Method for obtaining polydeoxyribonucleotide from fish testis tissue and application of polydeoxyribonucleotide

Abstract

The invention belongs to the technical field of biotechnology, and discloses a method for obtaining polydeoxyribonucleotide from fish testis tissue, which comprises the following steps: removing fascia from fish testis, and treating the fish testis into blocks; carrying out two-stage homogenization treatment on the blocky fish testis tissues to obtain homogenized precipitates; treating the homogenized precipitate with a compound enzyme enzymatic hydrolysate to obtain a system after enzymolysis; and performing deproteinization, alcohol precipitation, filtration, impurity removal, freeze drying and grinding on the system after enzymolysis to obtain the polydeoxyribonucleotide. The polydeoxyribonucleotide has the characteristics of high product homogeneity, small and concentrated molecular weight and high purity and yield, and has the effects of resisting inflammation and repairing damage.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a method for obtaining polydeoxyribonucleotides from fish sperm nest tissue and application thereof. BACKGROUND

[0002] Polydeoxyribonucleotides (PDRN) is a kind of natural bioactive ingredient, and its small molecule form (usually refers to fragments with a molecular weight of ≤60kDa) has high bioavailability and transdermal absorption, and has become a research hotspot. Small molecule PDRN can activate adenosine A2A receptor to promote the synthesis of extracellular matrix, and has the effect of regulating inflammatory response.

[0003] Currently, PDRN is generally prepared from fish sperm nest tissue. The process usually includes homogenization, lysis, salting-out, ethanol precipitation and ultrasonic disruption, so as to realize the extraction of DNA and the preparation of PDRN. For example, patent application files CN202211007267.4, CN202410166757.1, CN202211695344.X, CN201911191251.1 and CN202110391188.7 disclose a series of methods for preparing PDRN. However, the homogenate or lysis solution used in the above-mentioned application files generally contains one or more of organic chemical reagents such as Tris-HCl, SDS, disodium EDTA, phenol and chloroform. The introduction of such reagents poses a safety risk to the final product, and there are defects such as low product yield, yellowing and wide molecular weight distribution (50-500bp). Patent application file CN202110802304.X discloses a safe and edible fish sperm DNA extraction method by using edible alkaline solution and other solutions, but it needs to be pre-cooled at 4℃ for at least 48h, and the overall operation time is as long as 4-5d, which has the defect of high time cost. Patent application file CN202210451511.X discloses a method for preparing salmon genomic DNA fragments by introducing exogenous Phi DNA polymerase for multiplex displacement amplification, and using ultrasonic disruption and restriction endonuclease Sau3AI to break it to obtain PDRN. Patent application file CN202011188974.9 discloses a method for directly using restriction endonuclease Sau3 AI and ribonuclease to degrade DNA. However, these two methods have the defects of high enzyme cutting efficiency affected by environmental factors, poor product uniformity, high production cost, complex process, high PDRN loss and long time consumption. Therefore, it is necessary to provide a method for preparing small molecule PDRN with concentrated molecular weight and high uniformity, which is the main way to solve the above problems. SUMMARY

[0004] The technical problem solved by the present application is to provide a method for obtaining polydeoxyribonucleotides from fish sperm tissue and application thereof, aiming at the above-mentioned deficiencies of the prior art.

[0005] To solve the above technical problems, the technical scheme adopted by the present application is:

[0006] On the one hand, a method for obtaining polydeoxyribonucleotides from fish sperm tissue is provided, comprising:

[0007] The fish sperm is treated into blocks after the fascia is removed;

[0008] The blocky salmon sperm tissue is treated by two-stage homogenization to obtain a homogenate sediment;

[0009] The homogenate sediment is treated with a complex enzyme hydrolysis solution to obtain an enzyme hydrolysis system;

[0010] The enzyme hydrolysis system is subjected to protein removal, alcohol precipitation, filtration, impurity removal, freeze-drying and grinding to obtain polydeoxyribonucleotides.

[0011] On the other hand, the application of the polydeoxyribonucleotides obtained by the above method in anti-inflammatory and / or damage repair is provided.

[0012] Compared with the prior art, the present application has the following advantages:

[0013] 1. The method for obtaining polydeoxyribonucleotides from fish sperm tissue of the present application comprises two-stage homogenization and complex enzyme hydrolysis, and the obtained PDRN has a yield of up to 12% and a purity of up to 2.47, which is much higher than the yield and purity of PDRN obtained by single enzyme hydrolysis or non-two-stage homogenization, and the molecular weight of the obtained PDRN is concentrated in the range of 100-250 bp, which has the characteristics of small and concentrated fragments, significantly improved concentration and uniformity. The polydeoxyribonucleotides have the characteristics of anti-inflammatory and damage repair.

[0014] 2. The method for obtaining polydeoxyribonucleotides from fish sperm tissue of the present application is carried out in a safe and mild aqueous buffer system, which can effectively avoid the use of traditional Tris-HCl, SDS, EDTA disodium, phenol, chloroform and other chemical reagents, and the final product is non-toxic and harmless, which can effectively avoid the harm of immunogenicity risk to the human body, and has the characteristics of biocompatibility and high application safety.

