KR101739409B1 - Composition comprising mixture of DNA fragments separated from fish's testis for regenerating cartilage - Google Patents

Abstract

The present invention relates to a composition for regenerating cartilage comprising a DNA fragment mixture having a molecular weight of 3,000 to 10,000 kDa isolated from a fish testimony, and a preparation method thereof. The composition for regenerating cartilage of the present invention not only has an effect of raising cartilage lubrication or buffering action upon administration into the joints but also has an effect of proliferating cartilage cells, repairing damaged cartilage cells and improving arthritis, And can be used as a therapeutic agent for osteoarthritis.

Description

[0001] The present invention relates to a composition for regenerating cartilage comprising a mixture of DNA fragments separated from a fish testimony,

The present invention relates to a composition for regenerating cartilage comprising a DNA fragment mixture. More particularly, the present invention relates to a composition for regenerating cartilage for the treatment of osteoarthritis, a composition comprising a DNA fragment mixture separated from a fish testimony, and a preparation method thereof.

Osteoarthritis is the most common arthritis of the joint disease. It is also called degenerative arthritis. The articular cartilage that wraps around the joint surface of the bone is worn and the bone under the cartilage is exposed. The synovium around the joint is inflamed and painful and deformed . Osteoarthritis is a gradual degeneration of cartilage tissue in joints such as knees and ankles, fingers, elbows, and hip joints due to aging, obesity, trauma of the joints, dysplasia of the joints, history of arthritis, The process proceeds to destruction.

Cartilage is a resilient tissue that connects two bones, imparts mobility to the joints, and acts as an impact relief. In particular, articular cartilage includes cartilage cells embedded in a cartilage matrix, which is an intercellular material including proteoglycan and collagen fibrils such as type II collagen as a connective tissue, and other proteins and water. Damage to the cartilage tissue results in direct friction between the bones, stiffness of the joint, gradual decrease in articular cartilage movement, and frequent pain at the cartilage site.

Osteoarthritis is a chronic disease that requires long-term treatment due to the nature of the disease, long-term drug complications and side effects will occur.

More specifically, as a method of treating osteoarthritis, when there is only pain in the early stage, the focus is on pain relief by taking a simple analgesic agent. However, if the pain continues after the pain, the anti-inflammatory analgesic agent do. However, such anti-inflammatory analgesics use a corticosteroid-type anti-inflammatory substance. When long-term use of hydrocortisone and betamethasone, which are one of the analgesic agents, And inhibits the ability to regenerate chondrocytes. Therefore, it is the principle of intra-articular injection instead of oral administration with continuous observation.

Therefore, many medicines have been developed which have analgesic and antiinflammatory effects and lower the above-mentioned side effects in the most common method for treating osteoarthritis. Typical examples thereof include diclofenac, aspirin and ibuprofen Of nonsteroidal anti-inflammatory drugs (NSAIDs). Nonsteroidal anti-inflammatory drugs, however, also have side effects that cause side effects in the digestive system, especially the stomach.

Accordingly, the present inventors have been intensively studying a therapeutic method for reducing the breakage of cartilage cells or regenerating osteoarthritis through regeneration of cartilage cells, and found that a mixture of DNA fragments derived from fish testimonies It has been found that when a composition containing a mixture is applied to the treatment of osteoarthritis, the physical function as a lubricant is improved due to an increase in viscosity, and the osteoarthritis exhibits an excellent effect on cartilage regeneration while reducing the number of administrations.

In the present invention, the above-mentioned fish testimony-derived DNA fragment mixture is a mixture of DNA fragments consisting of phosphoric acid, four kinds of bases and deoxyribose extracted and purified from fish testimonials, Quot; and " mixed "

On the other hand, International Patent Publication No. 2010-049898 discloses that polydeoxyribonucleotide derived from fish can be used as an injection for treating osteoarthritis. However, the above-mentioned prior art discloses that the molecular weight of the polydeoxyribonucleotide is 20 to 2500 kDa, It has been disclosed that a low molecular weight polydeoxyribonucleotide of 240 kDa can be used as an injection for treating osteoarthritis and no method for purifying a DNA fragment mixture at high yield and high purity from fish testicles has been disclosed.

