KR20240026602A - Composition comprising DNA fraction and collagen and uses thereof - Google Patents

Abstract

The present invention relates to compositions comprising DNA fractions and collagen and uses thereof. The composition is preferably a mixture of DNA fraction and collagen, and has viscoelasticity, restoration ability, joint pain/inflammation inhibition ability, and intra-articular cell protection function, and can be used as a composition for lubricating joints, a composition for improving arthritis, a composition for skin injection, or wound healing. It can be used for various purposes such as compositions, tissue replenishment compositions, and viscosity replenishment compositions.

Description

Composition comprising DNA fraction and collagen and uses thereof {Composition comprising DNA fraction and collagen and uses thereof}

The present invention relates to compositions comprising DNA fractions and collagen and uses thereof.

The composition is preferably a mixture of DNA fraction and collagen, and is characterized by having viscoelasticity and restoration ability, and is used as a composition for lubricating joints, a composition for improving arthritis, a composition for skin injection, a composition for wound healing, a composition for tissue replenishment, and a viscous composition. It can be used for various purposes such as supplementary compositions.

Collagen is the main component of connective tissue, and is mainly found in bones and skin, but is distributed throughout the human body, including joints, membranes of each organ, and hair. Because collagen accounts for a high proportion of approximately 25 to 30% in the human body, it is used as a medical, beauty, and health functional food. Collagen used for medical purposes is biodegradable and is used in various formulations such as sponge, film, membrane, and gel for the purpose of replenishing collagen deficiency in the human body, for tissue regeneration, and as a drug delivery support.

In general, DNA fractions are composed of biopolymers such as phosphoric acid, four types of bases, and deoxyribose, and the mixture of these components is an essential component of cells. This fraction is injected into the wound area, etc. It is used for various purposes, including medicines for treatment and improvement, and cosmetics for the purpose of improving wrinkles related to cell activity. However, among products widely used for medical and cosmetic purposes, it is known that there are no products that have a mixed formulation of collagen and DNA fractions.

An articulation is an area where bones are connected, and on the surfaces where the two bones that make up the joint face each other, there is a thin layer of hyaline cartilage called articular cartilage. The area around the joint is surrounded by a connective tissue membrane that is a continuation of the periosteum, which is called the joint capsule, and its lumen is called the articular cavity. The inner surface of the joint capsule has a thin membrane called synovial membrane, which constantly secretes a small amount of synovial fluid into the joint space to smooth joint movements. When problems occur in these joints, joint diseases appear, and the most prevalent disease among these is osteoarthritis. The areas most commonly affected by osteoarthritis are the fingers, knees, hips, and lumbar spine. Pain and limitation of movement are common symptoms, although they vary slightly depending on the affected joint. The goal of treating osteoarthritis is to relieve pain and minimize disability by maintaining and improving joint mobility. Osteoarthritis is a chronic disease and requires long-term treatment along with increased life expectancy. Treatment options can be broadly divided into non-pharmacological conservative treatment, drug therapy, and surgical treatment. The treatment plan is based on the severity and duration of symptoms, radiological findings, patient's age and comorbidities, lifestyle, and socioeconomic level, It must be established individually depending on the level of activity before the onset of the disease. Unlike other joint diseases, osteoarthritis is a disease in which intra-articular drug administration is possible, and intra-articular administration of hyaluronic acid (HA) has been approved and used in many countries to treat osteoarthritis. However, hyaluronic acid itself has not been confirmed to have an effect on the regeneration of joint cartilage, etc., so it is necessary to develop various substances that not only have an analgesic effect but also have a regenerative function such as cartilage in bone joints.

Accordingly, while studying the various physiological activities of DNA fractions and collagen, the present inventors delayed the progression of osteoarthritis through supplementation through joint injection among the various collagen formulation methods, and added the anti-inflammatory and lubricating effects of the DNA fraction to this. An ideal arthritis injection was developed.

Republic of Korea Patent No. 10-1916193 (Title of the invention: Composition for intraarticular injection containing nucleic acid and chitosan, Applicant: Pharma Research Products Co., Ltd., Registration date: November 1, 2018) Republic of Korea Patent Publication No. 10-2018-0065113 (Title of the invention: Food composition using nucleic acid extract, Applicant: IG Bio Co., Ltd., Publication date: June 18, 2018)

The object of the present invention is a DNA fraction and collagen (cross-linked collagen powder) that have viscoelasticity and restoration ability and can be used for various purposes such as compositions for improving arthritis, fillers for skin injection, compositions for wound healing, compositions for tissue replenishment, and compositions for viscous replenishment. The object is to provide a composition comprising a mixture of.

