Extraction method of polydeoxyribonucleotide

Abstract

The invention belongs to the technical field of biology, and particularly relates to an extraction method of polydeoxyribonucleotide, which comprises the following steps: carrying out homogenate treatment on pretreated in-vitro testis tissue, filtering the obtained homogenate, adding a pancreas extract into the filtrate, and stirring to obtain enzymatic hydrolysate; and precipitating protein in the enzymatic hydrolysate, removing the protein, adding sodium acetate for treatment, adding absolute ethyl alcohol for precipitation, centrifuging, washing the obtained precipitate, and drying to obtain the product. Common trypsin and lipase in fish pancreas are beneficial to release of DNA, the fish pancreas extract is applied to extraction of the polydeoxyribonucleotide, the extraction time can be remarkably shortened, the yield can be increased, meanwhile, nuclease in the fish pancreas extract can effectively degrade DNA, and small-fragment polydeoxyribonucleotide is obtained.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for extracting polydeoxyribonucleotides. Background Art

[0002] Polydeoxyribonucletide (PDRN) is a low molecular weight DNA derivative from natural sources. Currently, it is mainly extracted from the testes of trout and salmon, which are rich in nucleic acids. The molecular weight is between 50kDa and 1500kDa, and the most representative molecular weight is 80kDa to 200kDa. The base pair length is between 50bp and 2000bp. It is a linear polymer of deoxyribonucleotides with phosphodiester bonds.

[0003] The base composition of PDRN is as similar as 98% to human DNA. After strict treatment and purification, PDRN is non-immunogenic and has high safety and stability. PDRN is an A2A receptor agonist. The stimulation of this receptor can reduce the secretion of inflammatory cytokines, effectively inhibit the expression of IL-6 and TNF-α, and reduce the formation of scars. In addition, it can also stimulate fibroblast proliferation, migration, angiogenesis and nucleic acid synthesis by activating purinergic receptors, thereby promoting wound healing. Based on this biological effect, its clinical application range has gradually expanded, and it has shown significant effects in tissue repair, wound healing, anti-ischemia, and anti-inflammation. PDRN of different molecular weights can be applied to different fields to play a powerful anti-inflammatory and repairing and regenerating effect: 700KDa and above are used for skin regeneration, articular cartilage regeneration and bone formation promotion; 350KDa~500KDa are used in pharmaceutical treatment to achieve tissue regeneration, corneal regeneration and treatment of musculoskeletal diseases; 40KDa~60KDa are used in cosmetics and food to achieve the effects of repairing barriers, improving wrinkles and skin beauty, etc. Based on this, it is of great significance to prepare PDRN with different molecular weights.

[0004] PDRN is a small fragment product after the degradation of macromolecular DNA. At present, its extraction can be roughly summarized into four categories; ① Cation exchange resin method: This method uses cation exchange resin to bind nucleic acid and uses different buffers for separation in the elution step. ② Saturated NaCl method: Based on the different solubility of DNA and protein in sodium chloride solution, the DNA is dissolved in a solution with a high salt concentration, which can remove impurities that cannot be dissolved in high salt; the DNA is precipitated with a low salt concentration, which can remove impurities dissolved in a low salt solution. Therefore, by repeatedly dissolving and precipitating DNA, a variety of impurities with different solubility from DNA can be removed. ③ Alkaline lysis method: It mainly uses the denaturation and renaturation of DNA. In an alkaline solution, the hydrogen bonds of double-stranded DNA are broken, and the double helix structure of DNA is destroyed and denatured. When a neutralization buffer is added, the denatured DNA can be restored to its original configuration. ④ Enzyme cleavage method: Use a specific enzyme to shear DNA to obtain DNA fragments of different molecular weights.

[0005] The existing preparation method mainly relies on enzyme preparations or lysates to release DNA from tissue cells, and then relies on restriction endonucleases to digest DNA or use ultrasound to interrupt DNA to obtain PDRN. For example, CN202211007267 and CN202410166757 both use ultrasound to interrupt to obtain PDRN, and CN202210451511 uses ultrasound and enzyme cutting to obtain PDRN. Ultrasonic interruption is the most common way of DNA fragmentation, and the shear force generated by ultrasound can shear the DNA fragments in the solution. Commonly used ultrasonic interrupters are divided into contact and non-contact types, among which the contact interrupter is relatively low in price, but it is necessary to insert an ultrasonic probe into the sample for interruption, which is prone to uneven interruption, sample loss and cross-contamination. The non-contact interrupter does not need to insert the probe into the sample, so it can effectively avoid the above problems, but the non-contact interrupter is expensive. Restriction endonucleases can only cut on specific DNA sequences, so the selected enzyme must have a recognition site for the target sequence. At the same time, the cutting efficiency of restriction endonucleases may be affected by factors such as temperature, pH, and salt concentration. Under undesirable reaction conditions, the enzyme digestion reaction may be inefficient, thus affecting the success rate of the experiment. The source and purity of the enzyme may also affect the effect of the enzyme digestion reaction, especially when high-purity enzymes are required, and commercially available enzymes cannot fully meet the requirements. Summary of the invention

