A method for extracting pdrn from salmon by salting out

Abstract

This invention discloses a method for salting out salmon PDRN. The method includes: adding a soluble salt and an organic solvent to a salmon ooze enzymatic hydrolysate, mixing thoroughly, allowing the mixture to stand and separate into three phases, forming a three-phase salting-out extraction system consisting of an upper phase, an intermediate phase, and a lower phase; collecting the intermediate phase when the organic solvent is hydrophilic, and collecting the lower phase when the organic solvent is hydrophobic; purifying and drying the collected intermediate or lower phase to obtain PDRN powder. This invention uses salmon ooze enzymatic hydrolysate as raw material and extracts salmon PDRN product in one step through salting-out extraction. The PDRN product yield is greater than 5.0%, A260 / A280 is 1.8–2.0, A260 / A230 is ≥2.0, endotoxin content is <0.03 EU / mg, protein content is ≤0.5%, and molecular weight is 200–4000 bp. The method of this invention has the advantages of simple process, low cost, high yield, and high product purity, making it suitable for large-scale industrial production.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and to a method for separating and purifying polydeoxyribonucleic acid (PDRN) and removing endotoxins and proteins from salmon leucovorin hydrolysate using salting-out extraction technology. Background Technology

[0002] Polydeoxyribonucleotide (PDRN) is a mixture of DNA fragments extracted from salmon milt, with a molecular weight typically ranging from 50,000 to 1.5 million Daltons. As an agonist of adenosine A2A receptors, PDRN participates in salvage pathways in vivo, exhibiting significant effects in promoting cell proliferation, angiogenesis, and anti-inflammation. Therefore, it has wide applications in cosmetic raw materials, high-end medical aesthetics (such as "baby injections"), and the pharmaceutical field (such as tissue repair and ulcer treatment).

[0003] Salmon PDRN shares a 98% similarity with human DNA base sequences, exhibiting extremely high biocompatibility and safety. However, the industrial production of this highly active, high-value-added raw material faces significant challenges, with the core difficulty lying in the deep control of impurities, particularly the removal of proteins and endotoxins. Firstly, PDRN originates from animal tissues; impure extraction can lead to residual foreign proteins that may cause immune rejection or allergic reactions in humans. Secondly, salmon spores inevitably come into contact with Gram-negative bacteria during harvesting and processing, resulting in endotoxin (lipopolysaccharide) contamination. Endotoxins are potent pyrogens; even trace amounts injected into the human body can cause fever, shock, or even death. High purity (low endotoxin) is a key indicator for PDRN to be used as an injectable-grade raw material, directly determining the product's market value. Existing separation and purification methods mainly include traditional alcohol precipitation, membrane separation technologies (ultrafiltration / nanofiltration), and ion exchange / affinity chromatography. While traditional alcohol precipitation is simple to operate, its ability to remove endotoxins is limited. Endotoxins are prone to co-precipitate with DNA; although membrane separation technology can retain some impurities, the high viscosity of the enzymatic hydrolysate can easily lead to membrane pore blockage, resulting in high cleaning and maintenance costs, and it is difficult to completely separate endotoxins and PDRN with similar molecular weights; ion exchange / affinity chromatography has high resolution, but the resin is expensive and has low loading capacity, making it unsuitable for large-scale industrial coarse separation. Summary of the Invention

[0004] In view of the problems existing in the prior art, the purpose of the present invention is to provide a new method for extracting PDRN from salmon milt. This method utilizes salting-out extraction technology to extract PDRN from salmon milt enzymatic hydrolysate in high yield, while simultaneously removing endotoxins and proteins.

[0005] The objective of this invention is achieved through the following technical solution.

[0006] In a first aspect, the present invention provides a method for salting out PDRN from salmon, comprising the following steps:

[0007] S1: Add soluble salt and organic solvent to the salmon bladder enzymatic hydrolysate, mix well, let stand and separate phases to form a three-phase salting-out extraction system consisting of upper phase, middle phase and lower phase. S2: When the organic solvent is a hydrophilic organic solvent, collect the intermediate phase; when the organic solvent is a hydrophobic organic solvent, collect the lower phase. S3: The intermediate phase or lower phase collected in S2 is purified and dried to obtain PDRN powder.