The present invention relates to a method for producing a composition comprising a DNA fraction and collagen.

In the above manufacturing method, (step 1) the pulverized mammalian skin is immersed in an ethanol aqueous solution, the pulverized skin is transferred to purified water, pepsin is added, stirred, and NaCl aqueous solution is added to aggregate the collagen, and the aggregated Obtaining a collagen extraction liquid by dissolving collagen in purified water;

(Second step) adding Na ₂ HPO ₄ aqueous solution to the collagen extraction liquid to fibrousize collagen into a slurry state and centrifuging to obtain concentrated collagen;

(Third step) freeze-drying the concentrated collagen to form a sponge, vacuum heating and cross-linking the sponged collagen, and then freeze-grinding the cross-linked collagen to prepare collagen powder;

(Step 4) Mixing the collagen powder obtained in the third step with the DNA fraction aqueous solution;

may include.

In the first step, more preferably, the washed mammalian skin pulverized material is immersed in a 50-90% (v/v) ethanol aqueous solution for 12 to 48 hours, then the skin pulverized material is transferred to purified water and pepsin is added. After stirring for 72 to 96 hours, a 1 to 3 N NaCl aqueous solution may be added to aggregate the collagen, and then the aggregated collagen may be dissolved in purified water to obtain a collagen-extracted liquid.

In the first step, the mammal is preferably a pig or cow. The skin can be used with the hair and fat layer removed.

In the first step, the concentration of pepsin is appropriate to be 0.05 to 0.2 wt%, and it is preferably mixed with purified water at the same weight or less than twice the weight.

In the second step, the concentration of the Na ₂ HPO ₄ aqueous solution may be 10 to 1000 mM.

When fibrosis of collagen occurs in the second step, the reaction can be carried out at 30-37°C for 4-30 hours.

Between the second and third steps, the slurried collagen is dried as is or diluted with purified water in a dry oven, and the dried collagen is diluted with purified water to prepare a 0.5 to 3.0 wt% fibrotic collagen solution. and the solution can be freeze-dried in the third step. It is preferable to use the purified water that has been sterilized and filtered.

In the third step, vacuum heating is performed at 90° C. or more for 4 hours or more. More preferably, in the third step, vacuum heating is performed at 90 to 110 ° C. for 4 to 48 hours.

In the third step, vacuum heating can be performed using a conventional vacuum oven. The freeze grinding can be performed using a conventional freeze grinder (freeze grinder). Liquid nitrogen is used during freeze grinding.

In the fourth step, the final concentration of the DNA fraction may be 1.0 to 3.0 wt%.

In the fourth step, collagen powder can be added so that the final concentration is 0.5 to 2.0 wt%.

The DNA fraction may be selected from the group consisting of polynucleotide (PN) and polydeoxyrbonucleotide (PDRN). The DNA fraction may have a molecular weight of 1,600 kDa to 2,500 kDa. At this time, if the molecular weight is less than 1,600 kDa, the viscosity is low when mixed with cross-linked collagen, so it is not suitable for use for the intended purpose of cushioning, lubrication, pain relief, etc., and if the molecular weight is more than 2,500 kDa, the viscosity is low. If it is too high, it may aggregate on its own or may not mix well when mixed with cross-linked collagen, which may also be undesirable.

The composition may be a joint lubrication and anti-inflammatory composition.

Additionally, the present invention may relate to a composition that can help relieve joint pain and inflammation. Preferably, the composition may be a composition for injection into the joint space.

The composition is used for wound treatment, replenishment of tissue defects, and regeneration purposes, and can also be used in the field of skin care. It can also be applied as a composition for lubricating joints, a composition for improving arthritis, a composition for skin injection, a composition for wound healing, a composition for tissue replenishment, and a composition for viscous replenishment.

Hereinafter, the present invention will be described in detail.

The composition may contain water-soluble polymers as additional ingredients. Water-soluble polymers include hyaluronic acid, chondroitin sulfate, glycogen, dextrin, dextran, dextran sulfate, and hydroxypropylmethylcellulose. methylcellulose, alginic acid, chitin, pullulan, chitosan, gelatin and their hydrolysates, polyvinyl alcohol, polyvinylpyrrolidone It may be one or more selected from the group consisting of pyrrolidone, polyacrylic acid, and carboxyvinylpolymer.

The composition may be in the form of a solution, cream, gel, sponge, or powder.