[0006] Based on the above technical problems, the present invention utilizes a variety of natural enzymes rich in fish pancreatic extract to achieve the extraction of PDRN: trypsin and lipase commonly found in fish pancreas help release DNA, so the application of fish pancreatic extract in the extraction of PDRN can significantly reduce the extraction time and improve the yield. At the same time, the nuclease therein can effectively degrade DNA to obtain small fragments of PDRN.

[0007] The specific technical solutions provided by the present invention are as follows:

[0008] The present invention provides a method for extracting polydeoxyribonucleotides, comprising the following steps:

[0009] homogenizing the pretreated isolated testicular tissue, filtering the obtained homogenate, adding a pancreatic extract to the filtrate, stirring, and obtaining an enzymatic hydrolyzate; the pancreatic extract and the isolated testicular tissue are derived from the same species;

[0010] The protein in the enzymatic hydrolysate is precipitated, sodium acetate is added after the protein is removed, and then anhydrous ethanol is added thereto for precipitation, and the precipitate is washed and dried to obtain the polydeoxyribonucleotide.

[0011] The addition of sodium acetate reduces the repulsion of like charges between DNA molecules, making them easier to aggregate. The addition of anhydrous ethanol captures the water molecules around the DNA, causing the DNA molecules to precipitate.

[0012] As a preferred embodiment of the present invention, the amount of the pancreatic extract added is 10% to 15% based on the total mass of the filtrate.

[0013] As a preferred embodiment of the present invention, the testicular tissue is fresh testicular tissue isolated from the body or testicular tissue cryopreserved at a temperature below -20°C.

[0014] As a preferred embodiment of the present invention, the final concentration of sodium acetate in the system is 0.3M.

[0015] As a preferred embodiment of the present invention, the amount of anhydrous ethanol added is 1 to 2 times the volume of the system before addition.

[0016] As a preferred embodiment of the present invention, after adding the pancreatic extract, stirring is carried out at room temperature and 1000 rpm to 2000 rpm for 1 hour to 3 hours to obtain the enzymatic hydrolyzate.

[0017] As a preferred embodiment of the present invention, the process for obtaining the pancreatic extract is as follows: the fish pancreas is homogenized with physiological saline and then centrifuged at 15000g~17700g for 10min~30min. The obtained sample is divided into four layers, namely, an oily layer, a fat layer, a pancreatic extract stock liquid layer, and an impurity layer from top to bottom. The pancreatic extract stock liquid layer is collected and centrifuged, and the obtained supernatant is the pancreatic extract.

[0018] Further preferably, the pancreatic extract is a frozen product of the supernatant at -20°C or a lyophilized powder obtained by lyophilizing the supernatant.

[0019] As a preferred embodiment of the present invention, the specific operation process of precipitating the protein in the enzymatic hydrolyzate and removing the protein is: adding sodium chloride solution, sodium dodecyl sulfate and ethylenediaminetetraacetic acid to the enzymatic hydrolyzate, stirring at 25°C to 40°C and 1000rpm to 2000rpm for 0.5h to 2h, centrifuging, collecting the supernatant, adding proteinase K to the supernatant, stirring at 25°C to 40°C and 1000rpm to 2000rpm for 30min to 60min, centrifuging, and collecting the supernatant.

[0020] Further preferably, the mass concentration of the sodium chloride solution is 5M, and the volume ratio of the enzymatic hydrolyzate to the sodium chloride solution is 1-1.5:1;

[0021] The mass ratio of the enzymatic hydrolysate, sodium lauryl sulfate and ethylenediaminetetraacetic acid is 100:1-2:0.5-1;

[0022] The final concentration of the proteinase K is 20 μg / mL to 50 μg / mL.

[0023] As a preferred embodiment of the present invention, the time for adding anhydrous ethanol for precipitation is at least 3 minutes.