[0008] In this invention, a three-phase salting-out extraction system consisting of an upper phase, an intermediate phase, and a lower phase is formed by adding soluble salts and organic solvents to the salmon bladder enzymatic hydrolysate. Based on the properties of the organic solvents, salmon PDRN contained in the salmon bladder enzymatic hydrolysate is enriched in one phase, while endotoxins and proteins are distributed to the other phases. This achieves high-yield extraction of salmon PDRN and simultaneous removal of endotoxins and proteins. Furthermore, the natural pH of salmon bladder enzymatic hydrolysate is typically 6.0–6.4. This invention eliminates the need for pH adjustment and allows for direct use; PDRN can be obtained directly from the salmon bladder enzymatic hydrolysate through salting-out extraction, eliminating the need for filtration to remove endotoxins and proteins, thus reducing costs and losses.

[0009] In this invention, the salmon bladder enzymatic hydrolysate is obtained by enzymatically hydrolyzing salmon bladder tissue. A complex enzyme containing both proteases and nucleases commonly used in the art for protein degradation and nucleic acid degradation can be selected for enzymatic hydrolysis, resulting in a salmon bladder enzymatic hydrolysate containing PDRN. In some embodiments, the salmon bladder tissue is added to an enzymatic hydrolysis system containing the complex enzyme for enzymatic hydrolysis. After hydrolysis, the tissue is centrifuged, and the supernatant is collected to obtain the salmon bladder enzymatic hydrolysate. Specifically, the salmon bladder is thawed, washed with water or PBS buffer, homogenized, and then the complex enzyme is added to form an enzymatic hydrolysis system. Enzymatic hydrolysis is carried out under appropriate reaction conditions for 5–10 hours. After hydrolysis, the tissue is centrifuged at 4°C and 8000 rpm, and the supernatant is collected as the salmon bladder enzymatic hydrolysate. The end of the hydrolysis can be determined by the following method: the hydrolysis process continues until the pH of the hydrolysate stabilizes, the viscosity no longer decreases, and the fluidity stabilizes.

[0010] In some embodiments, the complex enzyme comprises a protease and a nuclease. Preferably, the mass ratio of the protease to the nuclease is (0.5-1):1. The protease may be one or more of papain, flavor protease, and neutral protease, and the nuclease may be one or more of restriction endonuclease Sau3AI and ribonuclease. Preferably, the complex enzyme may be a combination of flavor protease and restriction endonuclease Sau3AI in a mass ratio of 0.6:1, or a combination of papain and restriction endonuclease Sau3AI in a mass ratio of 0.5:1.

[0011] In some embodiments, when enzymatically hydrolyzing salmon calcareous tissue, the pH of the hydrolysis system is 6.0–6.4, and the hydrolysis temperature is 50–55°C. In some embodiments, the concentration of salmon calcareous tissue in the hydrolysis system is 50–150 g / L, preferably 80–120 g / L, more preferably 90–110 g / L; the amount of the compound enzyme added is 0.4–1% of the total mass of the hydrolysis system, preferably 0.5–0.9%, more preferably 0.5–0.7%.