[0015] 3、The method for obtaining the polydeoxyribonucleotide from the fish sperm nest tissue has the characteristics of small and concentrated PDRN fragments, high activity and soothing anti-inflammatory effect, the method has a short extraction period, high product yield and purity, reaches the food-grade safety standard, and can be widely applied in the health field.

[0016] The technical solutions of the present application will be further described in detail below in combination with the drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Picture of appearance of the polydeoxyribonucleotide obtained in Example 1;

[0018] Figure 2 Picture of appearance of the polydeoxyribonucleotide obtained in Example 2;

[0019] Figure 3 Picture of appearance of the polydeoxyribonucleotide obtained in Example 3;

[0020] Figure 4 Picture of appearance of the polydeoxyribonucleotide obtained in Comparative Example 1;

[0021] Figure 5 Electrophoretogram of the polydeoxyribonucleotide obtained in each example and comparative example;

[0022] Figure 6 Schematic diagram of the detection results of each factor gene level of the PDRN in Example 1 in the anti-inflammatory performance test;

[0023] Figure 7 Picture of immunofluorescence staining of the PDRN in Example 1 in the damage repair test. DETAILED DESCRIPTION

[0024] The technical solutions will be described in detail below in combination with the examples of the present application. Obviously, the described examples are part of the examples of the present application, rather than all the examples. Based on the examples in the present application, all other examples obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0025] In the following description, the term "and / or" is used to describe the association relationship of the associated objects, which means that there can be three relationships, for example, A and / or B, which can represent the cases of A alone, B alone and A and B together. Wherein A and B can be singular or plural.

[0026] In the following description, the terms "include", "contain", "have" and "contain" and the like are all open terms, that is, they mean to include but not limited to.

[0027] Those skilled in the art shall understand that, in the following description of the embodiments of the present application, the order of the serial numbers does not mean the order of execution, and part or all of the steps can be executed in parallel or in sequence, and the execution order of each process shall be determined according to its function and inherent logic, and shall not constitute any limitation on the implementation process of the embodiments of the present application.

[0028] Those skilled in the art shall understand that the numerical ranges in the embodiments of the present application shall be understood as also specifically disclosing each intermediate value between the upper limit and the lower limit of the range. Each smaller range between any stated value or stated range of values and any other stated value or stated range of values within the stated range is also included in the present application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range.

[0029] Unless otherwise defined, technical / scientific terms used herein have the same meaning to those generally understood by a person of ordinary skill in the art to which the present application belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present application. All documents mentioned in the specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of conflict between the content of the specification and any incorporated document, the content of the specification shall prevail.

[0030] The technical principle adopted by the present application is that, by subjecting the blocky fish milt tissue after removing fascia to two-stage homogenization treatment to fully destroy the biological structure, and then subjecting to complex enzyme enzymolysis and protein removal and other operations, the DNA is fully released and the collagen is fully degraded, so that small molecule PDRN with a molecular weight of 100-250 bp is obtained, which has the characteristics of high yield and purity.

[0031] In one aspect, a method for obtaining polydeoxyribonucleotides from fish milt tissue is provided, comprising:

[0032] The fish milt is processed into a block after removing fascia;

[0033] The blocky fish milt tissue is subjected to two-stage homogenization treatment to obtain a homogenate sediment;

[0034] The homogenate sediment is treated with a complex enzyme enzyme solution to obtain an enzyme hydrolysis system;

[0035] The enzyme hydrolysis system is subjected to protein removal, alcohol precipitation, filtration, impurity removal, freeze-drying and grinding to obtain polydeoxyribonucleotides.

[0036] In some embodiments, the fish milt tissue is fresh salmon milt tissue or salmon milt tissue stored at-20℃.

[0037] The salmon sperm sac tissue is preferably fresh salmon sperm sac tissue or salmon sperm sac tissue stored at-20 DEG C, the fresh salmon sperm sac tissue is full, delicate and elastic, the frozen salmon sperm sac tissue is hard, complete in structure and increased in brittleness, and the fresh salmon sperm sac tissue or the salmon sperm sac tissue stored at-20 DEG C can effectively realize complete removal of fascia and connective tissue and avoid the influence of impurity residues on subsequent PDRN acquisition.

[0038] In some embodiments, the two-stage homogenization treatment comprises a first-stage homogenization treatment and a second-stage homogenization treatment performed in sequence, and the first-stage homogenization treatment and the second-stage homogenization treatment each comprises mixing the material with a homogenization solution, intermittent wall breaking treatment and centrifugal removal of supernatant.

[0039] In some specific embodiments, the homogenization solution is a mixed solution of sodium chloride and sodium citrate, and the concentration of sodium chloride and the concentration of sodium citrate in the homogenization solution are each 0.05-0.15 M.