The present inventors have previously disclosed a technique for obtaining a DNA fragment mixture (DNA polymer fragment complex) from a semen of a fish (Korean Patent No. 986603), but there was a limit to obtaining fish semen.

On the other hand, as a conventional method of separating the DNA fragment mixture, there is a method (Korean Patent Registration No. 390529) of separating and extracting a nucleic acid complex material from the testis of a tuna using an ultrafiltration membrane. However, A method of isolating protamine sulfate from tuna testis (Korean Unexamined Patent Publication No. 1993-0008087) is disclosed as a method of separating and extracting from salmon, herring, and yeast, However, this method is not intended to separate the DNA fragment mixture fragments, but merely to separate and extract proteins called protamine. In addition, extraction of nucleic acid complex materials from fish such as salmon and herring by extraction with saline (J. Biol. Chem., 203, 167-171, 1953) or by treatment with surfactants such as organic solvents or SDS (J. Chem. Soc., 1129, 1947; J. Biol. Chem., 190, 165-176, 1951). The aqueous solution thus obtained was precipitated with an acid or precipitated with alcohol such as ethanol However, these methods have a low yield of a DNA fragment mixture and are difficult to be industrially used because organic solvents are used. In addition, other ion exchange methods and ultrafiltration methods have been known as methods for extracting DNA fragment mixtures, but they have been difficult to be industrially used because of low yield of DNA fragments compared to high equipment cost.

Disclosure of the Invention The present invention has been made to solve the above-mentioned problems of the prior art, and it is an object of the present invention to provide a DNA fragment mixture in high purity and high yield from a testis of a fish and to produce a DNA fragment mixture having a specific molecular weight range, And thus the present invention has been completed.

Korean Patent No. 986603 (DNA polymer fragment complex isolated from fish semen or eggs and preparation method thereof, 2010.10.08) International Patent Publication No. 2010-049898 (INJECTABLE POLYDEOXYRIBONUCLEOTIDE COMPOSITION FOR THE TREATMENT OF OSTEOARTICULAR DISEASES, Oct. 28, 2009) Korean Patent No. 390529 (method for extracting nucleic acid complex material from tuna testis and nucleic acid complex material obtained by this method, published on July 7, 2003) Korean Patent Publication No. 1993-0008087 (Method for Separation of Protamine Sulfate from Testicular Tuna, published Aug. 25, 1993)

J. Biol. Chem., 203, 167-171, 1953: The large scale preparation of sodium desoxyribonucleate from ripe salmon testes J. Chem. Soc., 1129, 1947: Deoxypentose nucleic acids; preparation of the tetrasodium salt of the deoxypentose nucleic acid of calf thymus J. Biol. Chem., 190, 165-176, 1951: The isolation of sodium desoxyribonucleate with sodium dodecyl sulfate.

It is an object of the present invention to provide a composition for regenerating cartilage comprising a DNA fragment mixture separated from a fish testimony.

It is still another object of the present invention to provide a method for producing a composition for regenerating cartilage comprising a DNA fragment mixture from a fish testimony.

According to an aspect of the present invention, there is provided a composition for regenerating cartilage comprising a DNA fragment mixture having a molecular weight of 3,000 to 10,000 kDa separated from a fish testimony.

According to another aspect of the present invention, the fish is preferably a salmonaceous fish, more preferably a salmon or trout, and most preferably a salmon.

A method for producing a composition for regenerating cartilage comprising a DNA fragment mixture having a molecular weight of 3,000 to 10,000 kDa from fish testis according to the present invention comprises the following steps:

(1 step) Thawing fish testimony, removing blood and foreign matter;

(Step 2) A step of preparing a testis pulverized liquid by adding purified water to a thawed testis;

(Step 3) Mixing the testis pulverized liquid of the two steps and aqueous sodium chloride solution, reacting and cooling while stirring;

(Step 4) To the cooled reaction solution of the above three steps, an aqueous solution of sodium hydroxide is added to adjust the pH to 10 to 12, followed by stirring and filtration to obtain a filtrate;