Collagen used in the present invention is bio-derived collagen and may be derived from cows, pigs, fish, etc.

The pharmaceutical composition can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions according to conventional methods. Carriers, excipients, and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, and erythritol ( erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl Cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc ), magnesium stearate, and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient, such as starch or calcium carbonate, in the composition of the present invention. ), sucrose or lactose, gelatin, etc. are mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, syrups, etc. In addition to the commonly used simple diluents such as water and liquid paraffin, they contain various excipients such as wetting agents, sweeteners, and fragrances. , preservatives, etc. may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin fat, glycerogelatin, etc. can be used.

The dosage of the pharmaceutical composition of the present invention will vary depending on the age, gender, and weight of the subject to be treated, the specific disease or pathological state to be treated, the severity of the disease or pathological state, the route of administration, and the judgment of the prescriber. Dosage determinations based on these factors are within the level of one skilled in the art, and dosages generally range from 0.01 mg/kg/day to approximately 2000 mg/kg/day. A more preferred dosage is 1 mg/kg/day to 500 mg/kg/day. Administration may be administered once a day, or may be administered several times. The above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, livestock, and humans through various routes. All modes of administration are contemplated, for example, oral, rectal or by intravenous, intramuscular, subcutaneous, intrathecal or intracerebrovascular injection.

The present invention relates to compositions comprising DNA fractions and collagen and uses thereof. The composition is preferably a mixture of DNA fraction and collagen, has minimal cytotoxicity, has viscoelasticity, restoration ability, joint pain/inflammation inhibition ability, and intraarticular cell protection function, and is used as a composition for lubricating joints, a composition for improving arthritis, and for skin injection. It can be used for various purposes such as compositions, wound healing compositions, tissue replenishment compositions, and viscosity replenishment compositions.

Figure 1 is a diagram explaining the state of the mixed solution of Preparation Example 5 of the present invention.
Figure 2 shows a graph of FT-IR analysis results for the freeze-dried samples of Comparative Example 1, Preparation Example 2, and Preparation Example 5 of the present invention, and each graph peak represents the following: (A) Amide A and OH, (B) Amide B, (C) Amide Ⅰ, (D) Amide Ⅱ, (E) Amide Ⅲ, (F) Carbohydrater

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, it is provided to ensure that the content introduced here is thorough and complete, and to sufficiently convey the spirit of the present invention to those skilled in the art.

<Preparation Example 1: Preparation of fibrotic collagen solution and collagen powder>

Preparation Example 1-1: Preparation of collagen solution

Pig skin from which the hair and fat layer has been removed is soaked in 70% (v/v) ethanol aqueous solution for 24 hours and then washed with purified water. Washed pig skin is treated with 0.1 wt% pepsin aqueous solution and filtered to obtain collagen. Viruses are also inactivated during collagen extraction. Collagen was aggregated using a salting-out process (adding equal volumes of 2 M NaCl aqueous solution) and dissolved in purified water. 3 wt% was prepared as a stock solution, which was diluted to prepare a 0.5 wt% collagen solution (pepsin soluble collagen solution; PSC solution) and filtered with a 0.2 μm filter. At this time, the filter process may be performed first after preparing a collagen solution with a high concentration of 3 wt%, and then diluted to 0.5 wt%.

Preparation Example 1-2: Preparation of fibrotic collagen solution

A solution with a pH adjusted to 7.3-7.45 was prepared by dissolving 66 mM (9.369 g/L) of Na ₂ HPO ₄ in purified water, which was then filtered for sterilization through a 0.2 μm filter. This solution was mixed with the solution of Preparation Example 1-1 at a weight ratio of 1:1 (w/w) and reacted at 37° C. for 30 hours to prepare fibrous collagen (collagen slurry).

The fibrous collagen is centrifuged, recovered, and diluted with purified water that has been sterilized through a 0.2 μm filter. Dry the diluted fibrous collagen solution using a dry oven and check the weight of the solute (collagen). The final concentration of this solution is A fibrotic collagen solution was prepared by diluting the solution to 3 wt% with a bacteriostatically filtered diluent (20 mM dibasic phosphate buffer, pH 7.3 to pH 7.4).

Preparation Example 1-3: Preparation of non-crosslinked collagen powder

The fibrotic collagen solution of Preparation Example 1-2 is prepared into a sponge using freeze-drying, and non-cross-linked collagen powder (NCP; Non-Cross-linked collagen powder) is recovered using a freeze grinder.