[0040] In some specific embodiments, in the first-stage homogenization treatment, the volume of the homogenization solution is 2-3 times the mass of the material; in the second-stage homogenization treatment, the volume of the homogenization solution is 3-5 times the mass of the material; the intermittent wall breaking treatment is repeated in a wall breaking-resting mode, the ratio of wall breaking time to resting time is 4-5, and the number of repetitions is 4-5; the second-stage homogenization treatment comprises multiple times of mixing the material with a homogenization solution, intermittent wall breaking treatment and centrifugal removal of supernatant, and the number of times is 3-4; and the centrifugal removal of supernatant each comprises centrifugation at 10000-15000 g for 10-15 min at a temperature of 4 DEG C, and discarding the supernatant.

[0041] The two-stage homogenization treatment preferably fully destroys the structure of the sperm sac tissue and fully exposes the cell contents to a subsequent enzymatic environment.

[0042] In some embodiments, the treatment of the post-homogenization precipitate with a complex enzyme enzymolysis solution specifically comprises: mixing the post-homogenization precipitate with a complex enzyme enzymolysis solution, intermittent wall breaking treatment, and stirring to obtain an enzymolysis system.

[0043] The present application comprises treatment of the post-homogenization precipitate with a complex enzyme enzymolysis solution, the trypsin in the complex enzyme can specifically hydrolyze the peptide bond of arginine and lysine, efficiently degrading histone, protamine and the like combined with DNA, so that the DNA is fully released, the collagenase specifically decomposes collagen in the connective tissue, collapses the tough fascia network, releases the cell contents and reduces impurity residues, and the trypsin and collagenase cooperate to realize efficient separation of PDRN from impurities such as proteins, polysaccharides and salts.

[0044] In some specific embodiments, the complex enzyme enzymolysis solution is a sodium chloride solution of trypsin and collagenase, wherein the concentration of trypsin and the concentration of collagenase are both 1wt%-3wt%, and the mass of the complex enzyme enzymolysis solution is 8-10 times the mass of the post-homogenization precipitate.

[0045] In some specific embodiments, the intermittent cell wall breaking process is repeated in a cell wall breaking-resting mode, the ratio of the cell wall breaking time to the resting time is 4-5, and the number of repetitions is 2-3; the stirring rate is 800-1200 rpm, and the stirring time is 3h.

[0046] The inventors have found that, by using a complex enzyme enzymolysis solution with a trypsin concentration and a collagenase concentration both being 1%-3% and a mass being 8-10 times the mass of the post-homogenization precipitate, and by using a cell wall breaking-resting mode as the enzymolysis condition, the complex enzyme enzymolysis solution and the post-homogenization precipitate can be thoroughly mixed, and the DNA can be released and the collagen can be degraded maximally, which can effectively avoid the formation of fibrous impurities containing collagen and other incompletely decomposed fibers, which can affect the subsequent DNA purification process, and which can help to obtain PDRN with high yield, purity, and uniformity of small molecular fragments.

[0047] In some embodiments, the protein removal includes three separation treatments of the post-enzymolysis system; each of the separation treatments includes adjusting the pH and centrifugation, the pH is adjusted to 11.0-13.0 in the first separation treatment, the pH is adjusted to 4.5-5.0 in the second separation treatment, and the pH is adjusted to 5.0-7.0 in the third separation treatment, and the centrifugation includes centrifugation at 15000-17000g for 10-15min at a temperature of 4℃; the first separation treatment and the second separation treatment further include stirring at 500-800rpm for 3-5min after adjusting the pH and before centrifugation.

[0048] The present application preferably includes three separation treatments to remove proteins in the post-enzymolysis system, and the post-enzymolysis system of the complex enzyme is subjected to three separation treatments, so that the product does not contain fibrous impurities co-precipitated with DNA, and PDRN with high yield and purity is obtained.

[0049] In some embodiments, the alcohol precipitation is performed using pre-cooled anhydrous ethanol, the volume of the pre-cooled anhydrous ethanol is 2-3 times the volume of the post-protein removal system, and the alcohol precipitation is performed at -20℃ for 3-5h.

[0050] In some specific embodiments, the pre-cooled anhydrous ethanol is -20℃ pre-cooled anhydrous ethanol.

[0051] In still another aspect, the application provides a use of the polydeoxyribonucleotides obtained by the above method in anti-inflammatory and / or damage repair.

[0052] The present application has undergone a series of experiments before the application, now a part of the test results are listed below to further describe the application in detail, the following embodiments are described in detail.

