosan complexes prepared using chitosan of 12K, 300K and 1,000K also showed similar ¹ H-NMR spectra.

The gallic acid-chitosan complex was prepared by changing the amount of gallic acid to 1 to 30 wt% by the same method as described above.

Figure 2 shows the And the TGA curve of the gallic acid-chitosan complex. The TGA curve shows that the physical properties of chitosan vary greatly even when the amount of gallic acid added is as small as 1%.

Comparative Example: Preparation of gallic acid-chitosanamide conjugate

The gallic acid-chitosan complex in which gallic acid is bound to chitosan by amide linkage was prepared by EDC-NHS coupling from gallic acid and chitosan.

More specifically, 1 g of chitosan (KITO LIFE, Korea, number average molecular weight 300K Da) was dispersed in 100 ml of purified water, 5 M HCl was dissolved by dropping, and the pH was adjusted to 5 to 6 using 5M NaOH.

84 mg of gallic acid and 163.2 mg of EDC were dissolved in purified water (100 ml and 60 ml, respectively), mixed and reacted at room temperature for 10 minutes. A solution of 113.68 mg of NHS dissolved in 35 ml of purified water was added to the reaction solution, followed by further reaction at room temperature for 10 minutes.

The chitosan solution dissolved by the above method was added to the reaction solution of gallic acid and the reaction was carried out at room temperature for 24 hours. After 24 hours, the reaction solution was dialyzed with MWCO 3500 membrane for 48 hours and lyophilized.

Experimental Example 1: Evaluation of stickiness

0.2 g of the gallic acid-chitosan complex powder prepared in the Example was dissolved in 10 mL of PBS buffer solution of pH 7.4 to prepare a hydrogel. The prepared hydrogel was coated on a PET film support to prepare a hydrogel sheet. For comparison, a hydrogel sheet was prepared by the same method using chitosan instead of gallic acid-chitosan complex or gallic acid-chitosanamide bond prepared in Comparative Example. In the case of the gallic acid-chitosan amide conjugate prepared by the method of the comparative example, when the gallic acid concentration is 10% or more, the solubility in the buffer solution is low and hydrogel production is difficult, and the gallic acid (8%) -chitosan (300K) Respectively.

Then, the hydrogel sheets were adhered to the same positions of the upper and lower arms in a size of 3 x 4 cm 2 in 20 healthy adult men and the degree of detachment after 8 hours was measured to evaluate the adhesiveness. After 8 hours, 5 points for not peeling, 4 points for peeling 10% or less, 3 points for peeling 25% or less, 2 points for peeling 50% or less and 1 point for peeling 50% . Table 1 below is the average stickiness evaluation index measured by the above method.

As can be seen in Table 1, it was confirmed that the gallic acid-chitosan complex increased the adhesion more than the chitosan. Especially, as the content of gallic acid increased, the adhesion increased and the introduction of the gallic acid group affected the increase of the adhesion . From this, it was confirmed that the gallic acid-chitosan complex can be stably fixed on the wound area without using a separate support such as gauze. In contrast, the gallic acid-chitosan amide conjugate showed increased adherence rather than chitosan, but showed no adherence to the extent possible without using a separate support due to the low content of gallic acid.

Experimental Example 2: Evaluation of hemostatic effect

The blood hemostatic effect was evaluated by adding a gallic acid-chitosan conjugate or chitosan to the blood and measuring the blood coagulation induction time. More specifically, 1 ml of blood was dropped in a Petri dish, and then 1 g of a gallic acid-chinosanic acid binding powder or chitosan powder was sprayed on the petri dish. Thereafter, the change in blood was observed, and the time required for blood coagulation was measured, and the results are shown in Table 2.

Both the gallic acid - chitosan complex and chitosan started to coagulate immediately when added to the blood, and it was confirmed that the blood was completely clotted 1 to 3 minutes later. The gallate - chitosan conjugate showed a slightly faster effect than the chitosan hemostasis.

Experimental Example 3: Evaluation of antibacterial activity

The antimicrobial effect of the gallic acid-chitosan complex or chitosan prepared in the above examples was measured for four individual strains by MIC (minimum inhibitory concentration) test and MBC (minimum bactericidal concentration) test. Escherichia coli (ATCC 11229), S. aureus ATCC 6538, Candida albicans ATCC 10231, and A. niger ATCC 9642 were used as the strains.

For the MIC test, the gallic acid-chitosan complex or chitosan prepared in the examples was diluted by a 2-fold serial dilution method in a 24-well plate and dispensed. The microorganisms were inoculated at a concentration of 10 ⁵ CFU / ml in each well and cultured at 30 ° C for 48 hours. The microbial growth was visually judged based on the turbidity. The medium used was TrypticSoyBroth (DifcoCo.) From Dipcon.

The MBC test was carried out by immersing a sample of each well subjected to the MIC test on a solid medium and determining the concentration showing a killing rate of 99.9% of the initial concentration as MBC.

Table 3 and Table 4 show MIC and MBC test results, respectively. Chitosan also showed antibacterial effect, but it was confirmed that antibacterial effect was further increased when gallic acid was combined.

Experimental Example 4: Evaluation of cytotoxicity

Fibroblast was dispensed into a 24-well plate at a concentration of 10 ⁴ cells / well, and a glacial-chitosan complex or 0.1 g of chitosan was added to each well. After 1.5 ml of serum-free medium was added, the cells were incubated at 37 ° C and CO ₂ for 24 hours. Cell viability was measured by absorbance at 590 nm by MTT assay to evaluate cytotoxicity.

Table 5 below shows the results. It can be confirmed that the gallic acid-chitosan complexes prepared in the Examples have low cytotoxicity like chitosan and can be safely used.

Experimental Example 5: Evaluation of biodegradability

In order to evaluate the biodegradability of the gallic acid-chitosan complex, 0.1 g of the gallic acid-chitosan complex prepared in Example was administered to 3 ml of PBS (pH 7.4) buffer in which 2 mg of lysozyme (Sigma L-6876) ≪ / RTI > Every 5 days, 1 ml of lysozyme-dissolved PBS buffer was added to maintain the enzyme activity.

After 6 weeks, the buffer was centrifuged, and the residue was washed and dried to obtain the undegraded gallic-chitosan conjugate and weighed. For comparison, the same experiment was carried out on chitosan, and the weight of the residue remained without decomposition was measured.

Table 6 shows the results. It was confirmed that the weight of residual gallic acid-chitosan complex and chitosan was 5-30 mg and 5-10 mg, respectively, and 70-90% of biodegradation was observed.

Claims (6)

Wherein the gallic acid is selectively bound to the hydroxyl group of the chitosan by ester bonding.
The method according to claim 1,
Wherein the gallic acid-chitosan complex has a number average molecular weight of 4,000 to 1,000,000.
3. The method according to claim 1 or 2,
Wherein the gallic acid group of the gallic acid-chitosan complex is contained in an amount of 0.1 to 30% by weight based on the chitosan back-bone.
A composition for treating wounds comprising the gallic acid-chitosan conjugate of claim 1.
5. The method of claim 4,
Wherein the composition is powder, granule or hydrogel.
5. The method of claim 4,
Wherein the composition is applied to a gauze, band or patch.

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