KR101274608B1 - Anti-adhesion agent comprising curdlan and gellan gum, and process for producing the same - Google Patents

Abstract

The present invention relates to a biodegradable, biodegradable and excellent anti-adhesion composition using curdlan and gellan gum as active ingredients, and a method for preparing an anti-adhesion agent using the same. The anti-adhesion agent according to the present invention can prevent the adhesion phenomenon caused by the surgical operation, infection and trauma.

Description

Anti-adhesion composition comprising curdlan and gellan gum as an active ingredient and a method for manufacturing the same {ANTI-ADHESION AGENT COMPRISING CURDLAN AND GELLAN GUM, AND PROCESS FOR PRODUCING THE SAME}

The present invention relates to a biodegradable, biodegradable and excellent anti-adhesion composition using curdlan and gellan gum as active ingredients, and a method for preparing an anti-adhesion agent using the same. The anti-adhesion agent according to the present invention can prevent the adhesion phenomenon caused by the surgical operation, infection and trauma.

Adhesion (adhesion) refers to a phenomenon in which the tissues or organs of the human body that should be separated from each other to form a fibrous tissue by the wound does not fall from each other.

Frequent tissue adhesion after surgery occurs in most surgical procedures, and causes intestinal obstruction, chronic pain, sexual dysfunction, and infertility. If there is no symptom improvement by non-surgical treatment, surgical treatment should be considered actively. Car surgery presents many difficulties for doctors and patients.

The methods of preventing such adhesion include minimizing wounds during surgery, activating tissue plasminogen activator to prevent the formation of anti-inflammatory agents, or forming fibrin. Recently, a method of using a physical barrier has been developed. It is used. In particular, the physical adhesion barrier to apply and adhere to the wound site (physical adhesion barrier) directly blocks the occurrence of adhesion, so the effect is excellent, the physical adhesion inhibitor is mainly used in the current clinical.

Recently, studies on physical anti-adhesion agents using bioabsorbable polymers have been actively conducted. Bioabsorbable polymers are hydrated in vivo to isolate the wound from surrounding tissues during wound healing, thus preventing adhesions.After treatment is completed, the bioabsorbable polymer naturally absorbs and is removed and does not affect normal tissue. Attempt is in progress.

An example of such a bioabsorbable polymer is hyaluronic acid (HA). However, since HA is decomposed and absorbed in a relatively fast time in the living body and has high water solubility, there is a problem of diffusing and flowing from the surface of the wound in a short time, thereby limiting the performance as an anti-adhesion agent. Methyl cellulose and its derivatives are also known to prevent adhesion. However, solutions of such polymers are also absorbed too quickly and cannot exhibit the desired anti-adhesion effect.

The present invention is to solve the above problems of the conventional anti-adhesion agent and to develop an anti-adhesion agent having a function of maintaining the shape in vivo and as a protective film, the present inventors are a hydrogel based on the curdlan and gellan gum The present invention has been completed by discovering that the anti-adhesion agent may be used as an effective anti-adhesion agent.

It is an object of the present invention to provide an anti-adhesion composition comprising curdlan and gellan gum as active ingredients.

Another object of the present invention is to provide a method for preparing an anti-adhesion agent comprising curdlan and gellan gum as active ingredients.

The present invention can minimize or prevent adhesions occurring after surgery, prevent formation of adhesions after primary surgery, prevent remodeling of adhesions after secondary surgery intended for removal of adhesions, and disintegrate and / or Or relates to an anti-adhesion composition comprising an absorbable curdlan and gellan gum as an active ingredient. The present invention also relates to a method for preparing an anti-adhesion agent comprising curdlan and gellan gum as active ingredients.

Curdlan, which is used as an active ingredient in the present invention, is an insoluble β-glucan in which glucose is bound by β-1,3-glucoside bonds, which is one of the distinguishing characteristics of the polysaccharide. Heat coagulation to form a gel. The gel formed at this time has heat resistance which does not dissolve even when heated again. In addition, it has been confirmed that curdlan has a very low toxicity and high stability, and thus is widely used in food and various industrial fields.

