Low-endotoxin macromolecule PDRN, preparation method and application

Abstract

The invention discloses low-endotoxin macromolecule PDRN, a preparation method and application. A freeze-dried white powder sample is obtained through fish testis pretreatment, fish sperm cell enzymolysis, deproteinization, ultrasonic pretreatment, nuclease enzymolysis, primary alcohol precipitation, redissolution and filtration, endotoxin control, secondary alcohol precipitation, alcohol washing and freeze-drying. Fragmentation is carried out by combining ultrasonic waves with nuclease enzymolysis, the use amount of enzyme can be effectively reduced, the molecular weight of the product can be accurately controlled, operation is easy, and the industrialization cost is low. The method comprises the following steps: carrying out enzymolysis on protamine cells through protease K, releasing protamine DNA, carrying out fragmentation on a PDRN sample by using ultrasonic waves and nuclease, and carrying out deep filtration and triton 114 washing to obtain the protamine with the molecular weight controllable interval of 500bp-4000bp; the content of endotoxin is less than or equal to 0.5 EU / mg, and the content of protein is less than or equal to 1.0%. The finished product is obtained by freeze-drying the PDRN wet solid, and the quality and the additional value of the product are improved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a technology for efficiently preparing low-endotoxin macromolecular PDRN. Background Art

[0002] Polydeoxyribonucleotides (PDRN) are a class of active polynucleotides with a molecular weight range of 50-2000 bp. Small amounts of PDRN can be extracted from human placenta or sperm, but due to limited raw material availability, PDRN is currently primarily extracted from salmonid semen or testicular tissue. Studies have shown that PDRN binds to adenosine A2A receptors, initiating multiple signaling pathways that increase anti-inflammatory factors, reduce inflammatory factors, and inhibit inflammatory responses. It also provides purines and pyrimidines through salvage pathways, accelerating DNA synthesis and repairing damaged skin. It also promotes osteoblast activity, collagen synthesis, and angiogenesis. Currently, PDRN is being used as a cosmetic and pharmaceutical raw material in a wide range of applications, including medical aesthetics, cosmetics, medical devices, health foods, and pharmaceuticals. DNA fragments of different lengths have different effects. The length of cosmetic and food-grade PDRN fragments is about 50-100bp, while the length of pharmaceutical-grade PDRN fragments is about 500bp-800bp, and the length of medical device-grade PDRN fragments is greater than 1000bp. At the same time, pharmaceutical-grade and medical device-grade PDRN have higher requirements for endotoxin content.

[0003] Endotoxins are a general term for toxic substances present in Gram-negative bacteria. They are cell wall components of many Gram-negative bacteria, released upon bacterial lysis and also known as "pyrogens." Bacterial endotoxins are primarily composed of phospholipids, lipoproteins, and lipopolysaccharides. Lipopolysaccharides are the biologically active portion of the endotoxin complex, while lipid A is responsible for the toxic effects. The lipid A structures of different Gram-negative bacteria are largely similar, resulting in similar toxic effects associated with infections caused by these bacteria, primarily manifesting as fever, microcirculatory impairment, endotoxic shock, and disseminated intravascular coagulation. Endotoxins are released in large quantities upon bacterial death, lysis, or autolysis. Pharmaceuticals and medical devices that are susceptible to the introduction of endotoxins must undergo endotoxin testing. Endotoxin molecules have a low isoelectric point and carry a high negative charge in general solutions. Therefore, endotoxin removal can be achieved by selecting cation or anion exchange media to separate endotoxins from the target sample, depending on the properties of the target sample. Alternatively, specific affinity chromatography fillers can be used, which are usually based on certain polymers or agarose and coupled with ligands that specifically interact with endotoxins, such as polymyxin B, histamine, histidine, polylysine, and some polycationic ligands. The disadvantage of the chromatography method is that it requires the use of relatively expensive chromatography fillers and specialized chromatography equipment. Another commonly used method for endotoxin removal is phase separation, using commonly used reagents such as phenols, Triton X-114, and Triton X-100. Triton is a type of non-ionic surfactant with high solubility at low temperatures, and endotoxins will dissolve in it. As the temperature rises, the solubility of Triton gradually decreases. When the temperature rises to a very low level, Triton will form a precipitate together with the endotoxin and separate from the water phase. The precipitate is removed by centrifugation, and the endotoxin in the supernatant sample is removed.

