Medicinal PDRN and preparation method thereof

Abstract

The invention discloses medicinal PDRN and a preparation method thereof, and belongs to the technical field of biology. Comprising the following steps: (1) grinding salmon testis tissues in a pre-cooled grinding buffer solution to obtain homogenate; (2) inducing the ground homogenate by using Forskolin; the induction conditions are as follows: firstly adding Forskolin to 8-12 [mu] M, and standing for 1-3 hours at 4 DEG C; raising the temperature to 10 to 20 DEG C, supplementing Forskolin to 18 to 22 [mu] M and 2mM glutathione, and treating for 2 hours; and (3) extracting through SDS-sodium chloride to obtain the PDRN.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a pharmaceutical-grade PDRN and a preparation method thereof. Background Art

[0002] Polydeoxyribonucleotide (PDRN) is a bioactive substance composed of specific deoxyribonucleotide fragments. It has the effects of promoting tissue repair, anti-inflammation, regenerative medicine and skin barrier repair. It is widely used in medical cosmetology, wound healing and chronic disease treatment.

[0003] The current mainstream technology uses human chorionic gonadotropin (hCG) to induce sperm maturation in salmon testes, which carries significant risks: 1. Biosafety risks. hCG must be obtained from pregnant women's urine or through recombinant gene expression, which carries the risk of contamination with human viruses (such as HIV and HBV). This requires an additional viral inactivation step (S / D treatment or nanofiltration), resulting in a 15%-20% reduction in PDRN yield. The EU EMA guidelines (CPMP / BWP / 268 / 95) require TSE / BSE risk assessments for animal-derived products, placing the hCG method under additional regulatory scrutiny. 2. Hormone residual side effects. hCG has luteinizing hormone (LH) activity, and residual levels ≥0.1 IU / mg in the final product can cause endocrine disruption in humans. The FDA's requirements for hormonal residue limits in pharmaceutical excipients are becoming increasingly stringent (21 CFR 610.9). Traditional processes require the addition of immunoaffinity chromatography, which is costly. 3. Ethical and regulatory barriers.

[0004] Although forskolin, as a cAMP pathway activator, is theoretically feasible, its industrial application in fish sperm induction faces three major technical barriers: 1. Species-specific barriers: mammalian effective concentrations (10-50 μM) are significantly toxic to salmon testicular cells (apoptosis rate >40%); 2. Poor process compatibility: Traditional grinding causes cell membrane damage, and forskolin cannot effectively penetrate the target site; 3. Increased oxidative stress: Mechanical treatment produces reactive oxygen species (ROS), which offset the cAMP signaling effect.

[0005] Therefore, it is crucial to develop a hormone-free, highly safe, and low-cost PDRN large-scale preparation process, and to break through the technical and ethical bottlenecks of the traditional hCG program through the triple technological innovation of low-temperature grinding protection, sequential Forskolin induction, and antioxidant synergy. Summary of the Invention

[0006] The purpose of the present invention is to provide a pharmaceutical-grade polydeoxyribonucleotide (PDRN) and its preparation method to solve the problems existing in the above-mentioned prior art. The present invention provides a method for inducing in vitro maturation of salmon testicular cells based on the plant-derived adenylate cyclase activator Forskolin, and preparing ultra-low-risk pharmaceutical-grade polydeoxyribonucleotides (PDRN) by optimizing mechanical grinding and purification processes. This solution abandons hormone-like substances and meets the stringent requirements of regenerative medicine products for raw material safety.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] One of the technical solutions of the present invention is a method for preparing a pharmaceutical-grade PDRN, comprising the following steps:

[0009] (1) Grind salmon testis tissue in pre-cooled grinding buffer to obtain a homogenate;

[0010] (2) Forskolin was used to induce the ground homogenate; the induction conditions were as follows: first, forskolin was added to 8-12 μM, and the mixture was allowed to stand at 4°C for 1-3 hours; then the mixture was heated to 10-20°C, forskolin was added to 18-22 μM, and 2 mM glutathione was added, and the mixture was treated for 2 hours;

[0011] (3) PDRN was obtained by SDS-sodium chloride extraction.

