KR101702637B1 - Adhesive comprising extract of Yellowfin Tuna - Google Patents

Abstract

The present invention relates to an adhesive containing an extract of yellowfin tuna skin which is a marine material, and to a production method thereof. According to the present invention, the adhesive shows excellent biocompatibility and is effective in tissue adhesion, and thus enables the production of natural adhesives which can be easily used in medical fields.

Description

Adhesive comprising extract of Yellowfin Tuna < RTI ID = 0.0 >

The present invention relates to an adhesive comprising a marine extract having no toxicity and excellent biocompatibility.

Recently, researches on adhesives mimicking adhesive components derived from natural sources or secreted from organisms have been actively studied. These adhesives are less toxic than conventional adhesives containing synthetic compounds and can be used for various purposes, and most of them are expected to be suitable for medical use because of their excellent biocompatibility. For example, Korean Patent Laid-Open Publication No. 1998-0014365 discloses an adhesive containing a fish extract, and Korean Patent Publication No. 2012-0013626 discloses a composition utilizing an adhesive protein derived from a mussel.

The natural-derived adhesive can be used as a part of a medical or biomedical tissue such as a wound suture or an adherence of a living tissue. Natural adhesives can be easily applied to wound sites when used for medical purposes, and there is no need for a secondary suture such as a suture chamber or a stapler, less pain, and less scarring. Cyanoacrylate, a synthetic compound used for medical use, is a typical example. Natural adhesives such as fibrin glue and collagen glue are very effective in bonding some tissues, but they are expensive and have limited supply. In addition, fibrin glue has a weak adhesive strength and requires preparation before the procedure. In addition, natural polymers of the same nature as synthetic polymers that can be used for adhesives can be obtained from nature, but the types that have not yet been found are not various and their physical properties are often limited.

The present invention is to provide an economical adhesive that can replace conventional natural adhesive, which has excellent biocompatibility, low toxicity, low cost, and mass availability through research of various marine materials.

Korean Patent Publication No. 1998-0014365

The present invention is to provide a natural adhesive which can be supplied in a small quantity and in a large quantity using yellow tuna as a marine material.

The present invention provides an adhesive comprising a yellow tuna bark extract.

The present invention provides a method for producing an adhesive comprising separating yellow tuna shell from yellowfin tuna and treating acetic acid, treating with acetic acid, neutralizing with water and boiling, and dialyzing the hot solution.

The adhesive of the present invention is excellent in tensile strength and adhesive strength, and has no toxicity. In addition, it is rich in amino acids, proteins and chondroitin sulfate (Sodium Chondroitin Sulfate), so it has excellent biocompatibility and is very effective for tissue regeneration.

Fig. 1 shows a yellow tuna extract powder (A) and a fresh water extract powder (B).
FIG. 2 shows the results of measurement of chondroitin sulfate content (A) in HPLC graph and (B) in HPLC.
Fig. 3 shows a cowhide (A) and a birch specimen (B) prepared by applying an adhesive sample.
Figure 4 shows the adhesive strength of each adhesive sample applied to a cowhide.
Fig. 5 shows the adhesive strength of each adhesive sample applied to a birch specimen (birch).
FIG. 6 shows the cytotoxicity evaluation results of the concentration of yellowfin tuna bark extract.
FIG. 7 shows the cytotoxicity evaluation results of the concentration of freshwater fly extract.
Fig. 8 shows the cytotoxicity evaluation results for each concentration of the adhesive sample.

Hereinafter, the present invention will be described in detail. The terms used in the present specification should be construed as generally understood by a person having ordinary skill in the art unless otherwise defined. It is to be understood that the drawings and embodiments are not intended to limit the scope of the present invention, which is not intended to limit the scope of the present invention. It is not.

An object of the present invention is to provide a natural adhesive body which is free from toxicity, has excellent biocompatibility and adhesion to tissue using a marine material.