Preparation Example 1-4: Preparation of cross-linked collagen powder

The fibrous collagen solution of Preparation Example 1-2 was prepared into a sponge using freeze-drying, and subjected to a DHT (dehydrothermal) process of crosslinking by vacuum heating at 105°C for 48 hours using a vacuum oven, followed by post-freeze grinding. Collagen powder (CP; Cross-linked collagen powder) is recovered.

<Preparation Example 2: Preparation of PN solution>

Polynucleotide (PN) (HTL, France, molecular weight 2280 kDa, medical device grade), a DNA fraction extracted from salmon, was used. (In additional experiments, PN with molecular weights of 1990, 2080, and 1880 kDa were used, respectively, and it was confirmed that it was stable in the range of about 1,600 to 2,500 kDa).

A dilution (20 mM Dibasic phosphate buffer, pH 7.3 ~ pH 7.4) was prepared and filtered for sterilization through a 0.2 μm filter. After dissolving the sterilized PN powder in the diluted solution at 40°C for more than 12 hours, the PN content of this solution was measured using a UV detector at a wavelength of 260 nm, and then the diluted solution was added so that the final concentration was 2 wt%.

<Preparation Example 3: Preparation of mixed solution 1>

Mix the fibrotic collagen solution of Preparation Example 1-2 and the PN solution of Preparation Example 2 at a mixing ratio of 1:1 (w/w) and homogenize with a homogenizer.

<Preparation Example 4: Preparation of mixed solution 2>

1.5 g of non-crosslinked collagen powder (NCP) of Preparation Example 1-3 was mixed with 100 g of the PN solution of Preparation Example 2 and evenly dispersed in the solution using a homogenizer.

<Preparation Example 5: Preparation of mixed solution 3>

1.5 g of cross-linked collagen powder (CP) of Preparation Example 1-4 was mixed with 100 g of the PN solution of Preparation Example 2 and evenly dispersed in the solution using a homogenizer.

<Comparative Example 1>

As a comparative solution, the fibrotic collagen solution (total collagen content 3 wt%) of Preparation Example 1-2 was prepared.

<Experimental Example 1: Confirmation of formulation compatibility - Compositions of Preparation Examples 3 to 5>

In order to uniformly mix collagen and PN, the final formulation mixed with the PN solution of Preparation Example 2 was prepared according to the state of collagen (fibrosis, cross-linking, etc.), and the results of comparison are shown in Table 1. In Preparation Example 3, the fibrotic collagen solution and the PN solution were mixed at a weight ratio of 1:1, and in Preparation Examples 4 and 5, 1.5 g of non-crosslinked collagen powder and crosslinked collagen powder were mixed with 100 g of a 2.0 wt% PN solution. It is manufactured by putting it in.

composition mixing conditions Collagen and PN
Status after mixing Formulation
Whether Production example 3 Preparation Example 2 (PN) +
Preparation Example 1-3 (fibrotic collagen solution) Layer separation occurs x Production example 4 Preparation Example 2 (PN) +
Preparation Example 1-4 (NCP, non-crosslinked collagen powder) Collagen Powder Decomposition x Production example 5 Preparation Example 2 (PN) +
Preparation Example 1-5 (CP, cross-linked collagen powder) Evenly distributed without decomposition o

In Preparation Example 3, layer separation occurred when PN was mixed with fibrotic collagen, and in Preparation Example 4, when non-crosslinked collagen powder (NCP) was used, it was immediately decomposed in the mixing step with the PN solution, but was crosslinked as in Preparation Example 5. In the case of collagen powder (CP), it does not decompose in solution and remains in a dispersed solution state in the form of powder.

The mixed state of collagen and PN (Preparation Example 5) is as shown in the schematic diagram of FIG. 1.

Meanwhile, the state of Preparation Example 5 can be confirmed through Experimental Example 3, and this result can explain that collagen and PN are not in a chemical or ionic bond state.

Since this is not a chemical or ionic bond, there are no by-products from the combination of collagen and PN, indicating that there are no risk factors due to this specific combination, and it proves that no by-products that can cause side effects to the human body are created.

<Experimental Example 2: Confirmation of properties by concentration>

In order to confirm the formulation at each concentration of Preparation Example 5, the state after mixing at each concentration was compared through visual observation.