[0012] The second technical solution of the present invention is the pharmaceutical grade PDRN prepared by the preparation method.

[0013] Based on the above technical solution, the present invention has the following technical effects:

[0014] (1) Safety breakthrough: Zero hormone residue: Forskolin was not detected by HPLC (<0.1ppm), and hCG residue was <0.01IU / ml (ELISA); No pathogen risk: Plant-derived inducers avoid the risk of hormone residues.

[0015] (2) The pharmaceutical-grade PDRN prepared by the present invention has a shortened production cycle. Forskolin induction requires hours, while the hCG method requires 24 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1HPLC liquid chromatogram of Forskolin content (standard: Forskolin standard, sample: Forskolin residue in PDRN obtained in Example 1, negative control: solvent control). DETAILED DESCRIPTION

[0018] Various exemplary embodiments of the present invention are now described in detail. Unless otherwise specified, the methods in the examples are conventional methods. Unless otherwise specified, the reagents used are conventional commercially available reagents or reagents prepared using conventional methods. This detailed description should not be considered a limitation of the present invention, but rather should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0020] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0021] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0022] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0023] Unless otherwise specified, the technical solutions described in the present invention are all conventional solutions in the field, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0024] The present invention provides a method for preparing a pharmaceutical-grade PDRN, comprising the following steps:

[0025] (1) Grind salmon testis tissue in pre-cooled grinding buffer to obtain a homogenate;

[0026] (2) Forskolin was used to induce the ground homogenate; the induction conditions were as follows: first, forskolin was added to 8-12 μM, and the mixture was allowed to stand at 4°C for 1-3 hours; then the mixture was heated to 10-20°C, forskolin was added to 18-22 μM, and 2 mM glutathione was added, and the mixture was treated for 2 hours;

[0027] (3) PDRN was obtained by SDS-sodium chloride extraction.

[0028] In some specific embodiments, the pre-cooled grinding buffer comprises the following components: 5% to 10% by mass of BSA, 0.5-2 mM EDTA, and 10 mM ascorbic acid, and the temperature is 4°C.

[0029] In some specific embodiments, the grinding conditions are: 5s on / 10s off, grinding time is 30min, and cell disruption rate is controlled to be ≤30%.

[0030] In some specific embodiments, the SDS-sodium chloride extraction method is:

[0031] (1) Add TE buffer containing 5% SDS to 300 g of induced cell homogenate, dilute to 1800 ml, and heat with magnetic stirring at 55°C for 1 hour to obtain a lysate;

[0032] (2) Take 1800 ml of lysate, add 50 ml of 4 M NaCl, and stir magnetically at 1000 rpm for 20 minutes to dissolve the DNA;

[0033] (3) Add 1 / 10 volume of 5% SDS solution to the solution, stir until white protein precipitates, centrifuge at 4500 rpm for 30 min, and collect the filtrate;

[0034] (4) 1 / 10 volume of 5% SDS solution was added to the filtrate, mixed and allowed to stand for 10 min, centrifuged at 4500 rpm for 30 min, and the filtrate was collected;

[0035] (5) Add 1.5 times the volume of anhydrous ethanol to the filtrate, stir and mix, let it stand at -20°C for 30 minutes, and collect the DNA;

[0036] (6) Add 75% ethanol by volume, shake vigorously, wash thoroughly, remove the ethanol, and squeeze out the liquid in the white precipitate;

[0037] (7) Repeat step (5) to obtain a white precipitate.

[0038] The embodiments of the present invention also provide pharmaceutical-grade PDRN prepared by the preparation method.

[0039] Example 1

[0040] 1. Raw material pretreatment:

[0041] 100 kg of immature testis of cultured Atlantic salmon was minced and immersed in pre-cooled grinding buffer (containing 5% BSA + 1 mM EDTA + 10 mM ascorbic acid, 4°C);

[0042] The cells were intermittently ground (5 s on / 10 s off) for 30 min using an industrial-grade high-speed homogenizer (12,000 rpm). Microscopic examination confirmed that the cell disruption rate was 80%.