The present invention provides an adhesive comprising a yellowfin tuna bark extract which is a marine material. Yellow tuna bark extract has no cytotoxicity and has an adhesive power by itself without addition of other components such as compounds, so it can be used directly on the living body such as wound area. And, the yellow tuna bark extract is rich in amino acid, protein and chondroitin sulfate, so it has excellent regeneration effect as well as tissue adhesion when applied to the damaged area of the organism. For example, the amino acid extracted from tuna is a polypeptide that promotes tissue regeneration and has excellent cell proliferation effect. Chondroitin sulfate is the most important component of the extracellular matrix (ECM), and it can be used as an important material in the treatment of arthritis and cosmetics as it has the effect of regenerating or aging. According to one embodiment of the present invention, the yellowfin tuna extract extracted from yellowfin tuna bark for use as a natural adhesive has an amino acid content of 60 mg / g or more, a protein content of 5 mg / ml or more, and a chondroitin sulfate Fade. Therefore, it is more suitable for medical use particularly because it has excellent effects on tissue regeneration and the like, compared with a material used for a known natural adhesive agent.

The glue containing the yellow tuna bark extract of the present invention may further contain a butterfly extract. Adhesives that contain Minerubure extract may have a significantly higher adhesion than adhesives containing only yellow tuna bark extract. This increase in adhesive strength is a synergistic effect of the mixture of the Minerubure extract and the yellow tuna bark extract, resulting in an unexpected adhesive effect from the adhesive strength of each of the adhesives including the Minerubure extract or the yellow tuna bark extract alone. Yellow tuna bark extract: Minerubure extract is mixed in an amount of 1: 2 to 2: 1, 0.5: 1.5 to 1.5: 0.5, 0.8: 1.2 to 1.2: 0.8, 0.9: 1.1 to 1.1: But is not limited thereto.

The adhesive containing the yellow tuna bark extract of the present invention may further contain chitosan, gluten or a mixture thereof. Adhesives containing chitosan, gluten, or a mixture thereof may have a significantly higher adhesive strength than glue containing only yellow tuna bark extract. The adhesive strength may vary depending on the subject using the adhesive, but the adhesive strength may be increased at least 2 to 4 times. Yellow tuna bark extract: chitosan is present in a weight ratio of 10: 1 to 1: 1, 9: 1 to 2: 1, 8: 1 to 3: 1, 7: 1 to 4: 1, 6: 1 to 4: May be mixed in an adhesive, but not limited thereto, in an amount of 5: 1 to 1: 1, 5: 1 to 2: 1, 5: 1 to 3: Yellow tuna bark extract: gluten is present in a weight ratio of 5: 1 to 1: 1, 4: 1 to 1: 1, 3: 1 to 1: 1, 5: 1 to 2: 1, 4: 1 to 2: 3: 1 to 2: 1, may be mixed in the adhesive, but the present invention is not limited thereto. According to one embodiment of the present invention, in the evaluation of the adhesion strength of the biotissue, the adhesive including the yellowfin tuna extract and chitosan increased more than twice as much as the adhesive containing only the yellowfin tuna extract and the adhesive containing the yellowfin tuna extract and gluten The adhesive strength was increased four times or more than the adhesive containing only yellow tuna extract. In addition, the adhesive containing all of yellow tuna extract, chitosan and gluten increased the adhesion more than 8 times than the adhesive containing only yellow tuna extract. In case of chitosan, gluten may be used more than chitosan. In case of chitosan, it may be more adhesive force than gluten in birch. Therefore, it may be possible to selectively use different components depending on the animal or plant tissue .