[0012] In this invention, in step S1, soluble salts and organic solvents are added to the salmon ooze enzymatic hydrolysate, mixed thoroughly, and allowed to stand for phase separation, forming a three-phase salting-out extraction system consisting of an upper phase, an intermediate phase, and a lower phase. Specifically, the salmon ooze enzymatic hydrolysate is an aqueous solution. When appropriate concentrations of soluble salts and organic solvents are added, a three-phase salting-out extraction system can be obtained. For example, when the organic solvent is a hydrophilic organic solvent, the three phases formed, from top to bottom, are: an upper solvent phase rich in impurities (upper phase), an intermediate solid phase rich in PDRN (intermediate phase), and a lower salt phase rich in impurities (lower phase). When the organic solvent is a hydrophobic organic solvent, the three phases formed, from top to bottom, are: an upper solvent phase rich in impurities (upper phase), an intermediate emulsion phase rich in impurities (intermediate phase), and a lower salt phase rich in PDRN (lower phase). In this invention, two different phase-separation enrichment pathways for PDRN are constructed based on the properties of the selected organic solvents. When a hydrophilic organic solvent is used, due to the miscibility of the organic solvent and water, macroscopic phase separation occurs between the organic solvent and the aqueous phase under the strong salting-out effect of the soluble salt, forming an upper liquid phase rich in organic solvent. Simultaneously, due to the salting-out effect and the sharp drop in solubility near the isoelectric point, PDRN precipitates from the solution and accumulates in the intermediate layer under the synergistic effect of salting-out at the interface between the organic solvent and the aqueous phase and interfacial tension, forming a solid phase rich in PDRN. The lower layer is a PDRN-rich solid phase. The system uses a salt phase containing salt and hydrophilic impurities to selectively enrich PDRN in a three-phase system. When a hydrophobic organic solvent is used, the organic solvent and the aqueous phase are immiscible. Under the action of salt, the system forms an upper hydrophobic solvent phase and a lower salt phase. Since PDRN is an amphiphilic macromolecule, under the adsorption effect at the salt-solvent biphase interface, some impurities form a stable emulsion layer with the organic solvent. Due to the weak hydrophobic interaction and the influence of salting out, PDRN is mainly allocated to the lower salt phase, thereby achieving the enrichment of PDRN in the salt phase.

[0013] In some embodiments, in S1, in the three-phase salting-out extraction system, the mass percentage of soluble salt is 3-30 wt%, preferably 5-25 wt%, more preferably 10-25 wt%, and the mass percentage of organic solvent is 10-67 wt%, preferably 20-50 wt%, more preferably 20-33 wt%, based on the total mass percentage of the three-phase salting-out extraction system. In some embodiments, when the organic solvent is a hydrophilic organic solvent, the mass percentage of soluble salt is 3-30 wt%, preferably 5-25 wt%, more preferably 15-21 wt%; and the mass percentage of hydrophilic organic solvent is 10-67 wt%, preferably 20-50 wt%, more preferably 20-25 wt%. When the organic solvent is a hydrophobic organic solvent, the mass percentage of the soluble salt is 3-30 wt%, preferably 10-25 wt%, more preferably 20-25 wt%; the mass percentage of the hydrophobic organic solvent is 5-67 wt%, preferably 20-50 wt%, more preferably 25-33 wt%.

[0014] In some embodiments, in S1, the soluble salt is an inorganic salt or an organic salt: the inorganic salt is selected from one or more of sodium bisulfate, aluminum sulfate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, potassium phosphate, sodium carbonate, potassium carbonate, trisodium phosphate, sodium silicate, ammonium sulfate, sodium sulfate, sodium chloride, disodium hydrogen phosphate, dipotassium hydrogen phosphate, and ammonium chloride; the organic salt is selected from one or more of disodium hydrogen citrate, sodium dihydrogen citrate, potassium hydrogen tartrate, sodium formate, potassium formate, sodium oxalate, potassium oxalate, sodium acetate, potassium acetate, trisodium citrate, potassium citrate, ammonium citrate, ammonium acetate, sodium succinate, and sodium tartrate.

[0015] In some embodiments, in S2, the hydrophilic organic solvent is selected from one or more of ethanol, methanol, n-propanol, isopropanol, acetone, 1,3-propanediol, 1,2-butanediol, phenoxyethanol, propylene glycol, glycerol, butanediol, pentanediol, dipropylene glycol, propylene glycol methyl ether, propylene glycol ethyl ether, propylene glycol butyl ether, dipropylene glycol methyl ether, and polyethylene glycol.