(Step 5) adding hydrochloric acid to the filtrate of the above-mentioned four steps so as to have a pH of 6.5 to 8.5, reacting, cooling and then filtering the filtrate to obtain a filtrate containing the DNA fragments mixture;

(Step 6) A step of obtaining a fraction of a DNA fragment mixture having a molecular weight of 3,000 to 10,000 kDa by fractionating the filtrate of the above 5 steps by gel filtration;

(Step 7) Isopropyl alcohol or ethanol is added to the fraction of step 6 and stirred to precipitate a DNA fragment mixture; And

(Step 8) Washing the DNA fragment mixture of step 7 with isopropyl alcohol or ethanol, dehydrating it and drying.

According to another embodiment of the production method of the present invention, a process of fractionating and concentrating the fraction solution of the 6 steps by ultrafiltration can be added.

According to another embodiment of the method of the present invention, the step of washing with isopropyl alcohol or ethanol in the step 8 comprises washing the DNA fragment mixture with 40 to 80% (w / w) of isopropyl alcohol or ethanol, and 95 to 100% isopropyl alcohol or ethanol.

Hereinafter, the configuration of the present invention will be described in more detail.

The present invention relates to a composition for regenerating cartilage comprising a DNA fragment mixture having a molecular weight of 3,000 to 10,000 kDa isolated from a fish testimony.

The DNA fragment mixture is a white or light yellow crystalline powder which is slightly soluble in water and alkali, hardly soluble in alcohol, and almost insoluble in ether and acetone.

According to the present invention, by including a DNA fragment mixture having a molecular weight of 3,000 to 10,000 kDa, preferably an average molecular weight of 3,000 to 6,000 kDa, The physical function as a lubricant for reducing friction can be enhanced and the effect as such a lubricant is far superior to that of the polydeoxyribonucleotide of Patent Document 2 of the prior art. On the other hand, when the molecular weight of the DNA fragment mixture is more than 10,000 kDa, the efficiency of separation, purification, filtration and the like is low.

Further, according to the present invention, by including a DNA fragment mixture having a molecular weight of 3,000 to 10,000 kDa, preferably 3,000 to 6,000 kDa, the duration of action is prolonged, A total of three administrations can achieve an excellent cartilage regeneration effect.

Each step of the manufacturing method of the present invention will be described in detail as follows.

The first step and the second step are the steps of preparing the testis of the fish. After the fish testis is thawed and blood and foreign substances are removed, 300 to 500 parts by weight of purified water is added to 10 parts by weight of the thawed testis, .

Next, the third step and the fourth step are a step of removing proteins and fats contained in the testis, wherein the testis pulverized solution is mixed with a 30 to 50% (w / v) aqueous sodium chloride solution at a ratio of 1: 2 to 2: 1 The mixture was stirred at 90 to 110 ° C for 0.5 to 2 hours and then cooled. To the cooled reaction solution was added a 20 to 40% (w / v) aqueous solution of sodium hydroxide to adjust the pH to 10 to 12, The mixture is stirred for 2 hours and filtered to obtain a filtrate.

Then, as a fifth step corresponding to the step of reducing the molecular weight of the DNA fragment mixture, a 35% (w / v) hydrochloric acid solution was added to the filtrate so as to have a pH of 6.5 to 8.5 and the reaction was carried out at 90 to 121 ° C for 5 to 30 minutes After cooling to room temperature, it is filtered to obtain a filtrate containing the DNA fragment mixture.

Next, 6 steps of fractionation of a DNA fragment mixture of a specific molecular weight in a DNA fragment mixture was carried out by adding 20 to 40% (w / v) aqueous sodium hydroxide solution to the filtrate, making the pH 9 to 12 again, A fraction of the DNA fragment mixture of a specific molecular weight is obtained and then adjusted to pH 7.0 using a 35% (w / v) hydrochloric acid solution.