[0004] CN110747194A discloses a method for preparing PDRN. The molecular weight of the small molecule polydeoxyribonucleotide in this invention is 50bp-1000bp, and the mass of the 50bp-500bp fragment accounts for more than 85% of its total mass. This patent obtains the product of the target molecular weight by combining alkaline hydrolysis with a DNA fragmentation instrument. The preparation process involves alkali heating, acid-base neutralization, and Maillard reaction, which causes the product to turn yellow. The levels of key indicators such as protein content and endotoxin content are not mentioned. Patent CN106031709A discloses a method for preparing PDRN. The protein content of the PDRN product prepared by this method is as high as 2.5-5%, and the level of endotoxin is not mentioned. Runhui Biotechnology <Weihai> Co., Ltd. published an article titled "Process Improvement of PDRN and Research on Its Efficacy", which mainly describes the production of PDRN by enzymatic hydrolysis. The testes of salmon were pretreated to obtain a precipitate of fish sperm cells, and the fish sperm cells were hydrolyzed with trypsin to obtain a large-molecule PDRN product. The product was then hydrolyzed with nuclease to obtain a PDRN product of the target molecular weight. The protein content of the PDRN product prepared by this method is relatively high at 0.40-0.78%, and the endotoxin content is 2-3EU / mg, which is difficult to meet the quality requirements of pharmaceutical-grade and medical device-grade PDRN. In summary, there are few reports on the preparation process of low-endotoxin large-molecule PDRN in the existing technology. Therefore, it is necessary to develop a low-endotoxin large-molecule PDRN preparation process. Summary of the Invention

[0005] In order to obtain PDRN with a controllable molecular weight range and a PDRN wet solid with low endotoxin content and protein content, the present invention discloses a method for preparing low-endotoxin macromolecular PDRN, and the preparation method is as follows: (1) Fish testis pretreatment: Frozen salmon testis was thawed in pure water, rinsed, blood vessels removed, drained, and crushed into a paste; (2) Enzymatic hydrolysis of fish sperm cells: Take 1 kg of paste, add 10 L of enzymatic hydrolysis solution at a ratio of m:v = 1:10, add proteinase K at a concentration of 0.125 g / L, and react at 37°C, 180 rpm overnight; (3) Protein removal: Add 8-10% SDS (w / v) and a final concentration of 2.5 M NaCl solution, continue stirring for 3-5 h, then centrifuge to remove the precipitate and collect the supernatant; (4) Ultrasonic pretreatment: pre-cool the collected supernatant and use an ultrasonic disruptor to break the DNA fragments; (5) Nuclease hydrolysis: Nuclease hydrolysis is performed on the solution after ultrasonic pretreatment; (6) Primary alcohol precipitation: After the enzymatic hydrolysis is completed, use 1 volume of anhydrous ethanol for alcohol precipitation, stir evenly and let it stand, centrifuge at 4500 rpm for 10 min, and collect the wet solid; (7) Redissolution and filtration: Redissolve the wet solid in ultrapure water at 10 g / L and filter; (8) Endotoxin control: Sodium acetate and Triton 114 were added to the filtered solution, stirred in an ice bath, centrifuged, and separated into layers; (9) Secondary alcohol precipitation: Add 2 times of anhydrous ethanol to the upper sample solution for alcohol precipitation, centrifuge to obtain the precipitate, and obtain PDRN wet solid; Alcohol washing: The secondary alcohol precipitation is crushed with a crusher and the precipitation is washed; (11) Freeze-drying: The wet solid is directly freeze-dried and crushed to obtain a freeze-dried white powder sample.

[0006] A further improvement is that in step (4), an ultrasonic disruptor is used to break the DNA fragments, and PDRN of different molecular weights can be obtained by enzymatic hydrolysis, specifically 50-150 joules, 1 min-1.5 min treatment, for preparing 2000-4000 bp PDRN; 180-250 joules, 1 min-2 min treatment, for preparing 1000-2000 bp PDRN; 250-350 joules, 1 min-2.5 min treatment, for preparing 500-1000 bp PDRN.

[0007] A further improvement is that the nuclease used in step (5) is any one of deoxyribonuclease and restriction endonuclease.

[0008] A further improvement is that the enzymatic hydrolysis in step (5) uses deoxyribonuclease at a dosage of 10-50 mg / L and a hydrolysis time of 4 h to 10 h at 37° C., preferably 30 mg / L for 6 h.