The present invention may further comprise chitosan, gluten or a mixture thereof in an adhesive containing a yellow tuna bark extract and a minerubre extract. Adhesives that further include chitosan, gluten, or a mixture thereof may have significantly increased adhesive strengths compared to adhesives including yellow tuna bark extract and cider butter extract. In addition, the addition of chitosan or gluten may increase the adhesive strength depending on the biomaterials such as animal tissues or plant tissues depending on the added ingredients, but the adhesive strength may be increased at least 2 to 4 times. 0.1 to 2: 1: 0.1, 0.5: 1.5: 0.05 to 1.5: 0.5: 0.05, 0.8: 1.2: 0.08 to 1.2: 0.8: 0.08 in weight ratio to the extract of yellow tuna bark But it is not limited thereto. 0.5 to 1.5: 0.25 to 1.5: 0.5: 0.25, 0.8: 1.2: 0.4 to 1.2: 0.8: 0.4 in weight ratio of glutinous extract But it is not limited thereto. According to one embodiment of the present invention, the adhesion strength of yellow tuna extract, minerubure extract, and chitosan increased 1.5-8 times or more than that of the adhesive containing yellow tuna extract and minerubure extract, , Yellow tuna extract, glutinous extract, and gluten increased by 3 ~ 5 times more than glue containing yellow tuna extract and ginger extract. In case of chitosan, gluten may be used more than chitosan. In case of chitosan, it may be more adhesive force than gluten in birch. Therefore, it may be possible to selectively use different components depending on the animal or plant tissue .

According to the present invention, an adhesive containing a yellow tuna bark extract includes a step of treating acetic acid with a yellow tuna bark in a yellowfin tuna, a step of neutralizing with water after the treatment with acetic acid, a step of boiling the water, And the like. The yellow tuna bark extract can be stored in powder form by lyophilization after dialysis, and the yellow tuna bark powder can be used as an adhesive immediately after dissolving in water. The acetic acid treatment step can be carried out at 0 to 10 캜, 1 to 10 캜, 2 to 10 캜, 2 to 10 캜, 2 to 8 캜, preferably 4 to 8 캜 for 5 to 7 hours, . The yellow tuna bark swelled by acetic acid solution treatment is neutralized with flowing water for 10 hours or more and for 12 hours or more, preferably for 10 to 12 hours, and water is further added to the bath. The bathing step can be carried out at 50 to 80 캜, 50 to 70 캜, preferably 60 to 70 캜 for 3 to 4 hours, but is not limited thereto. The method may further include the step of removing impurities contained in the hot water solution by using a filter paper as needed before the concentration after the hot water bath step. The concentration process can be carried out by concentrating the solution, which has been filtered through a hot water solution or a hot water solution, at 50 to 80 DEG C, 50 to 70 DEG C, preferably 60 to 70 DEG C until the weight of the originally used yellowfin tuna shell. The yellowfin tuna bark extract obtained through the dialysis process after concentration is used as it has an adhesive strength and can be stored in powder form through freeze drying. Yellow tuna bark powder extract can be used as an adhesive by mixing with water or collagen without adding other compounds or chemical components.

The adhesive manufactured according to the manufacturing method of the present invention is a natural adhesive agent containing no compounds or chemical components in addition to the extract of yellow tuna bark, and is excellent in cytotoxicity and biocompatibility. According to one embodiment of the present invention, the yellow tuna bark extract or the adhesive containing the yellow tuna bark extract shows little cytotoxicity even when the concentration of yellow tuna bark extract is high. The natural material used as a natural adhesive agent may cause cytotoxicity due to the increase of the concentration, and could induce apoptosis of the biotissue when using an adhesive having a high concentration of ingredients to increase the adhesive strength. However, since the adhesive containing the yellow tuna bark extract of the present invention does not show cytotoxicity due to the increase in concentration, even if it is used at a high concentration to increase the adhesive strength, it does not cause cell damage at the wound area of the tissue.

In the manufacturing method of the present invention, it is possible to further include the step of adding the Minerubure extract, chitosan, gluten or a mixture thereof. The Minerubure extract is prepared by drying the broth and calling it distilled water, followed by heating and concentration. Minerubure extract can also be stored in the form of powder by freeze-drying after concentration. The chitosan or gluten may be prepared directly according to known methods, or a commercially available product may be used.

Hereinafter, embodiments for carrying out the present invention will be described. The present invention is not limited to the above-described embodiments, but may be embodied in many other specific forms without departing from the spirit or essential characteristics thereof.