Next, as the seventh step and the eighth step corresponding to the step of precipitating and drying the DNA fragment mixture, isopropyl alcohol or ethanol of 0.4 to 3 times the volume of the fraction liquid is added to the fraction having the above molecular weight fraction, After the mixture of the DNA fragments is precipitated by stirring for a time, the DNA fragment mixture is sequentially washed with 40 to 80% (w / w) of isopropyl alcohol or ethanol and 95 to 100% (w / w) of isopropyl alcohol or ethanol It is dehydrated and dried.

According to the present invention, it is possible to obtain a DNA fragment mixture having a molecular weight of 3,000 to 10,000 kDa from fish testimonies at a high yield of about 4% according to the purification method and the method including the molecular reduction process, . As in the case of the conventional invention, when the fish semen is used, the amount of the fish semen is much smaller than that of the fish testimonium (for example, about 100 g of the testis when the semen is about 20 mL per salmon) There is a problem in supplying a sufficient amount of raw materials to the artificial insemination of fish, but in the case of using testis of fish, it is not easy to obtain only a DNA fragment mixture after removing impurities such as proteins and other contained components , And according to the production method of the present invention, the DNA fragment mixture can be obtained much more easily from the testis of a fish.

The composition of the present invention induces proliferation or regeneration of cartilage cells, thereby inducing cartilage regeneration, thereby preventing or treating osteoarthritis.

The pharmaceutical composition for cartilage regeneration comprising the DNA fragment mixture according to the present invention can be formulated in the form of a sterile injection solution according to a conventional method. Also, the DNA fragment mixture may be dissolved in physiological saline (saline, pH 7.0 with phosphate buffer) and used as a sterile injectable solution.

The composition in the form of injection may be used in combination with an additional agent capable of proliferating or regenerating cartilage cells, and a pharmaceutical excipient conventionally used in injection may be used. In addition, the injectable composition can be injected directly into the joints for cartilage regeneration.

The amount of the composition containing the DNA fragment mixture when directly injected into a joint is determined according to the age, sex and weight of the subject to be treated, the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, It will be different. Dosage determinations based on these factors are within the level of those skilled in the art and generally the dosage is in the range of about 20 mg / 1 to 40 mg / 1 of the content of the DNA fragment mixture, which is the active ingredient. A more preferable dosage is 26 mg / 1 to 40 mg / 1. It is preferable to administer three times in total every 1 to 2 weeks, but the dose is not limited in any way to the scope of the present invention.

The composition containing the DNA fragment mixture of the present invention can be administered to mammals such as rats, livestock, and humans in various routes.

The composition comprising a DNA fragment mixture having a molecular weight of 3,000 to 10,000 kDa isolated from a fish testimony of the present invention is excellent in the proliferative effect of cartilage cells and the ability to recover damaged cartilage cells and can be usefully used for cartilage regeneration. In particular, the cartilage regeneration composition of the present invention can be used for the treatment or prevention of osteoarthritis.

In addition, since the composition for regeneration of cartilage of the present invention contains a DNA fragment mixture having a specific molecular weight range, it has an effect of raising the lubrication and buffering action of cartilage upon administration into the joints due to its high viscosity, So that the user's convenience can be obtained.

In addition, according to the method for producing a cartilage regeneration composition of the present invention, there is an effect of separating a DNA fragment mixture from a testis of a relatively large number of fishes other than a DNA polymer in high purity and high yield.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a flow chart showing a method for preparing a DNA fragment mixture of the present invention. FIG.
Fig. 2 is data showing the results of measuring viscosity and viscoelasticity of the DNA fragment mixture of the present invention.
3 is a graph showing the ability of the DNA fragment mixture of the present invention to repair damaged cartilage cells.
4 is data showing that the DNA fragment mixture of the present invention expressed a DNA repair-related gene that is damaged.
FIG. 5 is a photograph showing the degree of improvement of joint tissue when the DNA fragment mixture of the present invention is administered to an SD rat induced with arthritis. FIG.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the invention to those skilled in the art.