[0009] A further improvement is that in step (7), the solution is filtered using a 0.2-3 μm depth filter.

[0010] A further improvement is that the specific method of endotoxin control in step (8) is to add 1 / 30 volume of 3M sodium acetate to the filtered solution to a final concentration of 100 mM, control the pH to 5.2, and then add 2% of Triton 100 or Triton 114, stir in an ice bath for 1 hour, and keep at 4°C overnight; stir in a warm bath at 45°C for 1 hour, centrifuge, and separate the layers, with the upper layer being the sample solution and the lower layer being Triton 114, and the upper layer sample solution is collected.

[0011] A further improvement is that preferably Triton 114 is used for washing, and washing is performed twice.

[0012] A further improvement is that in step (10), the crushed precipitate is washed three times with 80% ethanol.

[0013] The present invention also discloses a low-endotoxin macromolecular PDRN, which is obtained by the above-mentioned preparation method.

[0014] The present invention also discloses the application of the above preparation method in the preparation of low-endotoxin macromolecular PDRN in the fields of food, skin care, medical beauty, and medicine.

[0015] The present invention has the following beneficial effects: 1) The present invention solves the problem of high endotoxin content in the existing macromolecular PDRN. By using ultrasound combined with nuclease enzymatic hydrolysis for fragmentation, the present invention can effectively reduce the amount of enzyme used and accurately control the molecular weight of the product, resulting in simple operation and low industrialization costs. 2) The present invention uses proteinase K to enzymatically hydrolyze fish sperm cells to release fish sperm DNA, then uses ultrasound combined with nuclease to fragment the PDRN sample. The fragmentation is then treated with deep filtration and Triton 114 to obtain a wet solid PDRN with a controllable molecular weight range of 500bp-4000bp, an endotoxin content of ≤0.5EU / mg and a protein content of ≤1.0%. The wet solid PDRN is then freeze-dried to obtain the finished product. 3) The product prepared by the present invention is a white powder that does not yellow and has a low protein content. It effectively avoids the Maillard reaction during acid or alkaline hydrolysis, thereby improving the quality and added value of the product. DETAILED DESCRIPTION

[0016] To further illustrate the technical means and effects of the present invention, the present invention is further described below with reference to the following examples. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention.

[0017] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0018] The following examples are included to illustrate preferred embodiments of the present invention. Those skilled in the art will recognize that the techniques disclosed in the following examples represent techniques discovered by the inventors to work well in the practice of the present invention. Based on this disclosure, those skilled in the art will recognize that many changes can be made to the specific embodiments disclosed without departing from the spirit and scope of the present invention, that is, any equivalent results obtained by utilizing the contents of the present specification, equivalent process changes, or direct or indirect application in other related technical fields should be included within the scope of protection of this patent. Example 1

[0019] This embodiment discloses a method for preparing a macromolecular low-endotoxin PDRN, comprising the following steps: (1) fish spermatozoa pretreatment: thawing frozen salmon spermatozoa in pure water, rinsing, removing blood vessels, draining water, and crushing into a paste; (2) fish sperm cell enzymatic hydrolysis: taking 1 kg of the paste, adding 10 L of enzymatic hydrolysis solution at a ratio of m:v=1:10, adding proteinase K at a concentration of 0.125 g / L, and reacting at 37°C, 180 rpm overnight; (3) protein removal: adding 8% SDS (w / v) and a NaCl solution with a final concentration of 2.5 M, continuing stirring for 3-5 hours, centrifuging to remove the precipitate, and collecting the supernatant; (4) ultrasonic pretreatment: precooling the collected supernatant, and using an ultrasonic disruptor to break the DNA fragments at 100 joules for 1 minute; (5) Enzymatic hydrolysis: The solution after ultrasonic pretreatment was enzymatically hydrolyzed at 30 mg / L of endonuclease at 37°C for 6 h; (6) Primary alcohol precipitation: After the enzymatic hydrolysis, 1 volume of anhydrous ethanol was used for alcohol precipitation, stirred evenly and allowed to stand, centrifuged at 4500 rpm for 10 min, and the wet solid was collected; (7) Redissolution and filtration: The wet solid was redissolved in ultrapure water at 10 g / L and the solution was filtered using a 0.2-3 μm depth filter; (8) Endotoxin control: After filtration, 1 / 30 volume of 3 M sodium acetate was added to the solution to a final concentration of 100 mM, the pH was controlled at 5.2, 2% Triton 114 was added, and the solution was stirred in an ice bath for 1 h and then incubated at 4°C overnight; stirred in a warm bath at 45°C for 1 h, centrifuged, separated into layers, and the upper sample solution was collected. After the end, repeat the washing with Triton 114 to obtain the upper sample solution after the second washing; (9) Secondary alcohol precipitation: add 2 times of anhydrous ethanol to the upper sample solution for alcohol precipitation, centrifuge to obtain a precipitate, and obtain PDRN wet solid; (10) Alcohol washing: the secondary alcohol precipitation precipitate is crushed with a crusher, and the crushed precipitate is washed 3 times with 80% ethanol; (11) Drying: the wet solid is directly freeze-dried and crushed to obtain a white powder sample.