[0024] As a preferred embodiment of the present invention, the washing process of the obtained precipitate is: add 70% ethanol solution and 95% ethanol solution in a mass fraction of 3 to 10 times the volume of the precipitate to the obtained precipitate in sequence, centrifuge, collect the precipitate, and dry to obtain the polydeoxyribonucleotide. Specifically: add 70% ethanol solution in a mass fraction equivalent to 3 to 10 times the volume of the precipitate to the obtained precipitate and centrifuge, then add 95% ethanol solution in a mass fraction equivalent to 3 to 10 times the volume of the precipitate to the precipitate obtained by centrifugation and centrifuge, and collect the final precipitate again. The volume of the precipitate is almost unchanged during the washing process.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] The invention utilizes pancreatic extract to process testicular tissue derived from the same species as the pancreatic extract, relies on enzymes therein to release DNA better, has higher extraction efficiency, shorter extraction time, and better enzyme cutting effect from the same species, and the extraction rate of the pancreatic extract stock solution for small molecular weight fragment PDRN is above 28%, and the purity is relatively high.

[0027] The pancreatic extract used in the present invention still has enzymatic hydrolysis ability under extreme acid-base conditions, but the storage method affects the performance of its enzymatic hydrolysis. The freshly prepared pancreatic extract has the best enzymatic hydrolysis effect and can obtain a small molecular weight fragment PDRN; the enzymatic hydrolysis ability of the pancreatic extract thawed after being stored at 4°C for one week or at -20°C for one week decreases, and only a medium molecular weight fragment PDRN can be obtained; and the pancreatic extract that has been freeze-dried can only obtain a high molecular weight fragment PDRN.

[0028] The invention effectively utilizes the pancreas, a byproduct in the process of fish processing, turns waste into treasure, and is economical and practical. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the alcohol precipitation diagram prepared in Example 1;

[0030] Figure 2 This is a picture of the sample after freeze-drying;

[0031] Figure 3 It is the electrophoresis detection diagram of samples obtained from different embodiments and comparative examples. DETAILED DESCRIPTION

[0032] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention but are merely intended to illustrate the present invention.

[0033] Unless otherwise specified, the experimental methods used in the following examples are generally carried out under conventional conditions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0034] Polydeoxyribonucleotide is a low molecular weight DNA derivative from natural sources. The base composition of PDRN is as similar as 98% to human DNA. After strict treatment and purification, PDRN is non-immunogenic and has high safety and stability. PDRN has shown significant effects in tissue repair, wound healing, anti-ischemia, and anti-inflammation. PDRN of different molecular weights can be applied to different fields to play a powerful anti-inflammatory and repairing and regenerating effect: 700KDa and above are used for skin regeneration, articular cartilage regeneration and bone formation promotion; 350KDa~500KDa are used in pharmaceutical treatment to achieve tissue regeneration, corneal regeneration and treatment of musculoskeletal diseases; 40KDa~60KDa are used in cosmetics and foods to achieve the effects of repairing barriers, improving wrinkles and skin beauty. Based on this, the preparation of PDRN of different molecular weights is of great significance.

[0035] The existing preparation methods of PDRN basically all use ultrasound. Ultrasonic interruption is the most common DNA fragmentation method. The shear force generated by ultrasound can shear the DNA fragments in the solution. Commonly used ultrasonic interrupters are divided into contact and non-contact types. Among them, the contact interrupter is relatively low in price, but the ultrasonic probe needs to be inserted into the sample for interruption, which is easy to cause uneven interruption, sample loss and cross-contamination. The non-contact interrupter does not need to insert the probe into the sample, so it can effectively avoid the above problems, but the non-contact interrupter is expensive. Restriction endonucleases can only cut on specific DNA sequences, so the selected enzyme must have a recognition site for the target sequence. At the same time, the cutting efficiency of restriction endonucleases may be affected by factors such as temperature, pH, and salt concentration. Under undesirable reaction conditions, the enzyme reaction may be inefficient, thereby affecting the success rate of the experiment. The source and purity of the enzyme may also affect the effect of the enzyme reaction, especially when high-purity enzymes are required, commercially available enzymes may not fully meet the requirements.

[0036] Based on this, the present invention provides a method for extracting polydeoxyribonucleotides, comprising the following steps:

[0037] homogenizing the pretreated isolated testicular tissue, filtering the obtained homogenate, adding a pancreatic extract to the filtrate, stirring, and obtaining an enzymatic hydrolyzate; the pancreatic extract and the isolated testicular tissue are derived from the same species;

[0038] The protein in the enzymatic hydrolysate is precipitated, sodium acetate is added after the protein is removed, and then anhydrous ethanol is added thereto to precipitate at -20°C, centrifuged, and the obtained precipitate is washed and dried to obtain polydeoxyribonucleotides.

[0039] Further, the method for extracting polydeoxyribonucleotides comprises the following steps:

[0040] S1: Pretreatment: The surface of the salmon testis was cleaned with ultrapure water, dried with absorbent paper, and the surface fascia was removed with tweezers. After weighing, it was cut into pieces of about 1 cm 3 The tissue blocks are reserved.