[0043] 2Forskolin induction:

[0044] Stage I: Add Forskolin to 10 μM and incubate at 4°C for 2 hours;

[0045] Phase II: The temperature was raised to 15°C, and Forskolin was added to 20 μM and 2 mM glutathione for 2 hours.

[0046] 3PDRN extraction:

[0047] 3.1 Pour 300 g of induced cell homogenate into a beaker, add TE buffer containing 5% SDS to make the volume 1800 ml, place on a magnetic stirrer, heat and stir at 55°C for 1 hour to obtain lysate.

[0048] 3.2 Add 50 ml of 4 M NaCl to the 1800 ml lysis buffer from 3.1 and stir on a magnetic stirrer at 1000 rpm for 20 minutes. The DNA should be dissolved.

[0049] 3.3 Add 1 / 10 volume of 5% SDS solution to the solution and stir for 10 minutes to precipitate white protein. Divide the liquid into centrifuge tubes or centrifuge cups and centrifuge at 4500 rpm for 30 minutes. Pour the supernatant onto the sieve to filter out some large particles of tissue fragments and collect the filtrate.

[0050] 3.4 Add 1 / 10 volume of 5% SDS solution to the filtrate, mix well with a glass rod, and then let it stand for 10 minutes.

[0051] 3.5 Divide the liquid into centrifuge tubes or centrifuge cups, centrifuge at 4500 rpm for 30 minutes, pour the supernatant onto the sieve to filter out some large particles of tissue fragments, and collect the filtrate.

[0052] 3.6 Add 1.5 times the volume of anhydrous ethanol to the filtrate, stir to mix, and place in a -20°C refrigerator for 30 minutes. Use a glass rod to scoop out the DNA and place it in a 50ml centrifuge tube. Discard the liquid.

[0053] 3.7 Add 10 ml of 75% ethanol, then cover the tube and shake vigorously up and down to wash thoroughly. Then pour out the ethanol. You can use a glass rod to squeeze out the liquid in the white precipitate.

[0054] 3.8 Repeat step 3.6. Use a glass rod to squeeze out the liquid from the obtained white precipitate, then pour it onto absorbent paper and wash off the excess liquid to obtain a white powder, which is the prepared PDRN.

[0055] 3.9 Place the white precipitate in a beaker, add 100 ml of TE buffer, and stir at 50°C at 1000 rpm until the precipitate dissolves.

[0056] 3.10 Use Nano Drop to detect and record the DNA concentration, A260 / A280, and A260 / A230 values. The test results are shown in Table 1.

[0057] Comparative Example 1

[0058] 1. Raw material pretreatment:

[0059] 100 kg of immature testis of cultured Atlantic salmon was minced and immersed in pre-cooled grinding buffer (containing 5% BSA + 1 mM EDTA + 10 mM ascorbic acid, 4°C);

[0060] The cells were intermittently ground (5 s on / 10 s off) for 30 min using an industrial-grade high-speed homogenizer (12,000 rpm). Microscopic examination confirmed that the cell disruption rate was 80%.

[0061] 2hCG induction:

[0062] Add hCG to a final concentration of 1 ng / ml and incubate at 37°C for 24 hours;

[0063] 3PDRN extraction:

[0064] 3.1 Pour 300 g of induced cell homogenate into a beaker, add TE buffer containing 5% SDS to make the volume 1800 ml, place on a magnetic stirrer, heat and stir at 55°C for 1 hour to obtain lysate.

[0065] 3.2 Add 50 ml of 4 M NaCl to the 1800 ml lysis buffer from 3.1 and stir on a magnetic stirrer at 1000 rpm for 20 minutes. The DNA should be dissolved.

[0066] 3.3 Add 1 / 10 volume of 5% SDS solution to the solution and stir for 10 minutes to precipitate white protein. Divide the liquid into centrifuge tubes or centrifuge cups and centrifuge at 4500 rpm for 30 minutes. Pour the supernatant onto the sieve to filter out some large particles of tissue fragments and collect the filtrate.

[0067] 3.4 Add 1 / 10 volume of 5% SDS solution to the filtrate, mix well with a glass rod, and then let it stand for 10 minutes.