Gellan gum used as an active ingredient in the present invention is an anionic polymer that forms a reversible gel, it is reported that heat and cations are required to form a gel, deacetylated gellan gum forms a gel similar to agar, It shows a unique melting point. Gellan gum is a linear anionic polysaccharide in which four sugars are repeated. In the natural state, glucose 6-position is o-acetylated per repeat unit. Gellan gum has a high stability to the human body and has a property that can be easily gelled, and researches are actively conducted to use it as a drug delivery raw material and a basic medical agent that combine with moisture of the human body.

The anti-adhesion composition according to the present invention comprises curdlan: gellan gum in a weight ratio of 1: 0.3 to 3, when the mixing ratio of the curdlan and gellan gum is out of the above range as appropriate adhesion, adhesion, coating properties Difficult to meet In addition, the gelation rate and gel strength of the gellan gum alone and gel strength is not high, the flexibility as a hydrogel was not good, but when using the mixed gelle and gellan gum as in the present invention, the gelation rate and gel strength is low, the decomposition rate is high hydrogel The flexibility was excellent and the adhesion prevention effect was also excellent.

The anti-adhesion composition according to the present invention can be prepared in a foam, powder or film formulation, bioactive proteins, functional peptides, anticancer agents, antithrombotic agents, steroids, nonsteroidal anti-inflammatory agents, hormonal chemotactic factors, analgesics Alternatively, one or more drugs selected from the group consisting of anesthetics may be further included.

The anti-adhesion agent according to the present invention is in the form of a hydrogel, wherein the hydrogel refers to a three-dimensional network structure in which hydrophilic polymers are crosslinked by covalent or non-covalent bonds. Hydrogel is a polymer material that can contain moisture, mainly hydroxyl group (-OH), carboxyl group (-COOH), amine group (-CONH ₂ ), amide group (-CONH-), sulfone group (-SO ₃ H Hydrophilic functional groups such as), capillaries, and osmotic pressures cause moisture to function. Hydrogel not only can be manufactured in the form of plate, fine particles, nanoparticles, coatings, films, etc., but also has excellent biocompatibility. It is widely used and widely applied in laboratory and clinical.

The anti-adhesion agent according to the present invention is prepared by mixing a curdlan, gellan gum and water to prepare an aqueous gel solution, and molding and drying the gel aqueous solution. The weight of the mixture is about 1 to 2 parts by weight of the curdlan and gellan gum. It comprises a part, the mixture of the curdlan and gellan gum is mixed in the weight ratio of the cudlan: gellan gum 1: 1: 0.3 to 3. The anti-adhesion agent of the present invention can be prepared in a hydrogel foam, hydrogel powder or hydrogel film formulation.

In addition, in the present invention, in order to prove the anti-adhesion effect of the composition prepared by the above method, the anti-adhesion efficacy test was performed on various types of formulations. Adhesion-forming animal models refer to rat adhesion models. Based on this animal model, by comparing and testing the anti-adhesion agent prepared by the method proposed in the present invention, it was confirmed that the result showed excellent anti-adhesion effect.

The anti-adhesion composition comprising the excellent biocompatibility according to the present invention, the curdlan and gellan gum as an active ingredient, the measured values for the degree of adhesion and adhesion strength is significantly reduced, the duration of the body is extended, and excellent adhesion prevention. In addition, the anti-adhesion agent of the present invention, unlike the anti-adhesion film made through chemical crosslinking with existing chemical materials or synthetic materials, is manufactured through non-chemical methods as a material suitable for living beings, which is more safe and prevents inflammation of wounds. Can be.

1-Gelation rate measurement result of Experimental Example 1
2-Decomposition rate measurement result of Experimental Example 2
3-Gel strength measurement result of Experimental Example 3
Figure 4-Results of confirming cytotoxicity of Experimental Example 4
5-Adhesion degree and adhesion strength results in the animal experiment of Experimental Example 5
6-photographs showing the results of the adhesion experiment in the animal experiment of Experimental Example 5 ((A) and (B): untreated group, (C) and (D) anti-adhesion treatment group of Example 1)

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited to the following examples.