[0041] S2: Homogenization: Add 3 to 10 times of physiological saline containing 3% sodium citrate to the pretreated salmon testis tissue block, and use a juicer to homogenize to obtain a homogenate. The juicer was purchased from Guangdong Xin'an Ya Trading Co., Ltd., and the product number is MJ301A.

[0042] S3: Enzymatic hydrolysis: The homogenate was filtered through an 80-mesh sieve, and then 10% to 15% of the mass of the filtrate system was added with salmon pancreatic extract, and the mixture was stirred at 1000 rpm to 2000 rpm at room temperature for 1 to 3 hours to obtain an enzymatic hydrolyzate; the agitator was purchased from Shanghai Lichen Bangxi Instrument Technology Co., Ltd.

[0043] S4: Precipitate protein: add an equal volume of 5M sodium chloride to the hydrolyzate, then add 1% to 2% SDS and 0.5% to 1% EDTA based on the mass of the hydrolyzate, and stir at 25℃ to 40℃, 1000rpm to 2000rpm for 0.5 to 2 hours. If the viscosity of the liquid after stirring is too high, add 1 to 5 times the volume of 2.5M sodium chloride; sodium chloride was purchased from McLean, the product number is S805275; SDS, i.e. sodium dodecyl sulfate, was purchased from McLean, the product number is S817788; EDTA, i.e. ethylenediaminetetraacetic acid, was purchased from McLean, the product number is E6228.

[0044] S5: Protein removal: Centrifuge the above liquid to collect the supernatant, add proteinase K with a final concentration of 20μg / mL to 50μg / mL to the supernatant, stir at 25℃ to 40℃, 1000rpm to 2000rpm for 30min to 60min, then centrifuge again to collect the supernatant. Proteinase K was purchased from Maclean with the product number P6157.

[0045] S6: Alcohol precipitation and washing: Add 3M sodium acetate equivalent to 10% of the total amount of the supernatant to the obtained supernatant, stir at room temperature for 10min to 30min, then add 1 to 2 times the volume of anhydrous ethanol, and precipitate at -20℃ for more than 3min. Use a glass rod to remove the filamentous precipitate or centrifuge all the liquid to collect the precipitate. Then add 70% ethanol and 95% ethanol by mass fraction equivalent to 3 to 10 times the volume of the precipitate, centrifuge and wash once, and collect the final precipitate.

[0046] S7: Sample drying: The final precipitate is freeze-dried or vacuum-dried to obtain the final product, which is polydeoxyribonucleotide, i.e., PDRN.

[0047] It should be noted that the salmon testis of the present invention can be freshly picked or frozen and stored at a temperature below -20°C.

[0048] The salmon pancreas extract of the present invention is obtained by extraction according to the following steps:

[0049] After washing the surface of the salmon pancreas with ultrapure water, weigh it, add 1 to 2 times the volume of physiological saline to homogenize it, and centrifuge it after homogenization. After centrifugation, the sample is divided into four layers, from top to bottom, oily layer, fat layer, pancreatic extract liquid layer, and impurity layer. The impurity layer is located at the bottom of the centrifuge tube. Use a pipette to carefully draw the pancreatic extract liquid layer into a new tube, centrifuge it again and draw it once to ensure that the obtained pancreatic extract liquid does not contain substances from other layers.

[0050] The pancreatic extract in the present invention can be added in the form of pancreatic extract stock solution or in the form of pancreatic extract lyophilized powder. When added in the form of pancreatic extract stock solution, the added amount is 10% to 15% of the volume of the homogenate after filtration; when added in the form of pancreatic extract lyophilized powder, the lyophilized powder is the lyophilized product of the required 10% to 15% pancreatic extract stock solution.

[0051] The pancreatic extract prepared by the invention can still exert enzymatic hydrolysis after acid or alkali treatment, the upper limit of acid treatment is: using 6M hydrochloric acid to adjust the pH of the pancreatic extract stock solution to 4.0; the upper limit of alkali treatment is: using 5M sodium hydroxide to adjust the pH to 10.0.

[0052] Unless otherwise specified, the 75% by mass ethanol, 95% by mass ethanol, and anhydrous ethanol mentioned in the present invention are all ethanol pre-cooled at -20°C.

[0053] The present invention utilizes a variety of natural enzymes rich in fish pancreatic extracts to achieve the extraction of PDRN: trypsin and lipase commonly found in fish pancreas contribute to the release of DNA, so the application of fish pancreatic extracts in the extraction of PDRN can significantly reduce the extraction time and improve the yield, while the nucleases therein can effectively degrade DNA to obtain small fragments of PDRN.