[0016] In some embodiments, in S2, the hydrophobic organic solvent is selected from one or more of ethyl acetate, triethyl citrate, tributyl citrate, trimethyl phosphate, tricresyl phosphate, triethyl phosphate, isopropyl palmitate, isopropyl myristate, ethylhexyl palmitate, caprylic triglyceride, capric triglyceride, caprylic / capric triglyceride, C12-15 alcohol benzoate, cyclopentamethoxysiloxane, polydimethylsiloxane, phenyl polytrimethylsiloxane, n-octanol, n-heptane, n-hexane, isododecane, isohexadecane, mineral oil, liquid paraffin, hydrogenated polyisobutylene, squalane, jojoba oil, almond oil, grape seed oil, nut oil, sunflower seed oil, octyldodecyl alcohol, petroleum ether, toluene, benzene, dichloromethane, chloroform, and methyl tert-butyl ether.

[0017] In this invention, in step S3, the intermediate phase or lower phase collected in step S2 is purified and dried to obtain PDRN powder. As described above, adding a soluble salt and an organic solvent to the salmon bladder enzymatic hydrolysate in step S1 forms a three-phase salting-out extraction system. When the organic solvent is hydrophilic, PDRN is enriched as a solid phase in the intermediate phase of the three-phase salting-out extraction system; when a hydrophobic organic solvent is used, PDRN is distributed to the lower salt phase (lower phase) of the three-phase salting-out extraction system. Thus, the PDRN-rich intermediate phase or lower phase is collected in step S2 and purified and dried in step S3 to obtain PDRN powder.

[0018] In some embodiments, in step S3, the intermediate phase collected in step S2 is washed with ethanol and then freeze-dried under vacuum to obtain PDRN powder. Preferably, the concentration of ethanol is 70% to 95%, and the vacuum freeze-drying conditions are: temperature -20°C to -40°C, vacuum degree below 10 Pa, and generally vacuum freeze-drying for 24 to 48 hours until constant weight is obtained, resulting in dry, fluffy powdered PDRN.

[0019] In some embodiments, in S3, the lower phase collected in S2 is subjected to dialysis desalting and ethanol precipitation, washed, and dried to obtain PDRN powder. The lower phase is a salt phase, which contains a large amount of salt components in addition to PDRN. Because the salt components (salt ions) are small molecules, a dialysis bag with a molecular weight cutoff of 10 kDa is used to remove the salts in the lower phase, thereby obtaining a PDRN-rich dialysis residue solution. 10 to 20 times the volume of ethanol is added to this residue solution to precipitate PDRN. The precipitate is washed with ethanol (70 wt%) and then freeze-dried under vacuum to constant weight to obtain powdered PDRN.

[0020] In a second aspect, the present invention provides salmon PDRN obtained by the above method, wherein the salmon PDRN has a yield greater than 5.0%, an A260 / A280 ratio of 1.8 to 2.0, an A260 / A230 ratio ≥ 2.0, an endotoxin content of < 0.03 EU / mg, a protein content ≤ 0.5%, and a molecular weight of 200 to 4000 bp.

[0021] A third aspect of this invention provides the application of salmon PDRN obtained by the above method in the preparation of medical aesthetic products and anti-inflammatory cosmetics. The salmon PDRN obtained by the method of this invention has high purity, with an endotoxin content of less than 0.03 EU / mg and a protein content of less than 0.5%, meeting the raw material standards for injectable / implantable biomaterials. It can be used as a core raw material in the production of medical aesthetic fillers, repair dressings, and functional anti-inflammatory skincare products.

[0022] The beneficial effects of this invention are: 1. This invention constructs a specific three-phase salting-out system, utilizing the slight differences in interfacial properties between endotoxin (lipopolysaccharide) and PDRN, to effectively partition endotoxin into the solvent phase or retain it in the intermediate emulsion layer, while PDRN precipitates as a solid or remains in the salt phase. Testing showed that the PDRN yield was greater than 5.0%, the purity A260 / A280 was 1.8–2.0, A260 / A230 ≥ 2.0, the endotoxin content was <0.03 EU / mg, the protein content was ≤0.5%, and the molecular weight was 200–4000 bp.