[0068] 3.5 Divide the liquid into centrifuge tubes or centrifuge cups, centrifuge at 4500 rpm for 30 minutes, pour the supernatant onto the sieve to filter out some large particles of tissue fragments, and collect the filtrate.

[0069] 3.6 Add 1.5 times the volume of anhydrous ethanol to the filtrate, stir to mix, and place in a -20°C refrigerator for 30 minutes. Use a glass rod to scoop out the DNA and place it in a 50ml centrifuge tube. Discard the liquid.

[0070] 3.7 Add 10 ml of 75% ethanol, then cover the tube and shake vigorously up and down to wash thoroughly. Then pour out the ethanol. You can use a glass rod to squeeze out the liquid in the white precipitate.

[0071] 3.8 Repeat step 3.6. Use a glass rod to squeeze out the liquid from the obtained white precipitate, then pour it onto absorbent paper and wash off the excess liquid to obtain a white powder, which is the prepared PDRN.

[0072] 3.9 Place the white precipitate in a beaker, add 100 ml of TE buffer, and stir at 50°C at 1000 rpm until the precipitate dissolves.

[0073] 3.10 Use Nano Drop to detect and record the DNA concentration, A260 / A280, and A260 / A230 values. The test results are shown in Table 1.

[0074] Effect Examples

[0075] The concentrations of PDRN prepared in Example 1 and Comparative Example 1 are shown in Tables 1 and 2.

[0076] Table 1 Concentration of PDRN extracted after Forskolin induction

[0077] Concentration (ug / ml) A260 / A280 A260 / A230 Dissolution volume (ml) Total amount (mg) 2295.08 1.82 1.93 2830 6495.08

[0078] Table 2 Concentration of PDRN extracted after hCG induction

[0079] Concentration (ug / ml) A260 / A280 A260 / A230 Dissolution volume (ml) Total amount (mg) 1908 1.82 1.93 2830 5399

[0080] As shown in Tables 1 and 2, the yield of PDRN after Forskolin induction was higher than that after hCG induction.

[0081] 2 The safety of the PDRN prepared in Example 1 and Comparative Example 1 was tested.

[0082] 2.1 Acute oral toxicity test

[0083] SPF-grade Sprague-Dawley (SD) rats, half male and half female, weighing 180-220 g, were used and provided by Beijing Weitong Lihua. The experiment was divided into three groups: Example 1 group (5000 mg / kg of the test substance was administered orally), Comparative Example 1 group (5000 mg / kg of the control substance was administered orally) and a blank control group (equal volume of normal saline), with 10 animals in each group (5 males and 5 females). After the animals were fasted for 12 hours, the test substance was administered orally with normal saline using a stainless steel gavage needle (16G), with a volume not exceeding 20 mL / kg. After administration, the mortality rate, weight change (accuracy ± 2 g) and neurotoxicity signs (such as disappearance of righting reflex, movement disorder, abnormal breathing) were closely observed at 0.5 h, 4 h, 24 h, 48 h, 72 h and on the 14th day. If there is no death and no symptoms of poisoning within 14 days, the LD is determined to be 50 >5000 mg / kg. Data were analyzed using Fisher's exact test to assess mortality and t-test to analyze differences in body weight changes.

[0084] 2.2 Subchronic toxicity test (90-day feeding test)

[0085] Select SD rats (40 male and female, body weight 60-80g) and cage raising (5 / cage), ambient temperature 22 ± 2 ℃, humidity 50 ± 10%, light and dark cycle 12h. Embodiment 1 group and comparative example 1 group respectively set low (300mg / kg), middle (1000mg / kg), high (3000mg / kg) three dosages, daily regular gavage, weekly dosage according to body weight adjustment. Weekly record body weight, food intake and water intake during the test, and at the 30th, 60th, 90th day, ALT, AST, creatinine and urea nitrogen (full-automatic biochemical analyzer) were taken by orbital blood sampling, and 24h urine test strip method was collected to detect protein, sugar and occult blood simultaneously. After 90 days, ether anesthesia was put to death, and liver, kidney, spleen, ovary / testis were weighed and calculated organ coefficients were obtained by dissection, and paraffin-embedded sections (4 μm) were sliced ​​after 10% formalin fixation, and HE staining was used to assess tissue pathology. Ovarian / testicular hyperplasia was semiquantitatively scored from 0 (normal) to 2 (structural abnormality), and liver / renal lesions were assessed for fatty degeneration, inflammatory cell infiltration, and other indicators. Data were compared between groups using one-way analysis of variance (ANOVA), with a significance threshold of p < 0.05.