[0054] The following describes the invention in conjunction with specific embodiments.

[0055] Example 1

[0056] A method for extracting polydeoxyribonucleotides comprises the following steps:

[0057] S1: Pretreatment: Take frozen salmon testis and thaw it at room temperature, wash the surface with ultrapure water, dry it with absorbent paper, and use tweezers to pick off the surface fascia, weigh 20g and cut it into about 1cm 3 organizational block.

[0058] S2: Homogenization: Add 160 mL of the extract to the pretreated tissue block and use a juicer to homogenize to obtain a homogenate. The homogenization conditions are: work 8 seconds, interval 2 seconds, and repeat 4 times.

[0059] S3: Enzyme hydrolysis: The homogenate was filtered using an 80-mesh sieve, and 18 mL of salmon pancreas extract a was added thereto after filtration. The mixture was stirred at room temperature at 1500 rpm for 3 hours to obtain an enzymatic hydrolyzate.

[0060] S4: Protein precipitation: Add 198 mL of 5 M sodium chloride, 7.92 g of SDS and 1.98 g of EDTA to the enzymatic hydrolyzate, stir at 37°C and 2000 rpm for 0.5 hour, and then add 396 mL of 2.5 M sodium chloride.

[0061] S5: Protein removal: centrifuge the above liquid at 4°C, 15000g for 30 min, collect the supernatant, add proteinase K with a final concentration of 20μg / mL to the supernatant, stir at 37°C, 2000rpm for 30 min, centrifuge again at 4°C, 15000g for 30 min, and collect the supernatant.

[0062] S6: Alcohol precipitation and washing: 7.92 mL of 3M sodium acetate was added to the obtained supernatant, stirred at room temperature for 10 min, 800 mL of anhydrous ethanol was added thereto, and precipitation was carried out at -20°C overnight. The obtained precipitate was as follows Figure 1 As shown. Centrifuge the entire liquid containing the precipitate at 4°C and 15000g for 30 min to collect the precipitate. Then add 50 mL of 70% ethanol and 95% ethanol in sequence and centrifuge at 4°C and 15000g for 30 min to wash once, and collect the final precipitate.

[0063] S7: Sample drying: The final precipitate is freeze-dried to obtain the final product, which is PDRN and is recorded as sample A. The actual product is shown in the figure Figure 2 shown.

[0064] Wherein, the preparation method of pancreatic extract a is as follows:

[0065] After washing the surface of the salmon pancreas with ultrapure water, it was weighed. 366 g of pancreas was homogenized by adding 500 mL of physiological saline. After homogenization, the sample was centrifuged at 4°C and 15000g for 30 minutes. After centrifugation, the sample was divided into four layers, from top to bottom, oily layer, fat layer, pancreatic extract liquid layer, and impurity layer. The impurity layer was located at the bottom of the centrifuge tube. The pancreatic extract liquid layer was carefully drawn into a new tube with a pipette, and then centrifuged at 4°C and 15000g for 30 minutes and drawn once to obtain the pancreatic extract liquid, which was recorded as pancreatic extract a.

[0066] Example 2

[0067] A method for extracting polydeoxyribonucleotides comprises the following steps:

[0068] S1: Pretreatment: Take frozen salmon testis and thaw it at room temperature, wash the surface with ultrapure water, dry it with absorbent paper, and use tweezers to pick off the surface fascia, weigh 20g and cut it into about 1cm 3 organizational block.

[0069] S2: Homogenization: Add 160 mL of the extract to the pretreated tissue block and use a juicer to homogenize to obtain a homogenate. The homogenization conditions are: work 8 seconds, interval 2 seconds, and repeat 5 times.

[0070] S3: Enzyme hydrolysis: The homogenate was filtered using an 80-mesh sieve, and 20 mL of salmon pancreas extract b was added thereto after filtration. The mixture was stirred at room temperature at 1500 rpm for 2 hours to obtain an enzymatic hydrolyzate.

[0071] S4: Protein precipitation: Add 200 mL of 5 M sodium chloride, 8 g of SDS and 2 g of EDTA to the enzymatic hydrolyzate, stir at 37°C and 2000 rpm for 1 hour, and then add 400 mL of 2.5 M sodium chloride.

[0072] S5: Protein removal: centrifuge the above liquid at 4°C, 17700g for 10 min, collect the supernatant, add proteinase K with a final concentration of 20μg / mL to the supernatant, stir at 37°C, 2000rpm for 30min, centrifuge again at 4°C, 17700g for 10min, and collect the supernatant.