[0020] By preparing by ultrasound and enzymatic hydrolysis in this protocol, PDRN of 2000-4000 bp can be obtained, and specifically 3000 bp PDRN can be obtained using the specific parameters of this embodiment. PDRN with an endotoxin content of 0.12 EU / mg and a protein content of 0.6% can be obtained. Example 2

[0021] This embodiment discloses a method for preparing a macromolecular low-endotoxin PDRN, comprising the following steps: (1) fish spermatozoa pretreatment: thawing frozen salmon spermatozoa in pure water, rinsing, removing blood vessels, draining, and crushing into a paste; (2) fish sperm cell enzymatic hydrolysis: taking 1 kg of the paste, adding 10 L of enzymatic hydrolysis solution at a ratio of m:v=1:10, adding proteinase K at a concentration of 0.125 g / L, and reacting at 37°C, 180 rpm overnight; (3) protein removal: adding 8% SDS (w / v) and a NaCl solution with a final concentration of 2.5 M, continuing stirring for 3-5 hours, centrifuging to remove the precipitate, and collecting the supernatant; (4) ultrasonic pretreatment: precooling the collected supernatant, and using an ultrasonic disruptor to break the DNA fragments at 200 joules for 1.5 minutes; (5) Enzymatic hydrolysis: The solution after ultrasonic pretreatment was enzymatically hydrolyzed with 30 mg / L endonuclease at 37°C for 6 h; (6) Primary alcohol precipitation: After the enzymatic hydrolysis, 1 volume of anhydrous ethanol was used for alcohol precipitation, stirred evenly and allowed to stand, centrifuged at 4500 rpm for 10 min, and the wet solid was collected; (7) Redissolution and filtration: The wet solid was re-dissolved with ultrapure water at 10 g / L and the solution was filtered with a 0.2-3 μm depth filter; (8) Endotoxin control: After filtration, 1 / 30 volume of 3 M sodium acetate was added to the solution to a final concentration of 100 mM, the pH was controlled at 5.2, 2% Triton 114 was added, and the solution was stirred in an ice bath for 1 minute. h, overnight at 4℃; incubate at 45℃ with stirring for 1h, centrifuge, separate layers, and collect the upper sample solution; (9) Secondary alcohol precipitation: add 2 times of anhydrous ethanol to the upper sample solution for alcohol precipitation, centrifuge to obtain a precipitate, and obtain PDRN wet solid; (10) Alcohol washing: the secondary alcohol precipitation precipitate is crushed with a crusher, and the crushed precipitate is washed 3 times with 80% ethanol; (11) Drying: the wet solid is directly freeze-dried and crushed to obtain a white powder sample.

[0022] By preparing by ultrasound and enzymatic hydrolysis in this protocol, a PDRN of 1000-2000 bp can be obtained. Specifically, the specific parameters of this embodiment can obtain a PDRN of 1500 bp. PDRN with an endotoxin content of 0.46 EU / mg and a protein content of 0.6% can be obtained. Example 3