≪ Example 1: Preparation of a DNA fragment mixture from salmon testes >

(1) Test preparation process

10 g of frozen testes were thawed and then blood was removed and foreign matter was removed. 400 ml of purified water was added to 10 g of the thawed testes and ground with a grinder to prepare a testis lysate

(2) Protein and fat removal process

Thereafter, sodium chloride solution was prepared by dissolving 200 g of sodium chloride in 500 ml of purified water. 500 ml of the sodium chloride solution and the testis solution were mixed and reacted with stirring at 100 캜 for 1 hour, followed by cooling. 30% sodium hydroxide (NaOH) was added to the cooled reaction solution to adjust the pH to 11.0, which was then stirred for 1 hour and filtered to obtain a filtrate from which protein and fat were removed.

(3) Molecular weight reduction process

Hydrochloric acid was added to the filtrate to adjust the pH to 7.0, and the mixture was reacted at 100 ° C for 10 minutes. After cooling, the filtrate was filtered to obtain a filtrate containing a DNA fragment mixture.

(4) Molecular weight fractionation process

To the filtrate, a 30% (w / v) aqueous sodium hydroxide solution was added to bring the pH to 10 again, and the resulting mixture was subjected to gel filtration to obtain a fraction of the DNA fragment mixture having a molecular weight of 3,000 to 10,000 kDa. ) Hydrochloric acid.

(5) Precipitation and drying process

Cooled ethanol (twice the volume of the filtrate) was added to the fraction solution, and the mixture was stirred for 10 minutes and centrifuged. The precipitated precipitate was recovered and washed with 70% ethanol. Dried under vacuum to obtain a DNA fragment mixture, and the process of filtering the 1-mm sieve was repeated to ensure uniformity of the powdered DNA fragment mixture. The final yield of the DNA fragment mixture was 4%, and the molecular weight was mainly distributed to 3,000 to 6,000 kDa as determined by a gel image analysis system.

Experimental Example 1. Increase of Viscosity and Viscoelasticity >

The viscosity and viscoelasticity of the DNA fragment mixture of the present invention were measured using a flow meter (Rheometer, Model AR 2000ex, TA Instruments).

1-1. Viscosity and viscoelasticity

To the 0.9% sodium chloride solution, the DNA fragment mixture prepared in Example 1 was added in an amount of 2%, and the mixture was heated to dissolve. When the DNA fragment mixture is completely dissolved, it is placed in a sealed container, and is subjected to wet sterilization at 121 ° C for 15 minutes, followed by cooling. Viscosity and viscoelasticity of the cooled contents were measured using a viscometer. The results are shown in FIG. As a control, a DNA fragment mixture having a molecular weight of 50 to 1,500 kDa was used.

In FIG. 2, it can be confirmed that the DNA fragment mixture of the present invention has increased viscosity and viscoelasticity as compared to a DNA fragment mixture having a molecular weight of 50 to 1,500 kDa. This is because the DNA fragment mixture of the present invention has higher viscosity and viscoelasticity than the DNA fragment mixture of the prior art having a molecular weight of 50 to 1,500 kDa, which can further increase the lubrication and buffering action of reducing the friction of the joints in the joints, It was confirmed that the pain caused by the friction was significantly reduced through administration to the patient.

≪ Experimental Example 2: Cell proliferation effect of chondrocytes >

The cell proliferation effect of the DNA fragment mixture of the present invention was measured by cell count and MTT assay [3- (4,5-dimethylthiazolyl-2) -2,5-diphenyltetrazolium bromide assay].

2-1. Cell number measurement

Human chondrocytes (CHON-001, ATTC No. CRL-2846) were cultured in DMEM (Dulbecco's Modified Eagle's Medium) containing 10% FBS (fetal bovine serum) and 0.1 mg / Cells were treated with trypsin / EDTA (trypsin / EDTA, Invitrogen), and the cells were detached with a single cell and the number of cells was measured using a hemacytometer. Thereafter, the cells were inoculated into a 100 mm culture dish at 4 x 10 < ⁵ > cells and stabilized for more than 24 hours. Then, the medium of each well was removed, and the DNA fragment mixture prepared in Example 1 was diluted with physiological saline solution (saline) at a concentration of 10, 50, and 100 μg / ml, ₂ in a 37 < 0 > C incubator. At this time, physiological saline solution was used as a control group as a non-treatment group of the DNA fragment mixture.