[0073] S6: Alcohol precipitation and washing: Add 8 mL of 3M sodium acetate to the obtained supernatant, stir at room temperature for 10 min, add 810 mL of anhydrous ethanol, and precipitate at -20°C overnight. Use a glass rod to remove the precipitate, centrifuge at 4°C and 17700g for 10 min, and collect the precipitate. Then add 50 mL of 70% ethanol and 95% ethanol in turn, centrifuge at 4°C and 17700g for 10 min, wash once, and collect the final precipitate.

[0074] S7: Sample drying: The final precipitate is freeze-dried to obtain the final product, which is PDRN and is recorded as sample B.

[0075] Wherein, the preparation method of pancreatic extract b is as follows:

[0076] After washing the surface of the salmon pancreas with ultrapure water, it was weighed. 366g of pancreas was homogenized by adding 500mL of physiological saline. After homogenization, the sample was centrifuged at 4℃ and 17700g for 10min. After centrifugation, the sample was divided into four layers, from top to bottom, oily layer, fat layer, pancreatic extract liquid layer, and impurity layer. The impurity layer was located at the bottom of the centrifuge tube. The pancreatic extract liquid layer was carefully pipetted into a new tube with a pipette, temporarily stored at 4℃ for one week, and then centrifuged at 4℃ and 17700g for 10min. The pancreatic extract liquid was pipetted once to obtain the pancreatic extract liquid, which was frozen at -20℃ for one week and used after thawing at room temperature. It was recorded as pancreatic extract b.

[0077] Example 3

[0078] A method for extracting polydeoxyribonucleotides comprises the following steps:

[0079] S1: Pretreatment: Take frozen salmon testis and thaw it at room temperature, wash the surface with ultrapure water, dry it with absorbent paper, and use tweezers to pick off the surface fascia, weigh 20g and cut it into about 1cm 3organizational block.

[0080] S2: Homogenization: Add 200 mL of the extract to the pretreated tissue block and use a juicer to homogenize to obtain a homogenate. The homogenization conditions are: work 8 seconds, interval 2 seconds, and repeat 5 times.

[0081] S3: Enzyme hydrolysis: The homogenate was filtered using an 80-mesh sieve, and 0.6 g of salmon pancreas extract c was added thereto after filtration. The mixture was stirred at room temperature at 2000 rpm for 3 hours to obtain an enzymatic hydrolyzate.

[0082] S4: Protein precipitation: Add 220 mL of 5 M sodium chloride, 8.8 g of SDS and 2.2 g of EDTA to the enzymatic solution, and stir at 37°C and 2000 rpm for 1 hour.

[0083] S5: Protein removal: centrifuge the above liquid at 4°C and 15000g for 30 min, collect the supernatant, add proteinase K with a final concentration of 20μg / mL to the supernatant, stir at 37°C and 2000rpm for 30min, centrifuge again at 4°C and 17700g for 10min, and collect the supernatant.

[0084] S6: Alcohol precipitation and washing: Add 4.4 mL of 3M sodium acetate to the obtained supernatant, stir at room temperature for 10 min, add 450 mL of anhydrous ethanol, and precipitate at -20°C overnight. Use a glass rod to remove the precipitate, centrifuge at 4°C and 15000g for 10 min, and collect the precipitate. Then add 50 mL of 70% ethanol and 95% ethanol in turn, centrifuge at 4°C and 17700g for 10 min, wash once, and collect the final precipitate.

[0085] S7: Sample drying: The final precipitate is freeze-dried to obtain the final product, which is PDRN and is recorded as sample C.

[0086] Wherein, the preparation method of pancreatic extract c is as follows:

[0087] After washing the surface of the salmon pancreas with ultrapure water, it was weighed. 366g of pancreas was homogenized by adding 500mL of physiological saline. After homogenization, the sample was centrifuged at 4°C and 15000g for 10min. After centrifugation, the sample was divided into four layers, from top to bottom, oily layer, fat layer, pancreatic extract stock layer, and impurity layer. The impurity layer was located at the bottom of the centrifuge tube. Carefully pipette the pancreatic extract stock layer into a new tube with a pipette, centrifuge it at 4°C and 15000g for 10min, and pipette it once to obtain the pancreatic extract stock solution. Take 20mL of the stock solution for freeze drying, collect the freeze-dried powder after drying, and obtain 0.6g of pancreatic extract in the form of freeze-dried powder, which is recorded as pancreatic extract c.