[0023] 2. In a hydrophilic solvent system, this invention enables PDRN to form a dense solid layer at the interface between two phases, while impurities such as proteins and endotoxins dissolve in the upper and lower phases, achieving a one-step solid-liquid separation with extremely high purity. In a hydrophobic solvent system, the formed intermediate emulsion layer effectively adsorbs a large amount of impurity proteins and endotoxins, preventing them from contaminating the lower PDRN solution.

[0024] 3. The method of this invention can directly process the enzymatic hydrolysate, which does not require precision filtration or ultrafiltration pretreatment, thus avoiding membrane clogging problems. The reagents used are inexpensive, readily available, and recyclable, making it suitable for large-scale industrial production. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to embodiments, but the embodiments of the present invention are not limited thereto. Unless otherwise specified in the embodiments, the experimental methods used are conventional methods, and the reagents and instruments used are all commercially available conventional products. All reagents used are of analytical grade; for example, the soluble salts used and the reagents used as organic solvents are all of analytical grade.

[0026] Materials used in the examples and testing of PDRN products: 1. Salmon bladder enzymatic hydrolysate: Thaw and wash salmon bladder tissue. Take 100 g of bladder tissue, add water, homogenize and break it up, and adjust the volume to 1 L. Add a compound enzyme (papain: restriction endonuclease Sau3 AI mass ratio of 0.5:1) at a certain ratio (0.4-1%) to form an enzymatic hydrolysis system. No pH adjustment is required. Incubate at 50℃ for 2-10 h with stirring. Centrifuge at 8000 rpm for 15 min and collect the supernatant, which is the salmon bladder enzymatic hydrolysate.

[0027] In the enzymatic hydrolysis system, when the content of the compound enzyme is 1% and the stirring time is 10 h, the molecular weight of PDRN contained in the salmon bladder hydrolysate is about 200 bp; when the stirring time is 6 h and the content of the compound enzyme is 0.5%, the molecular weight of PDRN contained in the salmon bladder hydrolysate is 800-1000 bp; when the stirring time is 2 h and the content of the compound enzyme is 0.4%, the molecular weight of PDRN contained in the salmon bladder hydrolysate is 3000-4000 bp.

[0028] Papain: Purchased from Nanning Pangbo Biotechnology Co., Ltd. (enzyme activity: 3 million U / g); Restriction endonuclease Sau3 AI: purchased from Shanghai Kanglang Biotechnology Co., Ltd. (Catalog No.: KL-D9346).

[0029] Salmon roe is the testes of frozen Pacific salmon.

[0030] 2. The PDRN product yield is calculated using the following formula: PDRN yield (%) = (PDRN product weight / fish shavings weight) × 100 3. PDRN Purity: The dried PDRN product was dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), and the absorbance at 230 nm, 260 nm, and 280 nm was measured using a spectrophotometer. The A260 / A280 and A260 / A230 ratios were calculated to determine the PDRN purity. A260 / A280 ratio between 1.8 and 2.0, and A260 / A230 ≥ 2.0, indicate high PDRN purity with minimal residual RNA and protein impurities.

[0031] 4. Detection of endotoxin content in PDRN products: The Limulus amebocyte lysate (LAL) reagent method was used. A commercially available bacterial endotoxin detection kit (manufacturer: Zhanjiang Andus Biotechnology Co., Ltd., model: KC-035) was used.

[0032] 5. Determination of protein content in PDRN products: The BCA method was used. A commercially available BCA protein quantification kit (manufacturer: Beyotime Biotechnology Co., Ltd., model: P0012S) was used. Working solutions and BSA standards were prepared according to the instructions. Appropriately diluted PDRN samples were added to 96-well plates, with working solution added to each well. The reaction was carried out in the dark, and absorbance was measured at 562 nm using a microplate reader. Protein concentration was calculated using the BSA standard curve.

[0033] 6. Dialysis desalting: The collected lower phase is placed into a dialysis bag (molecular weight cutoff of 10 kDa), and dialyzed with ultrapure water at 4°C under stirring conditions. The external solution is replaced every 2 hours, and the dialysis is repeated for 24 hours. The sample in the bag is then collected for later use.