Collagen (wt%) PN (wt%) Type 0.25 1.00 layer separation 3.00 0.5 0.25 layer separation 0.50 1.00 Gel 2.00 3.00 1.0 0.25 layer separation 0.50 1.00 Gel 2.00 3.00 1.5 0.25 layer separation 0.50 1.00 Gel 2.00 3.00 2.0 0.25 layer separation 0.50 1.00 Gel 2.00 3.00 3.0 1.00 layer separation 3.00

As shown in the results in Table 2, when mixing collagen and PN, layer separation occurred or a stable gel state was confirmed. The most stable gelation occurred when collagen was 0.5 to 2.0 wt% and PN was 1.0 to 3.0 wt% based on the total mixture. The solution state is shown.

<Experimental Example 3: FT-IR analysis>

In the formulation of Preparation Example 5, FT-IR analysis was performed using Comparative Example 1 and Preparation Example 2 as controls to confirm whether a chemical bond between collagen and PN exists.

Analysis conditions for FT-IR (Fourier transform IR spectroscopy)

(1) Model name: Thermo Scientific (Nicolet iS10)

(2) Measurement area: 7,800 - 350 cm ^-1 (KBr), 11,000-375 cm ^-1 (XT-KBr)

(3) Resolution: 0.5 cm ^-1

(4) S/N Ratio = 35,000:1 (Peak to Peak)

(5) Ordinate Linearity (ASTM E1421): Less than 0.1 %T

Each sample was freeze-dried and manufactured into a sheet form for analysis, and the FT-IR analysis results are shown in Figure 2.

As shown in Figure 2, the major peaks (amide A, B, Ⅰ, Ⅱ, Ⅲ) showed similar shapes. In addition, no new peaks were generated, and since there was only a difference in permeability, it was confirmed that there was no chemical bond between collagen and PN.

<Experimental Example 4: Viscoelasticity (Rheometer) measurement evaluation>

To determine the rheological properties of Preparation Example 5, Comparative Example 1, and Preparation Example 2, they were analyzed using a rheometer.

Analysis conditions for rotational rheometer

(1) Test equipment: Anton-Paar (MCR 302)

(2) Frequency: 1.0 Hz

(3) Temperature: 22℃

(4) Strain: 0.1%

The storage modulus and viscosity, which are the results of analysis under the above analysis conditions, are shown in Table 3.

storage modulus
(storage modulus) viscosity
(viscosity) Comparative Example 1 59Pa 62Pa·s Production example 2 128Pa 123Pa·s Production example 5 104Pa 103Pa·s

As shown in Table 3, Preparation Example 5 and Preparation Example 2 were confirmed to have similar storage modulus and viscosity, and Comparative Example 1 showed relatively low values.

That is, when comparing Preparation Example 5, which is a PN-collagen mixed formulation, with the collagen of Comparative Example 1, it can be seen that the PN-collagen mixed formulation prepared in the present invention is a material with significantly superior viscoelasticity than collagen.

<Experimental Example 4: Cytotoxicity evaluation>

Cytotoxicity evaluation was conducted to evaluate the safety of Preparation Example 5, Comparative Example 1, and Preparation Example 2.

(1) Sample elution conditions

Elute each sample at 4 g/20 ml (10% FBS, 1% antibiotics MEM medium) for 24 hours at 37°C.

(2) Cell culture

Incubate at 37°C for 24 hours at a ratio of 2 x 10 ⁵ Cells/well in a 6 well plate.

(3) Test material treatment

After centrifuging the test substance cultured for 24 hours (3,000 rpm, 15 min), replace the medium on the plate with elution medium and incubate at 37°C for 48 hours and then visually observe.

(4) Quantitative analysis (Cell counting)

After treatment with trypsin solution and incubation at 37°C for 3 minutes, the medium is treated and pipetted. The pipetted solution is loaded into a hematocymeter to count the number of cells. The cell viability of each test group is calculated using the formula below.

(Cell count _{test group} /Cell count _{negative control group)} x 100 = Cell viability (%)

The cell viability of each sample is shown in Table 4.

condition Cell survival rate (%) Negative control group 100.0 Production example 5 98.9 Comparative Example 1 93.0 Production example 2 65.0

As shown in Table 4, Preparation Example 5 and Comparative Example 1 showed similar values to the negative control, but Preparation Example 2 showed a value of less than 70% (ISO 10993-5 cytotoxicity safety standard → Cell viability 70% or more). . This shows that PN, when used alone at a high concentration, exhibits cytotoxicity and is therefore undesirable for use as an injectable agent in the body. However, when mixed with collagen, toxicity is significantly reduced, showing that it is advantageous in terms of biological safety.