[0088] Example 4

[0089] A method for extracting polydeoxyribonucleotides comprises the following steps:

[0090] The pancreatic extract stock solution was treated with acid and alkali respectively and then extracted by the method of Example 3. The specific preparation and treatment of the pancreatic extract was as follows: the surface of the salmon pancreas was washed with ultrapure water and then weighed, 366g of the pancreas was added with 500mL of physiological saline for homogenization, and the homogenization was centrifuged at 4°C and 15000g for 30min. After centrifugation, the sample was divided into four layers, from top to bottom, oily layer, fat layer, pancreatic extract stock solution layer, and impurity layer, and the impurity layer was located at the bottom of the centrifuge tube. Carefully draw the pancreatic extract stock solution layer into a new tube with a pipette, and then centrifuge it at 4°C and 15000g for 30min and draw it once to obtain the pancreatic extract stock solution. The stock solution was divided into 2 groups for further treatment, one group added 6M hydrochloric acid to adjust the pH to 4.0; the other group added 5M sodium hydroxide to adjust the pH to 10.0. 20mL of the stock solution was taken from each group for freeze drying, and the freeze-dried powder was collected after drying, and 0.6g of pancreatic extract in the form of freeze-dried powder was obtained, which were respectively recorded as pancreatic extract d and pancreatic extract e.

[0091] The PDRN extracted using pancreatic extract d was recorded as sample D, and the PDRN extracted using pancreatic extract e was recorded as sample E.

[0092] Comparative Example 1

[0093] A method for extracting polydeoxyribonucleotides comprises the following steps:

[0094] S1: Pretreatment: Take frozen salmon testis and thaw it at room temperature, wash the surface with ultrapure water, dry it with absorbent paper, and use tweezers to pick off the surface fascia, weigh 20g and cut it into about 1cm 3 organizational block.

[0095] S2: Homogenization: Add 160 mL of the extract to the pretreated tissue block and use a juicer to homogenize to obtain a homogenate. The homogenization conditions are: work 8 seconds, interval 2 seconds, and repeat 4 times.

[0096] S3: Enzymatic hydrolysis: The homogenate was filtered using an 80-mesh sieve, and 5.4 g of trypsin was added thereto after filtration. The mixture was stirred at room temperature at 1500 rpm for 3 hours to obtain an enzymatic hydrolyzate. Trypsin was purchased from McLean with a product number of T6325.

[0097] S4: Protein precipitation: Add 198 mL of 5 M sodium chloride, 7.92 g of SDS and 1.98 g of EDTA to the enzymatic hydrolyzate, stir at 37°C and 2000 rpm for 0.5 hour, and then add 396 mL of 2.5 M sodium chloride.

[0098] S5: Protein removal: Centrifuge the above liquid at 4°C and 15000g for 30 min, collect the supernatant, add proteinase K with a final concentration of 20 μg / mL to the supernatant, stir at 37°C and 2000 rpm for 30 min, and centrifuge again to collect the supernatant.

[0099] S6: Ultrasound: add the supernatant into a glass beaker, put it into an ultrasonic cleaning machine and perform ultrasonication at an ultrasonic power of 500 W and an ultrasonic frequency of 40 kHz. At the same time, add ice packs into the ultrasonic cleaning machine and replace them with new ice packs every hour for a total of 3 hours. The ultrasonic cleaning machine was purchased from Kunshan Jie Li Mei Ultrasonic Instrument Co., Ltd. with a product number of KQ-500B.

[0100] S7: Alcohol precipitation and washing: Add 7.92 mL of 3M sodium acetate to the supernatant after ultrasound, stir at room temperature for 10 min, add 800 mL of anhydrous ethanol, and precipitate at -20°C overnight. Centrifuge the liquid containing the precipitate at 4°C and 15000g for 30 min, and collect the precipitate. Then add 50 mL of 70% ethanol and 95% ethanol in turn, centrifuge at 4°C and 15000g for 30 min, wash once, and collect the final precipitate.

[0101] S8: Sample drying: The final precipitate is freeze-dried to obtain the final product, which is PDRN and is recorded as sample F.

[0102] Effect verification of PDRN prepared in each embodiment and comparative example

[0103] 1. Purity determination

[0104] A 260 / A 280 , A 260 / A 230 It is an indicator of nucleic acid purity. For pure PDRN samples, A 260 / A 280 =1.8, the normal value should be between 1.8 and 2.0, and A 260 / A 230 The ratio is greater than 2.0.

[0105] The present invention measured the purity of PDRN prepared in each embodiment and comparative example, and the results are shown in Table 1.

[0106] Table 1 Comparison of sample purity

[0107]

[0108]

[0109] As shown in Table 1, the purity of samples A to E obtained in the embodiments of the present invention meets the requirements and are pure PDRN samples.260 / A 280 <1.8, the purity is slightly low, and there is protein residue; and the yield of the final product of the present invention is much higher than that of the comparative example.