[0034] 7. Vacuum freeze drying: The temperature is -25℃, the vacuum degree is less than 10 Pa, and the vacuum freeze drying is carried out for 24 to 48 hours until constant weight is obtained to obtain powdered PDRN.

[0035] 8. PDRN molecular weight: determined by conventional agarose gel chromatography-electrophoresis.

[0036] Example 1 Extraction of PDRN from salmon using a hydrophilic organic solvent / inorganic salt system includes the following steps: Take 23.7 g of salmon leucine enzymatic hydrolysate, add 9.2 g of ammonium sulfate, vortex for 2 min to mix the salt and hydrolysate evenly to form a turbid liquid; slowly add 11.0 g of anhydrous ethanol, stir evenly, and let stand at room temperature to form a three-phase salting-out extraction system. The upper phase is a solvent phase rich in other components, the middle phase is a solid phase rich in PDRN, and the lower phase is a salt phase rich in other components. Carefully aspirate the upper and lower liquid layers and collect the white solid in the middle layer. Wash the collected solid with ethanol (70 wt%) to remove salt, and freeze-dry under vacuum to obtain PDRN powder.

[0037] PDRN product test results: yield 10.3%, A260 / A280 1.89, A260 / A230 2.20, endotoxin content <0.03 EU / mg, protein content 0.3%, molecular weight approximately 200 bp.

[0038] Example 2 Extraction of PDRN from salmon using a polymer-based hydrophilic organic solvent / inorganic salt system includes the following steps: Take 25.9 g of salmon oozema enzymatic hydrolysate, add 9.2 g of dipotassium hydrogen phosphate, vortex for 2 min to mix the salt and hydrolysate evenly and form a turbid liquid; slowly add 8.8 g of polyethylene glycol (PEG-600), stir evenly, and let stand at room temperature to form a three-phase salting-out extraction system. The upper phase is a solvent phase rich in polymer, the middle phase is a solid phase rich in PDRN, and the lower phase is a salt phase rich in other components. Carefully aspirate the upper and lower liquid layers and collect the solid in the middle layer. Wash the collected solid with ethanol (70 wt%) to remove salt, and freeze-dry under vacuum to obtain PDRN powder.

[0039] PDRN product test results: yield 8.5%, purity A260 / A280 1.87, A260 / A230 2.09, endotoxin content <0.03 EU / mg, protein content 0.5%, molecular weight 800-1000 bp.

[0040] Example 3 Extraction of PDRN from salmon using a hydrophobic organic solvent / inorganic salt system includes the following steps: Take 18.5 g of salmon ooze enzymatic hydrolysate, add 11.0 g of ammonium sulfate, vortex for 2 min to mix the salt and hydrolysate evenly to form a turbid liquid; slowly add 14.5 g of isopropyl myristate, stir evenly, and let stand at room temperature to form a three-phase salting-out extraction system. The upper phase is a clear solvent phase, the middle phase is a turbid emulsion layer, and the lower phase is a clear salt phase rich in PDRN. Carefully aspirate the upper and middle emulsion layers and collect the lower salt phase. After dialysis to desalt the lower salt phase, add 10 volumes of anhydrous ethanol to precipitate, centrifuge to collect the precipitate, and freeze-dry under vacuum to obtain PDRN powder.

[0041] PDRN product test results: yield 14.9%, A260 / A280 ratio 1.87, A260 / A230 ratio 2.24, endotoxin content <0.03 EU / mg, protein content 0.5%, molecular weight approximately 200 bp.

[0042] Example 4 Extraction of PDRN from salmon using a hydrophilic organic solvent / organic salt system includes the following steps: Take 12.9 g of salmon lecithin enzymatic hydrolysate, add 4.6 g of trisodium citrate, vortex for 2 min to mix the salt and hydrolysate evenly to form a turbid liquid; slowly add 4.4 g of methanol, stir evenly, and let stand at room temperature to form a three-phase salting-out extraction system. The upper phase is a solvent phase rich in other components, the middle phase is a solid phase rich in PDRN, and the lower phase is a salt phase rich in other components. Carefully aspirate the upper and lower liquid layers and collect the white solid in the middle layer. Wash the collected solid with ethanol (70 wt%) to remove salt, and freeze-dry under vacuum to obtain PDRN powder.