[0023] This embodiment discloses a method for preparing a macromolecular low-endotoxin PDRN, comprising the following steps: (1) fish spermatozoa pretreatment: thawing frozen salmon spermatozoa in pure water, rinsing, removing blood vessels, draining water, and crushing into a paste; (2) fish sperm cell enzymatic hydrolysis: taking 1 kg of the paste, adding 10 L of enzymatic hydrolysis solution at a ratio of m:v=1:10, adding proteinase K at a concentration of 0.125 g / L, and reacting at 37°C, 180 rpm overnight; (3) protein removal: adding 8% SDS (w / v) and a NaCl solution with a final concentration of 2.5 M, continuing stirring for 3-5 hours, centrifuging to remove the precipitate, and collecting the supernatant; (4) ultrasonic pretreatment: precooling the collected supernatant, and using an ultrasonic disruptor to break the DNA fragments at 200 joules for 1.5 minutes; (5) Enzymatic hydrolysis: The solution after ultrasonic pretreatment was enzymatically hydrolyzed at 30 mg / L of endonuclease at 37°C for 6 h; (6) Primary alcohol precipitation: After the enzymatic hydrolysis, 1 volume of anhydrous ethanol was used for alcohol precipitation, stirred evenly and allowed to stand, centrifuged at 4500 rpm for 10 min, and the wet solid was collected; (7) Redissolution and filtration: The wet solid was re-dissolved in ultrapure water at 10 g / L, and the solution was filtered using a 0.2-3 μm depth filter; (8) Endotoxin control: After filtration, 1 / 30 volume of 3 M sodium acetate was added to the solution to a final concentration of 100 mM, and the pH was controlled at 5.2. Then, 2% of Triton 114 was added, stirred in an ice bath for 1 h, and incubated at 4°C overnight; stirred in a warm bath at 45°C for 1 h, centrifuged, separated, and the upper sample solution was collected. After the end, the washing with Triton 114 was repeated once to obtain the upper sample solution after the second washing; (9) Secondary alcohol precipitation: 2 times of anhydrous ethanol was added to the upper sample solution for alcohol precipitation, and the precipitate was obtained by centrifugation to obtain PDRN wet solid; (10) Alcohol washing: The secondary alcohol precipitation precipitate was crushed with a crusher, and the crushed precipitate was washed with 80% ethanol for 3 times; (11) Drying: The wet solid was directly freeze-dried and crushed to obtain a white powder sample.

[0024] By preparing by ultrasound and enzymatic hydrolysis in this protocol, PDRN of 1000-2000 bp can be obtained. Specifically, the specific parameters of this embodiment can obtain PDRN of 1500 bp. The endotoxin content is 0.036 EU / mg and the protein content is 0.58%. Example 4

[0025] This embodiment discloses a method for preparing a macromolecular low-endotoxin PDRN, comprising the following steps: (1) fish spermatozoa pretreatment: thawing frozen salmon spermatozoa in pure water, rinsing, removing blood vessels, draining water, and crushing into a paste; (2) fish sperm cell enzymatic hydrolysis: taking 1 kg of the paste, adding 10 L of enzymatic hydrolysis solution at a ratio of m:v=1:10, adding proteinase K at a concentration of 0.125 g / L, and reacting at 37°C, 180 rpm overnight; (3) protein removal: adding 8% SDS (w / v) and a NaCl solution with a final concentration of 2.5 M, continuing stirring for 3-5 hours, centrifuging to remove the precipitate, and collecting the supernatant; (4) ultrasonic pretreatment: precooling the collected supernatant, and using an ultrasonic disruptor to break the DNA fragments at 200 joules for 1.5 minutes; (5) Enzymatic hydrolysis: The solution after ultrasonic pretreatment was enzymatically hydrolyzed at 30 mg / L of endonuclease at 37°C for 6 h; (6) Primary alcohol precipitation: After the enzymatic hydrolysis, 1 volume of anhydrous ethanol was used for alcohol precipitation, stirred evenly and allowed to stand, centrifuged at 4500 rpm for 10 min, and the wet solid was collected; (7) Redissolution and filtration: The wet solid was re-dissolved in ultrapure water at 10 g / L, and the solution was filtered using a 0.2-3 μm depth filter; (8) Endotoxin control: After filtration, 1 / 30 volume of 3 M sodium acetate was added to the solution to a final concentration of 100 mM, and the pH was controlled at 5.2. Then, 2% Triton 100 was added, stirred in an ice bath for 1 h, and incubated at 4°C overnight; stirred in a warm bath at 45°C for 1 h, centrifuged, separated, and the upper sample solution was collected. After the end, repeat the washing with Triton 100 to obtain the upper sample solution after the second washing; (9) Secondary alcohol precipitation: add 2 times of anhydrous ethanol to the upper sample solution for alcohol precipitation, centrifuge to obtain a precipitate, and obtain PDRN wet solid; (10) Alcohol washing: the secondary alcohol precipitation precipitate is crushed with a grinder, and the crushed precipitate is washed 3 times with 80% ethanol; (11) Drying: the wet solid is directly freeze-dried and crushed to obtain a white powder sample.