Example 1 Preparation of Hydrogel Adhesion Prohibitor Including Curdlan and Gellan Gum

Dissolve 0.75 parts of curdlan and 0.75 parts of gellan gum in 100 parts of distilled water, stir at 25 ° C. for 1 hour, pour into 50 mm × 12 mm of Petri dish, and dry in clean bench for 2 days. An inhibitor was prepared.

Comparative Example 1: Preparation of anti-adhesion agent containing curdlan

1.5 parts by weight of curdlan was dissolved in 100 parts by weight of distilled water, stirred at 25 ° C. for 1 hour, poured into a petri dish of 50 mm × 12 mm, and dried in a clean bench for 2 days to prepare a hydrogel anti-adhesion agent.

Comparative Example 2: Preparation of Anti-adhesion Agent Containing Gellan Gum

1.5 parts by weight of gellan gum was dissolved in 100 parts by weight of distilled water, stirred at 25 ° C. for 1 hour, poured into a petri dish of 50 mm × 12 mm, and dried in a clean bench for 2 days to prepare a hydrogel anti-adhesion agent.

Comparative Example 3: Preparation of Anti-Adhesion Agent Containing Chitosan

1.5 parts by weight of chitosan was dissolved in 100 parts by weight of distilled water, stirred at 25 ° C. for 1 hour, mixed with 0.075 parts by weight of Jenny Fin and 1 part by weight of ethanol, and then poured into a petri dish (50 mm × 12 mm) for 2 days in a clean bench. Drying to prepare a hydrogel anti-adhesion agent.

Experimental Example 1: Gelation Rate Experiment

In order to remove the polymer remaining without participating in the crosslinking reaction, the hydrogel type anti-adhesion agents prepared in Examples 1 and Comparative Examples 1 to 3 were immersed at 25 ° C. for 24 hours. The hydrogel type anti-adhesion agent prepared in Example 1 and Comparative Examples 1 to 3 subjected to the dipping process was taken out, and the water on the surface of the gel was wiped off and placed in a dryer at 55 ° C. and dried for 24 hours. The gelation rate was calculated using the following equation, and the results are shown in FIG. 1. It can be seen from FIG. 1 that the mixed gel of curdlan and gellan gum has a lower gelation rate than that of curdlan and gellan gum alone.

% Gelation = weight of dried gel / weight of initially used hydrogel × 100

Experimental Example 2: Degradation Rate Experiment

In order to determine the degree of decomposition of the anti-adhesion agent of the hydrogel prepared in Example 1 and Comparative Examples 1 to 3 was tested using the following method. The weight of the dried hydrogel was measured. The dried hydrogel was immersed in PBS (Phosphate buffered saline) at 37 ℃ and the weight change over time was measured. Before the measurement, the filter paper was used to remove the water on the surface of the hydrogel and weighed. The decomposition rate was calculated using the following equation, and the results are shown in FIG. 2. It can be seen from Fig. 2 that the mixed gel of the curdlan and gellan gum is better decomposed than the curdlan, gellan gum alone.

% Degradation = weight of gel when swollen / weight of gel when dried × 100

Experimental Example 3: gel strength measurement

Compressive strength was measured at room temperature using a universal property tester (Daego Co., RDE Ver 3.0) to determine the strength of the anti-adhesion agent in the hydrogel form prepared in Examples 1 and Comparative Examples 1 to 3. The thickness of the specimen is 3 mm ± 0.5 mm and the diameter is 10 mm. Gel strength was measured by preparing three specimens each. The results are shown in Fig. It can be seen from Fig. 3 that the gel strength of the mixed gel of curdlan and gellan gum is lower than that of the gellan gum alone.

That is, from the gelation rate, decomposition rate and gel strength measurement results of Experimental Examples 1, 2 and 3, when using a mixture of curdlan and gellan gum as in the present invention, the gelation rate and gel strength are low and the decomposition rate is excellent to prevent adhesion. It can be seen that the effect is excellent.