[0053] Example 1

[0054] The present embodiment provides a method for obtaining polydeoxyribonucleotides from fish sperm nest tissue, comprising:

[0055] S1: pretreatment: after the salmon sperm nest tissue frozen at-20℃ is thawed at room temperature, the surface is washed with deionized water, and after washing, the surface water is absorbed with a water-absorbing paper, the surface fascia of the sperm nest tissue is removed with a forceps, 100g of the salmon sperm nest tissue with the surface fascia removed is weighed, cut into 1cm 3 chunk, and the pretreated salmon sperm nest tissue is obtained;

[0056] S2: homogenate: the pretreated salmon sperm nest tissue is subjected to two-stage homogenate treatment to obtain a homogenate sediment; the two-stage homogenate treatment comprises sequentially performing first-stage homogenate treatment and second-stage homogenate treatment, the first-stage homogenate treatment comprises: mixing the pretreated salmon sperm nest tissue with 200mL of homogenate liquid, placing it in a wall-breaking machine, and performing intermittent wall-breaking treatment and centrifugal supernatant removal to obtain a homogenate system; the intermittent wall-breaking treatment comprises: working for 8s, stopping for 2s, and repeating the above working-stop mode for 5 times; the centrifugal supernatant removal is to centrifuge the broken wall system at 10000g for 10min at 4℃, and discard the supernatant to obtain a sediment; the second-stage homogenate treatment comprises: mixing the sediment obtained by the first-stage homogenate treatment with 300mL of the homogenate liquid, placing it in a wall-breaking machine, and performing intermittent wall-breaking treatment and centrifugal supernatant removal to obtain a sediment, and repeating the method of treating the sediment obtained by the first-stage homogenate treatment for 2 times; the intermittent wall-breaking treatment is the same as that in the first-stage homogenate treatment, and the centrifugal supernatant removal is the same as that in the first-stage homogenate treatment; the homogenate liquid is a mixed solution of sodium chloride and sodium citrate, the concentration of sodium chloride in the mixed solution is 0.15M, and the concentration of sodium citrate is 0.15M; the sodium chloride is derived from Maikelin, S805275, and the sodium citrate is derived from Maikelin, S818273;

[0057] S3: Enzymatic hydrolysis: 1000 mL of a complex enzyme hydrolysis solution was added to the homogenate post-deposits, and the working-stop mode of S2 was repeated three times. The post-crushing system was stirred at 1200 rpm at room temperature for 3 h to obtain the post-enzymatic hydrolysis system. The complex enzyme hydrolysis solution was a trypsin and collagenase sodium chloride solution, wherein the trypsin concentration was 1 wt%, the collagenase concentration was 1 wt%, and the sodium chloride concentration was 1 M. The trypsin was Xiasheng enzyme, which was a high-concentration trypsin, and the collagenase was derived from Nanning Pangbo Biological Engineering Co., Ltd., which was a collagen proteinase; the stirrer was purchased from Shanghai Licenbangxi Instrument Technology Co., Ltd., LC-DES-200SH;

[0058] S4: Protein removal: the post-enzymatic hydrolysis system was adjusted to pH 12.0 with 10 M NaOH solution, stirred at 500 rpm at room temperature for 3 min, then centrifuged at 15000 g at 4°C for 10 min, the supernatant was collected, the collected supernatant was adjusted to pH 4.5 with 6 M hydrochloric acid solution, stirred at 500 rpm at room temperature for 3 min, centrifuged at 15000 g at 4°C for 10 min, the supernatant was collected, the pH of the supernatant was adjusted to 7.0, and the supernatant was collected by centrifugation at 15000 g at 4°C for 10 min. The supernatant was the post-protein removal system; the NaOH solution was purchased from Maikelin, S817971, and the hydrochloric acid solution was purchased from Chengdu Kelong Chemical Co., Ltd., CAS: 7647-01-0;

[0059] S5: Alcohol precipitation: pre-cooled anhydrous ethanol was added to the post-protein removal system, and precipitated at -20°C for 3 h; the anhydrous ethanol was analytical pure and purchased from Guangdong Guanghua Science and Technology Co., Ltd.; the volume of the anhydrous ethanol was twice the volume of the post-protein removal system;

[0060] S6: Filtration: the post-alcohol precipitation system was filtered with 100 mesh filter cloth, and the precipitate was collected;

[0061] S7: Impurity removal: 500 mL of 75% ethanol was first added to the precipitate and centrifuged for 3 times, and then 200 mL of anhydrous ethanol was washed once, and the precipitate was collected; the centrifugation conditions were all 4°C, 15000 g, and 5 min; the 75% ethanol and the anhydrous ethanol were pre-cooled at -20°C, and were purchased from Guangdong Guanghua Science and Technology Co., Ltd., analytical pure;

[0062] S8: Freeze-drying: the collected precipitate was freeze-dried in a vacuum freeze-drier; the temperature of the vacuum freeze-drier was -40°C, and the time was 4 h;

[0063] S9: Weighing: After the freeze-dried material is ground into a powder without particles using a flour mill, the final product, edible salmon PDRN, is obtained. The flour mill is purchased from Cixi Naiou Electrical Appliance Co., Ltd., and the model is AQ-180E.

[0064] Example 2

[0065] This example provides a method for obtaining polydeoxyribonucleotides from fish sperm tissue. The same as example 1, the difference is that in S3, the enzyme solution used for enzymolysis is a trypsin sodium chloride solution, the concentration of trypsin in the enzyme solution is 1wt%, and the concentration of sodium chloride is 1M.