[0026] By preparing by ultrasound and enzymatic hydrolysis in this protocol, PDRN of 1000-2000 bp can be obtained. Specifically, the specific parameters of this embodiment can obtain PDRN of 1500 bp. The endotoxin content is 0.12 EU / mg and the protein content is 0.62%. Example 5

[0027] This embodiment discloses a method for preparing a macromolecular low-endotoxin PDRN, comprising the following steps: (1) fish spermatozoa pretreatment: thawing frozen salmon spermatozoa in pure water, rinsing, removing blood vessels, draining, and crushing into a paste; (2) fish sperm cell enzymatic hydrolysis: taking 1 kg of the paste, adding 10 L of enzymatic hydrolysis solution at a ratio of m:v=1:10, adding proteinase K at a concentration of 0.125 g / L, and reacting at 37°C, 180 rpm, and reacting overnight; (3) protein removal: adding 8% SDS (w / v) and a NaCl solution with a final concentration of 2.5 M, continuing stirring for 3-5 hours, and then centrifuging to remove the precipitate, and collecting the supernatant; (4) ultrasonic pretreatment: precooling the collected supernatant, and using an ultrasonic disruptor to break the DNA fragments, 300 joules, and working for 2 minutes; (5) Enzymatic hydrolysis: The solution after ultrasonic pretreatment was enzymatically hydrolyzed with 50 mg / L endonuclease at 37°C for 6 h; (6) Primary alcohol precipitation: After the enzymatic hydrolysis, 1 volume of anhydrous ethanol was used for alcohol precipitation, stirred evenly and allowed to stand, centrifuged at 4500 rpm for 10 min, and the wet solid was collected; (7) Redissolution and filtration: The wet solid was re-dissolved with ultrapure water at 10 g / L, and the solution was filtered with a 0.2-3 μm depth filter; (8) Endotoxin control: After filtration, 1 / 30 volume of 3 M sodium acetate was added to the solution to a final concentration of 100 mM, and the pH was controlled at 5.2. Then, 2% Triton 114 was added, stirred in an ice bath for 1 h, and incubated at 4°C overnight; stirred in a warm bath at 45°C for 1 h, centrifuged, separated, and the upper sample solution was collected. After the end, the washing with Triton 114 was repeated once to obtain the upper sample solution after the second washing; (9) Secondary alcohol precipitation: 2 times of anhydrous ethanol was added to the upper sample solution for alcohol precipitation, and the precipitate was obtained by centrifugation to obtain PDRN wet solid; (10) Alcohol washing: The secondary alcohol precipitation precipitate was crushed with a crusher, and the crushed precipitate was washed with 80% ethanol for 3 times; (11) Drying: The wet solid was directly freeze-dried and crushed to obtain a white powder sample.

[0028] By preparing by ultrasound and enzymatic hydrolysis in this protocol, a 500-1000 bp PDRN can be obtained, and specifically a 750 bp PDRN can be obtained using the specific parameters of this embodiment. The endotoxin content is 0.040 EU / mg, and the protein content is 0.38% PDRN.

[0029] The present invention respectively tested the bacterial endotoxin and protein content of the PDRN samples prepared in the above examples, and the specific tests are shown in Table 1 below.

[0030] Table 1. Detection of bacterial endotoxin and protein content of PDRN samples prepared in Example Test items Acceptable Standards Example 1 Example 2 Example 3 Example 4 Example 5 Appearance White or off-white powder White powder White powder White powder White powder White powder Molecular weight Report test value 2000-4000bp 1000-2000bp 1000-2000bp 1000-2000bp 500-1000bp bacterial endotoxins <0.5EU / mg 0.12 0.46 0.036 0.12 0.04 Protein content ≤1.0% 0.6% 0.6% 0.58% 0.62% 0.38% It can be seen from the examples and Table 1 thereof that the present technical solution can obtain a PDRN with a controllable molecular weight range of 500bp-4000bp; an endotoxin content of ≤0.5EU / mg and a protein content of ≤1.0% PDRN wet solid, with good stability.

[0031] The above descriptions are merely embodiments of the present invention and are not intended to limit the scope of the present invention. Any equivalent results or equivalent process transformations made using the contents of the present invention specification, or any direct or indirect application in other related technical fields, should be included in the scope of protection of this patent.