[0086] 2.3 Sensitization test (guinea pig maximization test, GPMT)

[0087] White guinea pigs (half male and half female, weighing 300-500 g) were selected, with 10 in each group. During the induction stage, 0.1 mL of the test substance (Example 1 or Comparative Example 1, 10% w / v) and an equal volume of Freund's complete adjuvant (FCA) were injected intradermally after depilation of the neck and back. After 48 hours, the area was covered with gauze to prevent licking. After 7 days, 0.05 mL of the test substance was applied to the same part and blocked for 24 hours. The erythema and edema reactions were observed 24h, 48h, and 72h after stimulation, and scored from 0 (no reaction) to 3 (severe ulceration). The formula for calculating the sensitization rate is the number of positive reactions / total number of cases × 100%, and the threshold value is set at ≥30% for strong sensitization and <30% for weak sensitization. The entire test was evaluated blindly by two experimenters, and a Kappa value of >0.8 was considered to be consistent.

[0088] The experimental results are shown in Table 3.

[0089] Table 3 Comparison of PDRN testing items

[0090]

[0091]

[0092] As shown in Table 3, the results show that Example 1 has no obvious toxicity and sensitization, while the comparative example exhibits certain subchronic toxicity and sensitization.

[0093] 2.4 Determination of Forskolin Content

[0094] Sample preparation

[0095] Positive control: Prepare a Forskolin standard solution (e.g., 1 mg / mL in methanol), filter, and inject to ensure peak elution under the set conditions. Record the peak elution time of the standard.

[0096] Sample preparation: Take 1g of the extracted PDRN sample, add 20mL of methanol and ultrasonically extract for 30 minutes, centrifuge (10,000rpm, 10 minutes), pass the supernatant through a 0.22μm filter membrane, filter and inject, and record the peak time

[0097] Negative control: An aqueous solution known to contain no Forskolin was filtered and injected, and the peak time was recorded.

[0098] The chromatographic parameters are shown in Table 4.

[0099] Table 4

[0100] parameter Setting value Chromatographic columns Reversed-phase C18 column (e.g., Agilent ZORBAXSB-C18, 250 × 4.6 mm, 5 μm) Mobile phase Acetonitrile: water = 50:50 (isocratic elution) or gradient optimization flow rate 1.0mL / min Column temperature 30℃ Detection wavelength 220nm (near the maximum absorption wavelength of Forskolin) Injection volume 20 μL Runtime 15 minutes (to ensure that the peak time of Forskolin is clear)

[0101] HPLC results showed that the Forskolin content in Example PDRN was 0.05 ppm.

[0102] 2.5 Determination of hCG content

[0103] ① Before beginning the experiment using the human chorionic gonadotropin (hCG) enzyme-linked immunosorbent assay kit (Kelu (Wuhan) Biotechnology Co., Ltd., ELK1364), all reagents should be equilibrated to room temperature and prepared in advance. When diluting reagents or samples, mix thoroughly and avoid foaming. If the sample concentration is too high, dilute it with sample diluent to bring it within the detection range of the kit.

[0104] ② Add 100 μL of the standard or sample to be tested, being careful not to create bubbles. Place the sample at the bottom of the plate well, avoiding contact with the well walls. Gently shake to mix. Cover the plate or apply film and incubate at 37°C for 80 minutes. To ensure the validity of the experimental results, use a fresh standard solution for each experiment.

[0105] ③ Discard the liquid in the wells, spin dry, and wash the plate three times. Wash each well with 200 ml of washing solution, soak for 1-2 minutes, and spin off the liquid in the ELISA plate (or wash the plate using a plate washer). After the final wash, pat the plate dry on absorbent paper.