[0066] Example 3

[0067] This example provides a method for obtaining polydeoxyribonucleotides from fish sperm tissue. The same as example 1, the difference is that in S3, the enzyme solution used for enzymolysis is a trypsin sodium chloride solution, the concentration of trypsin in the enzyme solution is 1wt%, and the concentration of sodium chloride is 1M.

[0068] Comparative Example 1

[0069] This comparative example examines the effect of the combination of homogenization treatment and enzymolysis on the product polydeoxyribonucleotides. The preparation method is the same as example 1, the difference is that,

[0070] S2 is to mix the pretreated salmon sperm tissue with 200mL of the homogenate, use a cell crusher to continuously break the wall for 2min, centrifuge at 4000rpm for 15min at 4℃, discard the supernatant, obtain the precipitate, and repeat the process of mixing the pretreated salmon sperm tissue with the homogenate, breaking the wall and centrifugation twice to obtain the homogenized precipitate;

[0071] S3 is to mix the homogenized precipitate with 1000mL of 10wt% sodium chloride solution, stir to dissolve completely, and stand at 4℃ for 48h.

[0072] Performance evaluation

[0073] 1. Product yield and purity

[0074] ① Small molecule PDRN yield: yield = small molecule PDRN sample / initial sperm tissue weight

[0075] ② Purity of small molecule PDRN: The products obtained in each example were dissolved in ddH2O at a preset concentration of 1 mg / mL to obtain the test solution. In nucleic acid purity evaluation, A260 / A280 and A260 / A230 are two key UV spectrophotometric detection indicators. For pure PDRN samples, the theoretical value of A260 / A280 is 1.8, and the actual acceptable range should be between 1.8 and 2.0. When the ratio is <1.8, it indicates possible protein contamination; when the ratio is >2.0, nucleic acid degradation may exist. Simultaneously, the A260 / A230 ratio needs to be greater than 2.0. This parameter is used to assess the residual levels of impurities such as salt ions, organic solvents (e.g., ethanol), or carbohydrates. A ratio that is too low usually indicates the presence of such contamination. Purity and yield are summarized in Table 1. Images of the sample appearance for each example and comparative example are shown below. Figures 1-4 As shown.

[0076] Table 1. Summary of PDRN purity and yield comparison for each example and comparative example.

[0077]

[0078] According to Table 1 and Figures 1-4 As observed, the product yield obtained in Example 1 was the highest, at 12%. When enzymatic hydrolysis was performed using a system without collagenase, the product yield was approximately 9% (Example 2). When enzymatic hydrolysis was performed using a system without trypsin, the product yield was approximately 8% (Example 3). In contrast, the product yield obtained by continuous homogenization and non-complex enzyme hydrolysis in the comparative example was only 5%. Furthermore, the product purity of Example 1 was 2.47, and the product color was white; the product purity of Example 2 was 2.21, and the product color was pale yellow; the product purity of Example 3 was 2.1, and the product color was pale yellow; and the product purity of Comparative Example 1 was 2.1, and the product color was slightly yellow. This indicates that the method of the present invention can prepare polydeoxyribonucleotides with high yield and high quality.

[0079] 2. Molecular weight

[0080] The molecular weight of small PDRN was determined by agarose gel electrophoresis. The principle is that agarose gel has a network structure, and molecules encounter resistance as they pass through. Large molecules experience greater resistance during migration, resulting in slower migration speeds during gel electrophoresis, while small molecules migrate faster. By comparing the position of different molecular weights in the sample with those in standards, the molecular weight of the sample molecule can be determined. Electrophoresis images of the products from various examples and comparative examples are shown below. Figure 5As shown in the figure, the molecular weight of the product in Example 1 is concentrated in the 100-250 bp range, meaning that 100% of the obtained PDRN has a molecular weight of 100-250 bp, and the bands are clear with relatively uniform concentration. The molecular weight of the product in Example 2 is in the 2000-5000 bp range, showing broad and bright bands with some tailing, and the fragment length is not uniform. The electrophoresis image of Example 3 also shows bands in the 2000-5000 bp range, in a position similar to that of Example 2, but the bands are narrower and slightly weaker, indicating that it mainly consists of large DNA fragments with a lower concentration. The band position of Comparative Example 1 is consistent with that of Examples 2 and 3 (2000-5000 bp), but the band brightness is between that of Examples 2 and 3, indicating that the DNA fragment size is similar to that of Examples 2 and 3, but the concentration and uniformity are poorer. This shows that the method of the present invention can prepare small PDRN fragments.

[0081] 3. Anti-inflammatory effects

[0082] IL-1α and IL-1β are members of the interleukin-1 (IL-1) family and are both typical pro-inflammatory cytokines that can induce the same pro-inflammatory effects. Tumor necrosis factor (TNF-α) is a major regulator of inflammatory responses and participates in the pathogenesis of some inflammatory and autoimmune diseases. Interleukin-6 (IL-6) is a member of the interleukin-6 (IL-6) family and is produced by monocytes and macrophages. It has pleiotropic effects in inflammation, immune response and hematopoiesis.