Claims

1. A method for preparing a low-endotoxin macromolecular PDRN, characterized in that: The preparation method is as follows: (1) Fish testis pretreatment: Frozen salmon testis was thawed in pure water, rinsed, blood vessels removed, drained, and crushed into a paste; (2) Enzymatic hydrolysis of fish sperm cells: Take 1 kg of paste, add 10 L of enzymatic hydrolysis solution at a ratio of m:v = 1:10, add proteinase K at a concentration of 0.125 g / L, and react at 37°C, 180 rpm overnight; (3) Protein removal: Add 8-10% SDS (w / v) and a final concentration of 2.5 M NaCl solution, continue stirring for 3-5 h, centrifuge to remove the precipitate, and collect the supernatant; (4) Ultrasonic pretreatment: precool the collected supernatant and use an ultrasonic disruptor to break the DNA fragments; (5) Nuclease hydrolysis: Nuclease hydrolysis is performed on the solution after ultrasonic pretreatment; (6) Primary alcohol precipitation: After the nuclease hydrolysis is completed, use 1 volume of anhydrous ethanol for alcohol precipitation, stir evenly and let stand, centrifuge at 4500 rpm for 10 min, and collect the wet solid; (7) Redissolution and filtration: Redissolve the wet solid in ultrapure water at 10 g / L and filter; (8) Endotoxin control: Sodium acetate and Triton 114 were added to the filtered solution, stirred in an ice bath, centrifuged, and separated into layers; (9) Secondary alcohol precipitation: Add 2 times of anhydrous ethanol to the upper sample solution for alcohol precipitation, centrifuge to obtain the precipitate, and obtain PDRN wet solid; (10) Alcohol washing: The secondary alcohol precipitation is crushed by a crusher and the precipitation is washed; Freeze-drying: The wet solid is directly freeze-dried and crushed to obtain a freeze-dried white powder sample.

2. A method for preparing a low-endotoxin macromolecular PDRN as claimed in claim 1, characterized in that, The ultrasonic pretreatment method in step (4) is: 50-150 joules, treatment for 1 min-1.5 min, for preparing 2000-4000 bp PDRN; 180-250 joules, treatment for 1 min-2 min, for preparing 1000-2000 bp PDRN; 250-350 joules, treatment for 1 min-2.5 min, for preparing 500-1000 bp PDRN.

3. A method for preparing a low-endotoxin macromolecular PDRN as claimed in claim 1, characterized in that, The nuclease used in the step (5) is any one of deoxyribonuclease and restriction endonuclease.

4. A method for preparing a low-endotoxin macromolecular PDRN as claimed in claim 3, characterized in that, The enzymatic hydrolysis in step (5) uses deoxyribonuclease at a dosage of 10-50 mg / L and a hydrolysis time of 4 h-10 h at 37° C. Preferably, the enzyme dosage is 30 mg / L and the hydrolysis time is 6 h.

5. A method for preparing a low-endotoxin macromolecular PDRN as claimed in claim 1, characterized in that, In step (7), the solution is filtered using a 0.2-3 μm depth filter.

6. A method for preparing a low-endotoxin macromolecular PDRN as claimed in claim 1, characterized in that, The specific method of endotoxin control in step (8) is to add 1 / 30 volume of 3M sodium acetate to the filtered solution to a final concentration of 100 mM, control the pH to 5.2, and then add 2% of Triton 100 or Triton 114, stir in an ice bath for 1 hour, and incubate at 4°C overnight; stir in a warm bath at 45°C for 1 hour, centrifuge, and separate the layers, with the upper layer being the sample solution and the lower layer being the Triton 114, and collect the upper layer of the sample solution.

7. A method for preparing a low-endotoxin macromolecular PDRN as claimed in claim 6, characterized in that, Preferably, Triton 114 is used for washing, and washing is performed twice.

8. A method for preparing a low-endotoxin macromolecular PDRN as claimed in claim 1, characterized in that, In step (10), the crushed precipitate is washed three times with 80% ethanol.

9. A low-endotoxin macromolecular PDRN, characterized in that: The method is obtained by any one of claims 1 to 8.

10. Application of the preparation method according to any one of claims 1 to 8 in the preparation of low-endotoxin macromolecular PDRN in the fields of food, skin care, medical beauty, and medicine.