[0043] PDRN product test results: yield 9.2%, purity A260 / A280 1.87, A260 / A230 2.18, endotoxin content <0.03 EU / mg, protein content 0.3%, molecular weight approximately 200 bp.

[0044] Example 5 Extraction of PDRN from salmon using a hydrophobic organic solvent / organic salt system includes the following steps: Take 9.2 g of salmon leucine enzymatic hydrolysate, add 3.9 g of ammonium citrate, vortex for 2 min to mix the salt and hydrolysate evenly to form a turbid liquid; slowly add 8.8 g of ethyl acetate, stir evenly, and let stand at room temperature to form a three-phase system: a clear solvent phase on top, a turbid emulsion layer in the middle, and a clear salt phase rich in PDRN on the bottom. Carefully remove the upper and middle emulsion layers and collect the bottom salt phase. After dialysis to desalt the bottom salt phase, add 10 volumes of anhydrous ethanol to precipitate, centrifuge to collect the precipitate, and freeze-dry under vacuum to obtain PDRN powder.

[0045] PDRN product test results: yield 9.6%, A260 / A280 ratio 1.91, A260 / A230 ratio 2.18, endotoxin content <0.03 EU / mg, protein content 0.2%, molecular weight approximately 200 bp.

[0046] Example 6 Effect of different salt concentrations on PDRN extraction efficiency: This embodiment uses Example 3 as an example. Based on Example 3 (ammonium sulfate / isopropyl myristate system), the effect of different salt concentrations on PDRN extraction efficiency was investigated, that is, the difference from Example 3 is the amount of ammonium sulfate added.

[0047] Table 1. Effect of different salt concentrations on PDRN extraction efficiency

[0048] As shown in Table 1, the PDRN yield gradually increases with increasing salt concentration. This is because as the salt concentration increases, the intermediate emulsion layer becomes thinner, allowing more PDRN to separate into the lower phase, while endotoxins and proteins remain in the intermediate emulsion layer. Table 1 shows that under salt concentrations of 5%–25%, the PDRN in the lower phase maintains high purity, and the contents of endotoxins and proteins remain at low levels. Further experimental results indicate that when the salt concentration is greater than 30%, a three-phase salting-out extraction system cannot be formed, and high-purity PDRN cannot be obtained. Similar results were obtained in experiments conducted in other examples; therefore, in the three-phase salting-out extraction system of this invention, the proportion of soluble salt is controlled below 30 wt%.

[0049] Example 7 The effect of different amounts of organic solvents (extractants) added on the PDRN extraction efficiency: This embodiment uses Example 3 as an example. Based on Example 3 (ammonium sulfate / isopropyl myristate system), the effect of different amounts of extractant added on the extraction effect was investigated, that is, the difference from Example 3 is the amount of isopropyl myristate added is different.

[0050] Table 2. Effect of different extractant dosages on PDRN extraction efficiency

[0051] As shown in Table 2, the PDRN yield initially increased and then decreased with increasing amounts of hydrophobic solvent. This is because insufficient hydrophobic solvent is insufficient to create an emulsion layer for separating endotoxin and PDRN, while excessive hydrophobic solvent (e.g., 70%) leads to a surge in the amount of intermediate emulsion layer, causing PDRN to be drawn into the intermediate emulsion layer and resulting in a decrease in yield. Similar results were obtained in experiments conducted in other examples. Therefore, in the three-phase salting-out extraction system of this invention, the solvent content is controlled below 67 wt%, preferably between 20 and 50 wt%.

[0052] The embodiments of the present invention are given for illustrative and descriptive purposes only, and are not intended to be exhaustive or to limit the invention to the forms disclosed. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described in order to better illustrate the principles and practical application of the invention, and to enable those skilled in the art to understand the invention and to design various embodiments with various modifications suitable for a particular purpose.