[0083] Experimental cells: The cells used in this test were Raw264.7 mouse macrophages (Wuhan Pronosai Biotechnology Co., Ltd.), and the PCR primers used were all synthesized by Qingke Biotechnology Co., Ltd.

[0084] Experimental steps:

[0085] Cell resuscitation and culture

[0086] Raw264.7 cells were taken out of liquid nitrogen and revived. After the cells grew to 70%-80% in the culture flask, they were digested and counted.

[0087] Cell inoculation

[0088] With 1x10 5 The cells were seeded into 6-well plates at a density of 2 mL of cell suspension per well. The seeded 6-well plates were then placed in a cell culture incubator (5% CO2, 37°C) and cultured for 24 h.

[0089] Experimental drug administration

[0090] The control group and sample group were set up as shown in Table 2. The dosage per well was 2 mL, and each group had 3 replicates. The samples were incubated in an incubator (37℃, 5% CO2).

[0091] Table 2 Sample Grouping

[0092]

[0093] Cell RNA extraction

[0094] (1) Determine the number of cells, thoroughly remove the culture medium supernatant, and immediately proceed with the second step of lysis.

[0095] (2) Add 350 μL of lysis buffer RLA to the well plate, let stand for 2 min, blow and aspirate the cells on the surface of the well plate, collect the lysis buffer and transfer it to a centrifuge tube, and vortex to mix.

[0096] (3) Centrifuge at 12000 rpm for 5 min, take the supernatant and place it in the genomic DNA removal column, centrifuge at 12000 rpm for 30 sec, and retain the filtrate.

[0097] (4) Slowly add 70 vt% ethanol (1 volume of supernatant) to the obtained filtrate and mix well (precipitation may occur at this time). Transfer the obtained solution and precipitate together into an RNase-Free adsorption column CR4, centrifuge at 12000 rpm for 30 seconds, discard the waste liquid, and put the adsorption column back into the collection tube.

[0098] (5) Add 700 μL of protein removal solution RW3 to the RNase-Free adsorption column CR4, centrifuge at 12000 rpm for 30 seconds, discard the waste liquid, and put the adsorption column back into the collection tube.

[0099] (6) Add 500 μL of washing solution RW to the RNase-Free adsorption column CR4, let it stand at room temperature for 2 min, centrifuge at 12000 rpm for 30-60 sec, discard the waste liquid, put the adsorption column back into the collection tube, and repeat this step once.

[0100] (7) Centrifuge at 12000 rpm for 2 min, discard the waste liquid, place the RNase-Free adsorption column CR4 at room temperature for 2 min, and thoroughly dry the remaining rinsing liquid.

[0101] (8) Transfer the RNase-Free adsorption column CR4 into a new RNase-Free centrifuge tube, add 35 μL of DEPC water to the middle of the adsorption membrane, incubate at room temperature for 2 min, centrifuge at 12000 rpm for 2 min to obtain RNA solution.

[0102] (9) Repeat step 8 to concentrate the RNA solution, measure the concentration of RNA using an ultra-micro nucleic acid protein analyzer, and store the extracted RNA in a -80℃ freezer.

[0103] cDNA cloning:

[0104] Following the system and reaction conditions in Table 3, obtain the cDNA template, which can be stored at -20°C.

[0105] Table 3 Reverse transcription system

[0106]

[0107] qRT-PCR:

[0108] The obtained cDNA was used as a template for real-time PCR amplification, with GAPDH as the internal reference gene and IL-1α, IL-1β, TNF-α, and IL-6 as the target genes. The primer sequences are shown in Table 4.

[0109] Table 4 Gene Information

[0110]

[0111] Prepare the mixture according to the number of reactions, aliquot into 8-tube strips, add cDNA from each sample, and perform three technical replicates for each sample. The reaction conditions are shown in Table 5. Results are expressed as follows: --Δ Δct This indicates that the results of gene level detection for each factor are as follows: Figure 6 As shown.

[0112] Table 5 PCR System and Conditions

[0113]

[0114] The IL-1α gene level detection results showed that, compared with the control group, the relative expression level of IL-1α gene in the model group was significantly increased, indicating that the LPS-induced inflammation model was successfully established. Compared with the model group, the relative expression level of IL-1α gene in Example 1 was significantly decreased, with a decrease rate of 78.70%, indicating that Example 1 has the effect of inhibiting the release of pro-inflammatory factors at a concentration of 0.05%.

[0115] The IL-1β gene level detection results showed that, compared with the control group, the relative expression level of IL-1β gene in the model group was significantly increased, indicating that the LPS-induced inflammation model was successfully established. Compared with the model group, the relative expression level of IL-1β gene in Example 1 was significantly decreased, with a decrease rate of 61.51%, indicating that Example 1 has the effect of inhibiting the release of pro-inflammatory factors at a concentration of 0.05%.

[0116] The results of TNF-α gene level detection showed that, compared with the control group, the relative expression level of TNF-α gene in the model group was significantly increased, indicating that the LPS-induced inflammation model was successfully established. Compared with the model group, the relative expression level of TNF-α gene in Example 1 was significantly decreased, with a decrease rate of 48.61%, indicating that Example 1 has the effect of inhibiting the release of pro-inflammatory factors at a concentration of 0.05%.