Thereafter, the cells were treated with trypsin / EDTA (trypsin / EDTA, Invitrogen) to remove the cells, and the number of cells was measured using a hemacytometer. Respectively.

Condition
Treated group The DNA fragment mixture of Example 1 (占 퐂 / ml) 10 50 100 Cell number 5.5 ± 2 (× 10 ⁵ ) 5.8 ± 1 (× 10 ⁵ pieces) 8.4 ± 3 (× 10 ⁵ ) 13.2 ± 0.4 (× 10 ⁵ )

Referring to the results of Table 1, it can be confirmed that the DNA fragment mixture of the present invention has an excellent chondrocyte proliferation effect as compared with the untreated group.

2-2. MTT assay

The chondrocytes used in the above 2-1 were cultured in DMEM (Dulbeco's Modified Eagle's Medium) containing 10% of fetal bovine serum (FBS), 100 μg / ml of streptomycin and 100 U / ml of penicillin, After treatment with trypsin / EDTA (trypsin / EDTA, Invitrogen), the cells were separated into single cells and the number of cells was measured using a hemacytometer. Then, the cells were inoculated into a 96-well plate at 1 × 10 ⁴ cells / 200 μl per well and stabilized for 12 hours or more. Then, the culture medium of each well was removed and 200 μl of the diluted DNA fragment mixture prepared in Example 1 was added at a concentration of 50, 100 μg / ml, followed by culturing in a 37 ° C. incubator containing CO ₂ for 48 hours Respectively. At this time, physiological saline solution was used as a control group as a non-treatment group of the DNA fragment mixture.

Then, for the MTT assay, 20 μl (final concentration: 5 μg / ml) of 5 mg / ml MTT reagent was added to the cells cultured in 1-1 of the above example, and cultured in an incubator for 2 hours The medium containing the post-MTT reagent was cleanly removed. After the medium containing the MTT reagent was removed, 200 μl of DMSO or isopropanol was added to each well, and the mixture was allowed to stand at room temperature for 15 minutes. The absorbance at a wavelength of 570 nm was measured using an ELISA meter (BioTek, Power Wave XS2) And the results are shown in Table 2. The results are shown in an increased value with the untreated group as 100%.

Condition
Untreated group The DNA fragment mixture of Example 1 (占 퐂 / ml) 10 50 100 Cell proliferation rate 100 ± 2.1% 1,372 ± 2.3% 1,657 ± 5.2% 2,024 ± 1.2%

The results of Table 2 also show that the DNA fragment mixture of the present invention has an excellent chondrocyte proliferation effect as compared with the untreated group.

2-3. Recovery ability of damaged cartilage cells

The chondrocytes used in the above 2-1 were cultured in the same manner as the above 2-2, and inoculated into a 96-well plate at 5 × 10 ³ cells / 200 μl per well and stabilized for 12 hours or more. These cells were irradiated with UVB at a wavelength of 315 nm at an intensity of 180 mJ / cm ² to induce cell damage, and 200 μl of a 100 μg / ml dilution of the DNA fragment mixture prepared in Example 1 And incubated in a 37 ° C incubator containing CO ₂ for 48 hours. At this time, physiological saline solution was used as a control group as a non-treated group of a DNA fragment mixture, and hyaluronic acid (HA) used as a therapeutic agent for osteoarthritis was added at the same concentration for comparison of efficacy. Thereafter, an MTT assay was carried out in the same manner as the above-mentioned 2-2. The results are shown in FIG. 3, and the results are shown in an increased value with the untreated group as 100%.

The result of FIG. 3 shows that the damaged cartilage tissue repair ability is superior to that of the DNA fragment mixture ("Control" in FIG. 3) and HA treatment group ("HA" It looked.