Manufacture of yellowfin tuna bark extract

Yellow tuna ( Thunnus) albacares ) were weighed and weighed (100 g), and then immersed in a 10-fold volume of 0.2 N acetic acid and immersed in a refrigerated room (4-8 ° C) for 6 hours. The yellowfin tuna shells swollen in solution were taken out and neutralized with running water for 12 hours. Four times as much water was added to 300 g of the neutralized shell, followed by bathing at 60 to 70 ° C for 3 to 4 hours. After the water bath, filtration was carried out using a filter paper to remove impurities contained in the solution. The filtered solution was heated again at 60-70 < 0 > C and finally concentrated to the trimmed shell weight. After concentration, dialysis was performed with distilled water (1 L, 2 times, 10 hours / times) at 4 ° C using a cutoff size 12 kDa to 14 kDa membrane tube. After completion of the dialysis, freeze-drying was carried out to remove most impurities except the protein and lyophilized to obtain a yellow tuna extract in powder form and stored in a desicator (Fig. 1 (A)). The extract of yellowfin tuna was obtained with a yield of 50% or more.

Minerubure  Extract preparation

In a hot air dryer, 100 g of Miichthys miiuy broth was placed and dried for 2 weeks at a temperature of 40 ° C. and a humidity of 10%. During the drying period, the blades were ventilated back and forth every 24 hours to prevent corruption. Dried Minerubure was soaked in distilled water for 2 ~ 3 days. The water was added to the broth called distilled water four times its original weight and heated at 60 to 70 ° C for 5 to 6 hours until the amount of the solution became one third of the initial weight. After concentration, the extract was lyophilized to obtain a powder of Mycorrhizae extract and stored in a desiccator (Fig. 1 (B)). The extract of Bombyx mori was obtained with a yield of about 35%.

Determination of constituent amino acids of yellowfin tuna skin and crustacea extract

0.5 ml of each of the yellow tuna bark and the minerubre extract powder obtained in Examples 1 and 2 was weighed into an 18 ml test tube, and 3 ml of 6 N HCl was added thereto. Then, the test tube was sealed with a vacuum pump. The sealed tube is hydrolyzed in a heating block at 121 ° C for 24 hours. Remove the acid with a rotary evaporator at 50 ° C and 40 psi, then dilute with 10 ml of sodium loading buffer. , And 1 ml of the solution was filtered through 0.2 쨉 l membrane filter and quantitatively analyzed with an amino acid analyzer (S433-H, SYKAM, Germany). The column used was a cation separation column (LCA K06 / Na), the column size was 4.6 × 150 mm, the column temperature was 57-74 ° C., the buffer was 0.45 ml / min at the flow rate, ) Was 0.25 ml / min. The pH range of the buffer was 3.45 ~ 10.85 and the wavelengths were 440 nm and 570 nm, respectively. As a result of the measurement, it was observed that 33 g of the Minerubure extract and 100 g of the constituent amino acid of the yellow tuna bark extract were contained per 100 g (Table 1).

sample
ingredient Minerubure
extract Yellowfin tuna skin
extract Aspartic acid 2,179,144 3,516,086 Threonine 1,096,597 3,115,244 Serine 987.071 1,873,037 Glutamic acid 3,787,700 3,972,885 Proline 4,600,581 5,247,638 Glycine 8,635,019 2,838,359 Alanine 4,039,095 3,775,695 Cystine 87.860 7,066,405 Valine 791.341 2,655,622 Methionine 705.376 4,528,139 Isoleucine 297.747 2,192,082 Leucine 828.167 2,561.001 Tyrosine 183.977 3,568,444 Phenylalanine 665.371 4,209,753 Histidine 281.804 1,661.987 Lysine 1,274.718 3,386,512 Arginine 2,392,264 5,755,024 Total 32,833,833 61,923,913 Unit: mg / 100g

Determination of protein content of yellowfin tuna bark and cider leaf extract

Protein content was measured using a BCA protein assay kit (Pierce, Rockford, IL, USA). Add 25 μl of standard and sample diluted by concentration (0.00625%, 0.0125%, 0.025%, 0.05%, 0.1% (w / v)) to a 1.5 ml tube and add compatible reagent stock solution And then mixed. After incubation at 37 ° C for 15 minutes, 1 ml of BCA working reagent was added, followed by incubation at 37 ° C for 30 minutes in a thermostat. After incubation at room temperature for 5 to 10 minutes, the absorbance was measured at 562 nm and a standard curve was prepared using bovine serum albumin standard. The content of 5.3mg / ml was determined from the extract of yellowfin tuna bark, and the content of 5.7mg / ml was found from the extract of subspecies.