[0117] The IL-6 gene level detection results showed that, compared with the control group, the relative expression level of IL-6 gene in the model group was significantly increased, indicating that the LPS-induced inflammation model was successfully established. Compared with the model group, the relative expression level of IL-6 gene in Example 1 was significantly decreased, with a decrease rate of 79.86%, indicating that Example 1 has the effect of inhibiting the release of pro-inflammatory factors at a concentration of 0.05%.

[0118] In summary, the PDRN prepared by this invention has a significant anti-inflammatory effect at a concentration level of 0.05%.

[0119] 4. Damage Repair

[0120] Photoaging of the skin is an important component of skin aging. UVB (ultraviolet light) with a wavelength range of 290-320 nm is the main inducing factor. In a skin fibroblast photoaging model, repeated UVB irradiation leads to excessive reactive oxygen species (ROS) causing a series of DNA damages, including DNA strand breaks (DSBS), DNA-protein cross-linking, and deletion mutations. When DNA is attacked, especially after DNA double-strand breaks (DSBS), serine 139 of histone H2AX is rapidly phosphorylated, generating phosphorylated H2AX, i.e., γ-H2AX. γ-H2AX accumulates in large quantities at DSB sites, forming focal points and participating in DNA damage repair. The number of γ-H2AX focal points is directly proportional to the number of DSBs. Therefore, γ-H2AX can be used as a quantitative marker for DNA double-strand breaks. After inducing damage in human skin fibroblasts through UVB irradiation, immunofluorescence staining is used to detect the effect of the sample on the cells, thereby measuring the level of the DNA damage marker γ-H2AX to confirm whether DNA damage has occurred.

[0121] Experimental cells: The cells used in this test were human skin fibroblasts (HFB), provided by Guangdong Boxi Biotechnology Co., Ltd., and the cell passage number was P6.

[0122] Experimental reagents: DMEM (Gibco), fetal bovine serum (Zhejiang Tianhang Biotechnology Co., Ltd.), PBS (Solepro), trypsin (Gibco), DNA damage detection kit (γ-H2AX immunofluorescence assay) (Beyotime), anti-fluorescence quenching solution (Beyotime).

[0123] Experimental equipment: CO2 incubator (Thermo, 150i), clean bench (Sujing Antai, SW-CJ-1F), inverted microscope (Leica Microsystems Shanghai Co., Ltd., DMI1), UVB lamp (Philips).

[0124] Experimental steps:

[0125] Cell resuscitation and passage: Take HFB cells with a substitution number of P6 from the liquid nitrogen tank, resuscitate and seed them into culture flasks, and culture them in a 37°C, 5% CO2 incubator. When the cells reach 70%-80% confluence, digest and passage them. After digestion, resuspend the cells in culture medium and count them for later use.

[0126] Cell seeding: 4 × 10 4 Inoculate 1 mL of cells per well into 12-well plates and incubate overnight in an incubator (37°C, 5% CO2).

[0127] Experimental grouping: When the cell plating rate reaches 70%-80%, the culture plates are removed and set up as control group, model group and sample group respectively.

[0128] Solution preparation: Prepare the working solution for the sample according to Table 6.

[0129] Table 6 Experimental Plan

[0130]

[0131] γ-H2AX immunofluorescence staining:

[0132] (1) Fixation: After culturing the cells in an incubator for 24 hours, discard the supernatant, add 1 mL of PBS to each well for washing, aspirate, add 500 μL of fixative to each well, fix for 5-15 minutes, aspirate the fixative, and wash 3 times with washing buffer for 3-5 minutes each time.

[0133] (2) Blocking: Add 500 μL of immunostaining blocking solution to each well and block at room temperature for 10-20 minutes.

[0134] (3) Incubation of primary antibody: Remove the immunostaining blocking solution, add 250 μL of γ-H2AX rabbit monoclonal antibody to each well, and incubate overnight at 4ºC.

[0135] (4) Washing: Remove the γ-H2AX rabbit monoclonal antibody and wash with washing solution 3 times, 5-10 minutes each time.

[0136] (5) Incubation of secondary antibody: Add 250 μL of anti-rabbit 488 to each well and incubate at room temperature in the dark for 1 hour.

[0137] (6) Washing: Remove anti-rabbit 488, wash twice with washing solution, 5-10 minutes each time.

[0138] (7) DAPI staining: Add 500 μL of nuclear staining solution (DAPI) to each well and stain at room temperature in the dark for about 5 minutes.

[0139] (8) Washing: Remove the staining solution from the cell nuclei and wash with washing solution 3 times, 3-5 minutes each time.

[0140] (9) Photographing: Add 300 μL of anti-fluorescence quenching solution to each well and take a picture under a 10x fluorescence inverted microscope to record the fluorescence image of each group of γ-H2AX.