2-4. DNA repair ability (RT-PCR)

DNA extracted from damaged chondrocytes as described in 2-3 above was put into a solution containing a 2X Taq-Tenuto DNA polymerase mixture, a target primer and a DNA fragment mixture prepared in Example 1, and reverse transcription PCR was performed . As a control group, physiological saline solution ("Control" in FIG. 4) and hyaluronic acid ("HA" in FIG. 4) were used as a positive control group and treated under the same conditions. RAD51, a gene associated with damaged DNA repair, P53, a gene that inhibits abnormal proliferation or mutation of a cell, and CHK1, an enzyme involved in repairing a cell cycle, are shown in FIG.

In FIG. 4, the DNA fragment mixture expresses RAD51, a gene associated with damaged DNA repair, and P53, a gene that inhibits abnormal proliferation or mutation of a cell. Especially, It is confirmed that the induction of the expression of CHK1, the enzyme, is maximized and an excellent effect is obtained in DNA repair of damaged cartilage cells.

<Experimental Example 3> Effect of growth of cartilage tissue through in vivo test>

3-1. Improvement of arthritis by arthritis injection of SD rats induced arthritis

After SD rats were anesthetized, the hair of one leg was shaved and the skin was disinfected. Then, 2 mg sodium monoiodoacetate was injected in a single intra-articular injection and arthritis was induced for 2 weeks. The SD rat was induced with arthritis, and the DNA fragment preparation prepared in Example 1 was administered once a week for 4 times. After 1 week, the degree of improvement of joint tissue was measured through tissue staining. The results are shown in Fig.

In FIG. 5, it can be confirmed that the damaged cartilage tissue after administration of the drug of the present invention has an improved degree of cartilage cartilage improvement compared to the control group, thereby improving arthritis.

&Lt; Use examples 1. Pharmaceutical formulations >

1-1. Injection preparation

2.0 g of the DNA fragment mixture of the present invention was dissolved in a physiological saline solution of pH 7.0 to make 100 ml. This solution was placed in a bottle and sterilized by heating at 121 DEG C for 20 minutes.

Claims (6)

A composition for regeneration of cartilage comprising a DNA fragment mixture of a molecular weight of 3,000 to 10,000 kDa isolated from a fish testis by a testis preparation process, a protein and fat removal process, a molecular weight reduction process, a molecular weight fractionation process and a precipitation and drying process . The method according to claim 1,
Wherein the fish is a fish of salmonid. A method for producing a composition for regenerating cartilage comprising a DNA fragment mixture having a molecular weight of 3,000 to 10,000 kDa from a fish testimony,
(1 step) Thawing fish testimony, removing blood and foreign matter;
(Step 2) A step of preparing a testis pulverized liquid by adding purified water to a thawed testis and pulverizing the same;
(Step 3) Mixing the testis pulverized liquid of the two steps and aqueous sodium chloride solution, reacting and cooling while stirring;
(Step 4) To the cooled reaction solution of the above three steps, an aqueous solution of sodium hydroxide is added to adjust the pH to 10 to 12, followed by stirring and filtration to obtain a filtrate;
(Step 5) adding hydrochloric acid to the filtrate of the above-mentioned four steps so as to have a pH of 6.5 to 8.5, reacting, cooling and then filtering the filtrate to obtain a filtrate containing the DNA fragments mixture;
(Step 6) A step of obtaining a fraction of a DNA fragment mixture having a molecular weight of 3,000 to 10,000 kDa through gel filtration of the filtrate of the above 5 steps;
(Step 7) Isopropyl alcohol or ethanol is added to the fraction of step 6 and stirred to precipitate a DNA fragment mixture;
(Step 8) Washing the DNA fragment mixture of step 7 with an aqueous solution of ethanol or isopropyl alcohol, dehydrating and drying it;
Wherein the composition for regeneration of cartilage is at least one selected from the group consisting of: The method of claim 3,
Wherein the fraction of step 6 is fractionated and concentrated by ultrafiltration. The method of claim 3,
The step of washing with the alcohol aqueous solution in the step 8 is a step of sequentially washing the DNA fragment mixture with 40 to 80% (w / w) of isopropyl alcohol or ethanol and 95 to 100% (w / w) of isopropyl alcohol or ethanol Wherein the composition for cartilage regeneration is a composition for regenerating cartilage. The method of claim 3,
Wherein the fish is a fish of salmonaceae.

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