Yellowfin tuna peel and cider leaf extract Chondroitin sulfate  Content measurement

Chondroitin sulfate (Cs sodium salt) of yellowfin tuna bark extract and minerubure extract was analyzed by HPLC, and the contents of the extracts were analyzed by graph. Chondroitin sulfate was dissolved in 0.1wt%, 0.2wt%, 0.4wt%, and 0.8wt% of water for HPLC and used for the analysis. , And it was observed that the area of the peak of the 4th minute was increased in a concentration dependent manner. The cone sulfate content was calculated as the area value by concentration. (Y = 1.213, 651x + 237, 435). The results showed that the content of chondroitin sulphide was 4.7% and the content of chondroitin sulphate was 22% in the yellowfin tuna bark extract (Fig. 2).

Identify the strength of adhesive containing yellow tuna bark extract

An adhesive sample was prepared to confirm the adhesive strength of the adhesive containing the yellow tuna bark extract. [Table 2] shows the preparation of adhesive samples.

Adhesive sample Ingredients Manufacturing method (weight ratio (g))
One Yellow tuna extract (TS, tuna skin) TS: water
1.0: 10 2 Extract of cider (CSB, croaker swim bladder) CSB: Water
1.0: 10 3 Yellowfin tuna extract and cider butter extract TS: CSB: Water
1.0: 1.0: 10 4 Yellowfin tuna extract and chitosan TS: chitosan: water
1.0: 0.2: 10 5 Yellowfin tuna extract and gluten TS: gluten: water
1.0: 0.5: 10 6 Minerubure extract and chitosan CSB: chitosan: water
1.0: 0.2: 10 7 Minerubure extract and gluten CSB: Gluten: Water
1.0: 0.5: 10 8 Yellowfin Tuna Extract, Minerubure Extract and Gluten TS: CSB: Gluten: Water
1.0: 1.0: 0.5: 10 9 Yellowfin tuna extract, Minerubure extract and chitosan TS: CSB: Chitosan: Water
1.0: 1.0: 0.2: 10

25 mm) 20mm만큼 겹쳐서 붙인 뒤 접착강도를 측정하기 위한 준비를 마쳤다(도 3). 로드셀(load cell)은 1000Lbs을 사용하였고, 측정속도는 5mm/min으로 설정하였다. 한 종류의 샘플마다 5회씩 접착강도를 측정하여 평균 및 표준편차를 계산하여 접착강도 계산에 이용하였다. 단위는 MPa를 사용하였다. 인장강도기(INSTRON, No5566)를 통하여 접착력을 측정하였다(도 4, 도 5, 표 3). 접착력 측정 결과, 황다랑어 껍질 추출물의 접착력이 민어 부레 추출물의 접착력보다 2배이상 높았고, 황다랑어 껍질 추출물과 키토산 또는 글루텐을 포함한 시료에서 접착력이 현저히 상승하였다. 황다랑어 껍질 추출물에 글루텐을 더 포함한 시료는 특히 자작나무에 사용하였을 때 접착력이 현저히 상승하였다. 황다랑어 껍질 추출물에 키토산을 더 포함한 시료는 특히 소가죽에 사용하였을 때 접착력이 현저히 상승하였다. The garlic adhesive was compared as a control. Prepare the adhesive sample of cowhide and birch specimens (100 mm

25 mm) 20 mm, and the preparation for measuring the adhesive strength was completed (FIG. 3). The load cell used was 1000 Lbs, and the measurement speed was set at 5 mm / min. The adhesive strength was measured five times for each kind of sample, and the average and standard deviation were calculated and used for calculating the adhesive strength. The unit was MPa. The adhesive strength was measured through a tensile strength device (INSTRON, No 5566) (Figs. 4, 5, and Table 3). As a result of the adhesion test, the adhesive strength of the yellow tuna bark extract was more than twice as high as that of the black broom extract, and the adhesion of yellow tuna bark extract and chitosan or gluten was significantly increased. Samples containing more gluten in the yellow tuna bark extracts showed a remarkable increase in adhesion when used in birch. Samples containing more chitosan in the extract of yellowfin tuna bark, especially when used in cowhide, increased significantly.