<Experimental Example 5: Animal experiment (confirmation of joint pain relief effect)>

Animal experiments were conducted to evaluate the efficacy of Preparation Example 5 and Preparation Example 2 for joint injection.

The test group consisted of a total of 4 groups, including a normal group, a negative control group, and a group administered Preparation Example 5 and Preparation Example 2 after damage treatment, with 6 animals per group.

To induce osteoarthritis, MIA (Monosodium iodoacetate) was administered at a dose of 3 mg/head into the joint space of the right leg in all groups except the normal group, and the normal group was administered the same amount of physiological saline injection as an excipient.

Preparation Example 5 and Preparation Example 2 were administered as a single dose into the joint space 3 days after MIA administration at a dose of 50 μL/head (dry weight, respectively, Preparation Example 5: 35 mg/mL, Preparation Example 2: 20 mg/mL), The normal group and negative control group were administered the same dose of phosphate buffer as an excipient. And WBI (weight bearing index) was measured before MIA administration (day 0) and on days 3, 7, 14, and 21 after administration.

※ Rate of change in weight placed on the right foot (administration) (WBI, %) =

[Weight on the right foot (administration) / (Weight on the right foot (administration) + Weight on the left foot (non-administration)] x 100

The WBI results are shown in Table 5.

Group WBI after administration (%) 0 days 3 days 7 days 14 days 21st Jeongsang-gun Mean 49.9 47.9 49.1 50.6 51.2 S.D. 1.2 4.6 1.8 0.9 1.9 Negative control group Mean 50.1 28.2 32.0 33.1 32.5 S.D. 1.2 5.2 2.8 5.1 3.0 Production example 5 Mean 49.3 27.4 31.9 34.4 37.0 S.D. 1.5 3.8 3.8 3.9 4.9 Production example 2 Mean 49.9 28.5 33.3 34.2 37.3 S.D. 1.7 6.8 5.1 3.9 4.6 * Weight change rate on the right foot (administration) → 50% when both feet are balanced

As shown in Table 5, it was confirmed that Preparation Example 5 showed a statistically significant difference from the 7th day of test substance administration compared to the negative control group, and that pain recovered by the 21st day. In addition, as in Preparation Example 2, it was confirmed that it had the same pain relief effect as when using PN alone.

Through these results, it can be confirmed that the composition prepared in the present invention exhibits excellent efficacy in reducing cytotoxicity while providing a pain-relieving effect in PN.

Claims (13)

(Step 1) After immersing the pulverized mammalian skin in an aqueous ethanol solution, the pulverized skin is transferred to purified water, added with pepsin, stirred, added with an aqueous NaCl solution to aggregate collagen, and the aggregated collagen is dissolved in purified water. Obtaining a collagen extraction liquid;
(Second step) adding Na ₂ HPO ₄ aqueous solution to the collagen extraction liquid to fibrousize collagen into a slurry state and centrifuging to obtain concentrated collagen;
(Third step) freeze-drying the concentrated collagen to form a sponge, vacuum heating and cross-linking the sponged collagen, and then freeze-grinding the cross-linked collagen to prepare collagen powder;
(Step 4) Mixing the collagen powder obtained in the third step with the DNA fraction aqueous solution;
A method for producing a composition containing a DNA fraction and collagen, comprising: According to paragraph 1,
A method for producing a composition containing a DNA fraction and collagen, characterized in that the concentration of the Na ₂ HPO ₄ aqueous solution in the second step is 10 to 1000 mM. According to paragraph 1,
A method for producing a composition containing a DNA fraction and collagen, characterized in that vacuum heating in the third step is performed at 90° C. or more for 4 hours or more. According to paragraph 1,
A method for producing a composition containing a DNA fraction and collagen, wherein the DNA fraction is selected from the group consisting of polynucleotide (PN) and polydeoxyrbonucleotide (PDRN). A composition containing a DNA fraction and collagen, characterized in that it is prepared by the method of claim 1. An injectable composition for joint space, characterized in that it contains the composition of claim 5. According to clause 5,
The composition is an injectable composition for joint space, characterized in that it has the effect of relieving joint pain or joint inflammation. A composition for joint lubrication, characterized in that it contains the composition of claim 5. A composition for improving arthritis, characterized in that it contains the composition of claim 5. A composition for viscosity supplementation, characterized in that it contains the composition of claim 5. A composition for skin injection, characterized in that it contains the composition of claim 5. A composition for wound healing, characterized in that it contains the composition of claim 5. A composition for tissue replenishment, characterized in that it contains the composition of claim 5.