[0106] ④ Add 100 μL of biotin antibody working solution to each well (can be prepared 15 minutes in advance), cover the ELISA plate with film, and incubate at 37°C for 50 minutes.

[0107] ⑤ Discard the liquid in the wells and wash the plate three times. Wash each well with 200 μl of washing solution, soak for 1-2 minutes, and then remove the liquid from the plate (or wash the plate using a plate washer). After the final wash, pat the plate dry on absorbent paper.

[0108] ⑥ Add 100 μL of enzyme conjugate working solution to each well (can be prepared 15 minutes in advance) and incubate at 37°C for 50 minutes.

[0109] ⑦ Discard the liquid in the wells and wash the plate 5 times. Wash each well with 200ml of washing solution, soak for 1-2 minutes, and then discard the liquid in the plate (or wash the plate with a plate washer). After the last wash, pat the plate dry on absorbent paper.

[0110] ⑧Add 90mL of TMB color development substrate solution to each well and incubate at 37℃ in the dark for 20 minutes (shorten or extend the time as appropriate based on the actual color development situation, but not more than 30 minutes).

[0111] 9. Add 50 μL of stop solution to each well to terminate the reaction (the blue color will immediately turn yellow). The order of adding the stop solution should be as similar as possible to the order of adding the color developer. To ensure the accuracy of the experimental results, the stop solution should be added as soon as possible after the substrate reaction time expires.

[0112] ⑩ Immediately measure the optical density (OD) of each well using a microplate reader at a wavelength of 450nm. The instrument should be preheated before use and the detection program should be set.

[0113] A standard curve was drawn, and the hCG concentration of the example was 0.001 IU / mL, while that of the control was 0.5 IU / mL.

[0114] The comparison table of concentration and OD value is shown in Table 5.

[0115] Table 5

[0116]

[0117]

[0118] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the implementation methods of the present invention. A person skilled in the art will be able to make other variations or modifications based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing pharmaceutical-grade PDRN, characterized in that: The following steps are involved: (1) Grind salmon testis tissue in pre-cooled grinding buffer to obtain a homogenate; (2) Forskolin was used to induce the ground homogenate; the induction conditions were as follows: first, forskolin was added to 8-12 μM, and the mixture was allowed to stand at 4°C for 1-3 hours; then the mixture was heated to 10-20°C, forskolin was added to 18-22 μM, and 2 mM glutathione was added, and the mixture was treated for 2 hours; (3) PDRN was obtained by SDS-sodium chloride extraction.

2. The preparation method according to claim 1, characterized in that The pre-cooled grinding buffer comprises the following components: 5% to 10% by mass of BSA, 0.5-2 mM EDTA and 10 mM ascorbic acid, and the temperature is 4°C.

3. The preparation method according to claim 1, characterized in that The grinding conditions are: 5s on / 10s off, grinding time is 30min, and cell disruption rate is controlled to be ≤30%.

4. The preparation method according to claim 1, characterized in that The method of described SDS-sodium chloride extraction is: (1) Add TE buffer containing 5% SDS to 300 g of induced cell homogenate, dilute to 1800 ml, and heat with magnetic stirring at 55°C for 1 hour to obtain a lysate; (2) Take 1800 ml of lysate, add 50 ml of 4 M NaCl, and stir magnetically at 1000 rpm for 20 minutes to dissolve the DNA; (3) Add 1 / 10 volume of 5% SDS solution to the solution, stir until white protein precipitates, centrifuge at 4500 rpm for 30 min, and collect the filtrate; (4) 1 / 10 volume of 5% SDS solution was added to the filtrate, mixed and allowed to stand for 10 min, centrifuged at 4500 rpm for 30 min, and the filtrate was collected; (5) Add 1.5 times the volume of anhydrous ethanol to the filtrate, stir and mix, let it stand at -20°C for 30 minutes, and collect the DNA; (6) Add 75% ethanol by volume, shake vigorously, wash thoroughly, remove the ethanol, and squeeze out the liquid in the white precipitate; (7) Repeat step (5) to obtain a white precipitate.

5. the pharmaceutical grade PDRN prepared by the preparation method as described in any one of claims 1-4.

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