Experimental Example 4: Cytotoxicity Test

MTT (3- (4,5-dimethyldiazol-2-yl) -2,5- in order to investigate the cytotoxicity of the hydrogel anti-adhesion agents prepared in Examples 1 and Comparative Examples 1 to 3 MTT search was performed using diphenyltetrazolium bromide). Raw 264.7 cells were put at 100 μl per well in a 96-well plate at 1 × 10 ⁵ cells / ml and incubated for 24 hours in a 37 ° C., 5% CO ₂ incubator. Hydrogel-type anti-adhesion agents prepared in Example 1 and Comparative Examples 1 to 3 were suspended in phosphate buffer solution (PBS) at a concentration of 1 mg / ml and diluted 1/10 until the concentration was 0.1 μg / ml. The dilution was continued and the final volume was adjusted to 200 μl, followed by incubation for 24 hours in the incubator in which the Raw 264.7 cells were incubated. Next, incubate for 4 hours by adding 20 μl of phosphate buffer solution containing 5 mg / ml of MTT reagent, remove the supernatant, add dimethyl sulfoxide (DMSO) solution, and then use 570 nm with ELISA Reader. Cell viability was measured by measuring the absorbance at.

Average survival rate (%) = absorbance of the sample treatment group / absorbance of the control group × 100

Hydrogel anti-adhesion agent including curdlan and gellan gum of the present invention of Example 1 did not show direct cytotoxicity was found to be a safe material (see Figure 4).

Experimental Example 5: Effect of preventing peritoneal membrane adhesion

In order to investigate the anti-adhesion effect of the anti-adhesion agent of the present invention, experiments were performed using Sprague-Dawley rat 4 weeks old, weighing about 250-300 g. Three of each comparative example and three of each example were used.

The abdomen of the rat anesthetized with zoletil and xylazine was opened and the cecum was removed to damage the intima with a size of 1 cm × 2 cm, and the same amount of damage was applied to the opposing peritoneal membrane, and then Comparative Examples 1, 2, 3 and After treating Example 1, the rat was euthanized after 1 week, and the rat was euthanized, and then the degree of adhesion and adhesion strength were examined (FIG. 5 and FIG. 6). Adhesion degree and adhesion strength was based on the following Table 1, the results are shown in Table 2.

[Table 1]

[Table 2]

As shown in Table 2, it can be seen that the anti-adhesion agent of Example 1 of the present invention has an excellent anti-adhesion effect compared to the non-treated group and Comparative Examples 1 to 3 (curdlan, gellan gum, chitosan alone treatment group). . The strength of the formed adhesion is also reduced, it can be seen that it is very effective in preventing the adhesion of internal organs after surgery. 6, (A) and (B) are photographs of the untreated group, (C) and (D) photographs of the anti-adhesion treatment group of Example 1, and (A) and (B) In the case of the adhesion to the cecum and peritoneum, while in (C) and (D) was treated with the anti-adhesion agent including the curdlan and gellan gum of Example 1 it can be seen that the adhesion to the cecum and peritoneum did not occur have.

Claims (7)

Anti-adhesion composition comprising a curdlan: gellan gum 1: 1: 0.3 to 3 by weight as an active ingredient. delete The method of claim 1,
The anti-adhesion composition is an anti-adhesion composition, characterized in that the foam, powder or film formulation. The method of claim 1,
The anti-adhesion composition further comprises one or more drugs selected from the group consisting of physiologically active proteins, functional peptides, anticancer agents, antithrombotic agents, steroids, nonsteroidal anti-inflammatory agents, hormone chemostat factors, analgesics or anesthetics Adhesion prevention composition, characterized in that. Curdlan: gellan gum is prepared by mixing 1 to 2 parts by weight of a mixture of cudlan and gellan gum and 100 parts by weight of water, which is mixed in a weight ratio of 1: 0.3 to 3, to prepare a gel aqueous solution and to form and dry the gel aqueous solution. Method for producing an anti-adhesion agent, characterized in that. delete delete

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