[0110] 2. Determination of PDRN by gel electrophoresis

[0111] Agarose gel electrophoresis is often used to separate and identify nucleic acids. Its separation of nucleic acids is mainly based on their relative molecular weight and molecular configuration. The larger the molecular weight, the slower it "runs".

[0112] like Figure 3 As shown, the comparative example sample F exhibits fragments of different sizes after degradation, that is, the fragments are unevenly broken, covering the entire molecular weight range, and it is difficult to obtain specific fragments with relatively concentrated molecular weights. The molecular weight fragment distribution range of each sample group in the embodiment is narrower.

[0113] The size of sample A in Example 1 is about 100 bp to 1000 bp. In Example 2, the pancreatic extract stored at 4°C for one week and -20°C for one week and then thawed was hydrolyzed to obtain a medium molecular weight fragment, i.e., sample B, with a size of about 500 bp to 2000 bp. In Examples 3 and 4, the pancreatic extracts that were freeze-dried were hydrolyzed to obtain high molecular weight fragments, i.e., samples C, D, and E, which were greater than 5000 bp.

[0114] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the protection scope of the present invention. It should be pointed out that, for those of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention, and the protection scope of the present invention shall be subject to the attached claims.

Claims

1. A method for extracting polydeoxyribonucleotides, characterized in that: The steps include: homogenizing the pretreated isolated testicular tissue, filtering the obtained homogenate, adding a pancreatic extract to the filtrate, stirring, and obtaining an enzymatic hydrolyzate; the pancreatic extract and the isolated testicular tissue are derived from the same species; The protein in the enzymatic hydrolysate is precipitated, sodium acetate is added after the protein is removed, and then anhydrous ethanol is added thereto for precipitation, and the precipitate is washed and dried to obtain the polydeoxyribonucleotide.

2. The method for extracting polydeoxyribonucleotides according to claim 1, characterized in that: The added amount of the pancreatic extract is 10% to 15% based on the total mass of the filtrate.

3. The method for extracting polydeoxyribonucleotides according to claim 1, characterized in that: The testicular tissue is fresh testicular tissue isolated from the body or testicular tissue cryopreserved below -20°C.

4. The method for extracting polydeoxyribonucleotides according to claim 1, characterized in that: After adding the pancreatic extract, stirring is performed at room temperature and 1000 rpm to 2000 rpm for 1 h to 3 h to obtain the enzymatic hydrolyzate.

5. The method for extracting polydeoxyribonucleotides according to claim 1, characterized in that: The pancreatic extract is obtained as follows: after the fish pancreas is homogenized with physiological saline, it is centrifuged at 15000g~17700g for 10min~30min. The obtained sample is divided into four layers, which are an oily layer, a fat layer, a pancreatic extract liquid layer, and an impurity layer from top to bottom. The pancreatic extract liquid layer is collected and centrifuged, and the obtained supernatant is the pancreatic extract.

6. The method for extracting polydeoxyribonucleotides according to claim 5, characterized in that: The pancreatic extract is a product of the supernatant frozen at -20°C or a lyophilized powder obtained by lyophilizing the supernatant.

7. The method for extracting polydeoxyribonucleotides according to claim 1, characterized in that: The specific operation process of precipitating the protein in the enzymatic hydrolysate and removing the protein is: adding sodium chloride solution, sodium dodecyl sulfate and ethylenediaminetetraacetic acid to the enzymatic hydrolysate, stirring at 25°C to 40°C and 1000rpm to 2000rpm for 0.5h to 2h, centrifuging, adding proteinase K to the obtained supernatant, stirring at 25°C to 40°C and 1000rpm to 2000rpm for 30min to 60min, centrifuging, and collecting the final supernatant.

8. The method for extracting polydeoxyribonucleotides according to claim 7, characterized in that: The mass concentration of the sodium chloride solution is 5M, and the volume ratio of the enzymatic hydrolyzate to the sodium chloride solution is 1-1.5:1; The mass ratio of the enzymatic hydrolysate, sodium lauryl sulfate and ethylenediaminetetraacetic acid is 100:1-2:0.5-1; The final concentration of the proteinase K is 20 μg / mL to 50 μg / mL.

9. The method for extracting polydeoxyribonucleotides according to claim 1, characterized in that: The precipitation time of adding anhydrous ethanol is at least 3 minutes.

10. The method for extracting polydeoxyribonucleotides according to claim 9, characterized in that: The washing process of the obtained precipitate is: adding 70% ethanol solution and 95% ethanol solution of 3 to 10 times the volume of the precipitate in sequence, washing, centrifuging, collecting the precipitate, and drying to obtain the polydeoxyribonucleotide.

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