Adhesive sample Cowhide Birch Garlic 0.987 ± 0.032 11.91 + 1.47 Yellow tuna extract (TS) 0.56 + 0.03 1.664 + 0.038 Minerubure extract (CSB) 0.23 ± 0.06 0.62 ± 0.105 Yellowfin tuna extract and
Minerubure extract 1.07 ± 0.053 3.12 + 0.0125 Yellowfin tuna extract and chitosan 0.896 ± 0.05325 15.025 ± 1.545 Yellowfin tuna extract and gluten 2.31 + 0.095 6.385 ± 2.805 Yellowfin tuna extract, Minerubure extract and chitosan 1.4267 0.0634 26.385 ± 2.805 Yellowfin Tuna Extract, Minerubure Extract and Gluten 3.2367 + 0.1621 17.325 + 2.535

Evaluation of cytotoxicity of yellowfin tuna bark extract and cider leaf extract

Cell culture media were DMEM medium (Dulbecco's modified Eagle's medium, GIBCO) containing 10% fetal bovine serum (FBS; GIBCO) and 1% penicillin. Cells were obtained from NIH 3T3 fibroblast cells purchased from Korean cell line bank (KCLB). NIH3T3 cells were cultured in DMEM medium containing 10% FBS and 1% penicilin at 37 ° C and 5% CO ₂ . MTT assay was performed to evaluate cytotoxicity. 100 [mu] l of each well was dispensed into a 96 well cell culture plate and cultured for 24 hours. The extract was prepared by diluting Yellowfin Tuna Extract and Minerubure extract in the concentrations of 125 ppm, 250 ppm, 500 ppm and 1,000 ppm. After 24 hours, the medium was removed. After removal of the medium, 100 μl of a medium containing 0.5% MTT (3- [4,5-dimethyl-Ithiazol-2-yl] -2,5-dipheyltera-zol- ium bromide] (MTT assay reagent) (DMSO) for 15 minutes, and the absorbance was measured at 570 nm using an ELISA reader (Biotex, USA). Cell viability was calculated three times in each experiment, and cytotoxicity was judged to be almost no toxicity when 80% or more of the cells were compared with the control.

As a result of the cytotoxicity evaluation, the extract of yellowfin tuna did not show any toxicity in the whole section when compared with the control group. As the concentration of yellow tuna extract increased, no significant change was found (Fig. 6). The cell extract was not cytotoxic at low concentrations (125-250 ppm) but at high concentrations (500-1000 ppm), cell viability was measured at about 80% (FIG. 7). Based on this, the cell viability was slightly lower than that of yellowfin tuna bark extract at high concentration and relatively cytotoxic was observed.

Assessment of cytotoxicity of adhesive

The cytotoxicity of the adhesive was evaluated through the adhesive sample prepared in Example 6. The method of Example 7 was used for the cytotoxicity evaluation method. When the samples were measured at concentrations of 10000 ppm, 1000 ppm, and 500 ppm, the cell viability was found to be 80% or more (FIG. 8).

Claims (4)

Yellow tuna bark extract containing chondroitin, amino acids and protein;
And glue.
The method according to claim 1,
Adhesives further containing Cider root extract.
The method according to claim 1,
Glue containing more chitosan.
Separating the yellowfin tuna bark from yellowfin tuna and treating acetic acid;
Acetic acid, neutralizing with water and warming;
Filtering and concentrating the water; Dialyzing the concentrated solution; And
A method for producing an adhesive according to claim 1, comprising mixing dialyzed yellow tuna extract and gluten.

Images