[0141] (10) Calculation of γ-H2AX fluorescence intensity: The average fluorescence intensity of each sample in each group was calculated using ImageJ software. The fluorescence images were observed under a microscope and the bar charts were as follows. Figure 7 As shown, γ-H2AX marks DNA damage, DAPI marks the cell nucleus, and Merge indicates overlap.

[0142] according to Figure 7 As can be seen, compared with the control group, the fluorescence intensity of γ-H2AX in the model group increased significantly, indicating that the UV-induced DNA damage model in HFB cells was successfully established. Compared with the model group, the fluorescence intensity of γ-H2AX in 1 mg / mL Example 1 decreased significantly, with a decrease rate of 13.72%. The results show that 1 mg / mL Example 1 can reduce the increase in γ-H2AX content caused by UVB, and the PDRN prepared in this invention has the effect of DNA damage repair.

[0143] 5. Toxicity of small molecule PDRN raw materials

[0144] The acute oral toxicity test was conducted by Shaanxi Kangruian Testing Service Co., Ltd., a special food verification and evaluation technical institution under the State Administration for Market Regulation. The dosage of the product PDRN was 12.0 g / kg BW. Rats were administered the product orally by gavage and observed for 14 consecutive days. The results showed no signs of poisoning and no animal deaths. Autopsies of the animals after the test revealed no gross abnormalities. This indicates that the LD50 of PDRN in male and female rats is >12.0 g / kg BW. According to the acute toxicity (LD50) grading standard in the National Food Safety Standard for Acute Oral Toxicity Test, this sample is practically non-toxic.

Claims

1. A method for obtaining polydeoxyribonucleotides from fish testicular tissue, characterized in that, include: The fish testes were processed into blocks after the fascia was removed; Blocky fish testis tissue was homogenized in two stages to obtain the homogenized precipitate; The homogenized precipitate was treated with a compound enzyme hydrolysate to obtain the hydrolyzed system. The enzymatically hydrolyzed system was subjected to protein removal, alcohol precipitation, filtration, impurity removal, freeze drying, and grinding to obtain polydeoxyribonucleotides.

2. The method for obtaining polydeoxyribonucleotides from fish testicular tissue according to claim 1, characterized in that, The fish testicular tissue is either fresh salmon testicular tissue or salmon testicular tissue frozen at -20℃.

3. The method for obtaining polydeoxyribonucleotides from fish testicular tissue according to claim 1, characterized in that, The two-stage homogenization process includes a first-stage homogenization process and a second-stage homogenization process performed sequentially. Both the first-stage and second-stage homogenization processes involve mixing the material with the homogenate, followed by intermittent cell disruption and centrifugation to remove the supernatant.

4. The method for obtaining polydeoxyribonucleotides from fish testicular tissue according to claim 3, characterized in that, The homogenate solutions are all mixed solutions of sodium chloride and sodium citrate, and the concentrations of sodium chloride and sodium citrate in the homogenate solutions are both 0.05~0.15M.

5. The method for obtaining polydeoxyribonucleotides from fish testicular tissue according to claim 3, characterized in that, In the first stage of homogenization, the volume of the homogenate (mL) is 2 to 3 times the mass (g) of the material; in the second stage of homogenization, the volume of the homogenate (mL) is 3 to 5 times the mass (g) of the material; the intermittent cell disruption process is repeated in a cell disruption-stop cycle, with a cell disruption time to stop time ratio of 4 to 5, and the number of repetitions is 4 to 5; the second stage of homogenization includes multiple mixing of the material with the homogenate, followed by intermittent cell disruption and centrifugation to remove the supernatant, the number of times being 3 to 4.

6. The method for obtaining polydeoxyribonucleotides from fish testicular tissue according to claim 1, characterized in that, The compound enzyme hydrolysate is a sodium chloride solution of trypsin and collagenase, wherein the concentrations of trypsin and collagenase are both 1wt% to 3wt%, and the mass of the compound enzyme hydrolysate is 8 to 10 times the mass of the precipitate after homogenization.

7. The method for obtaining polydeoxyribonucleotides from fish testicular tissue according to claim 1, characterized in that, The treatment of the homogenized precipitate with the compound enzyme hydrolysate specifically includes: mixing the homogenized precipitate with the compound enzyme hydrolysate, performing intermittent cell wall disruption treatment, and stirring to obtain the enzymatically hydrolyzed system.

8. The method for obtaining polydeoxyribonucleotides from fish testicular tissue according to claim 1, characterized in that, The protein removal process includes: subjecting the enzymatically hydrolyzed system to three separation processes; each separation process includes pH adjustment and centrifugation, wherein the pH is adjusted to 11.0-13.0 in the first separation process, to 4.5-5.0 in the second separation process, and to 5.0-7.0 in the third separation process.

9. The method for obtaining polydeoxyribonucleotides from fish testicular tissue according to claim 1, characterized in that, The alcohol precipitation is performed using pre-cooled anhydrous ethanol.

10. The use of polydeoxyribonucleic acid obtained by the method for obtaining polydeoxyribonucleic acid from fish testicular tissue as described in claim 1 in anti-inflammatory and / or damage repair.

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