Claims

1. A method for salting out PDRN from salmon, characterized in that, Includes the following steps: S1: Add soluble salt and organic solvent to the salmon bladder enzymatic hydrolysate, mix well, let stand and separate phases to form a three-phase salting-out extraction system consisting of upper phase, middle phase and lower phase. S2: When the organic solvent is a hydrophilic organic solvent, collect the intermediate phase; when the organic solvent is a hydrophobic organic solvent, collect the lower phase. S3: The intermediate phase or lower phase collected in S2 is purified and dried to obtain PDRN powder.

2. The method according to claim 1, characterized in that, In S1, the salmon ooze enzymatic hydrolysate is obtained by enzymatically hydrolyzing salmon ooze tissue with a complex enzyme, wherein the complex enzyme includes a protease and a nuclease, wherein the protease is one or more of papain, flavor protease, and neutral protease, and the nuclease is one or more of restriction endonuclease Sau3AI and ribonuclease.

3. The method according to claim 1, characterized in that, In S1, in the three-phase salting-out extraction system, the mass percentage of the soluble salt is 3-30 wt% and the mass percentage of the organic solvent is 10-67 wt%, based on the total mass percentage of the three-phase salting-out extraction system.

4. The method according to claim 1, characterized in that, In S1, the soluble salt is an inorganic salt or an organic salt: the inorganic salt is selected from one or more of sodium bisulfate, aluminum sulfate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, potassium phosphate, sodium carbonate, potassium carbonate, trisodium phosphate, sodium silicate, ammonium sulfate, sodium sulfate, sodium chloride, disodium hydrogen phosphate, dipotassium hydrogen phosphate, and ammonium chloride; the organic salt is selected from one or more of disodium hydrogen citrate, sodium dihydrogen citrate, potassium hydrogen tartrate, sodium formate, potassium formate, sodium oxalate, potassium oxalate, sodium acetate, potassium acetate, trisodium citrate, potassium citrate, ammonium citrate, ammonium acetate, sodium succinate, and sodium tartrate.

5. The method according to claim 1, characterized in that, In S2, the hydrophilic organic solvent is selected from one or more of ethanol, methanol, n-propanol, isopropanol, acetone, 1,3-propanediol, 1,2-butanediol, phenoxyethanol, propylene glycol, glycerol, butanediol, pentanediol, dipropylene glycol, propylene glycol methyl ether, propylene glycol ethyl ether, propylene glycol butyl ether, dipropylene glycol methyl ether, and polyethylene glycol; the hydrophobic organic solvent is selected from ethyl acetate, triethyl citrate, tributyl citrate, trimethyl phosphate, tricresyl phosphate, triethyl phosphate, isopropyl palmitate, and isopropyl myristate. The following are some of the following: ethylhexyl palmitate, caprylic triglyceride, caprylic / capric triglyceride, C12-15 alcohol benzoate, cyclopentamethoxysiloxane, polydimethylsiloxane, phenyl polytrimethylsiloxane, n-octanol, n-heptane, n-hexane, isododecane, isohexadecane, mineral oil, liquid paraffin, hydrogenated polyisobutylene, squalane, jojoba oil, almond oil, grape seed oil, nut oil, sunflower seed oil, octyldodecyl alcohol, petroleum ether, toluene, benzene, dichloromethane, chloroform, and methyl tert-butyl ether.

6. The method according to claim 1, characterized in that, In S3, the intermediate phase collected in S2 is washed with ethanol and then freeze-dried under vacuum to obtain PDRN powder; the lower phase collected in S2 is subjected to dialysis desalting and ethanol precipitation, washed, and then freeze-dried under vacuum to obtain PDRN powder.

7. A salmon PDRN obtained by the method according to any one of claims 1 to 6, characterized in that, The salmon PDRN yield is greater than 5.0%, A260 / A280 is 1.8-2.0, A260 / A230 is ≥2.0, endotoxin content is <0.03 EU / mg, protein content is ≤0.5%, and molecular weight is 200-4000 bp.

8. The application of salmon PDRN obtained by the method according to any one of claims 1 to 6 in the preparation of medical aesthetic products and anti-inflammatory cosmetics.

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