Preparation method of deoxyribonucleotide zinc salt with skin barrier repair effect

Abstract

The present invention relates to a preparation method of a deoxyribonucleotide zinc salt with skin barrier repair efficacy, the method comprising taking fish testis and washing it with SSC buffer; adding SSC buffer to the obtained milt, breaking it into a homogenate, adding SSC buffer to homogenize it, centrifuging it to clarify the supernatant after homogenization; then digesting it in a water bath, adding ethanol to the supernatant obtained after digestion, and obtaining PDRN sodium salt; adding PDRN sodium aqueous solution to zinc salt solution, adjusting pH, stirring, standing, centrifuging, and drying to obtain PDRN zinc salt. While reducing the steps of preparing PDRN, the present invention controls the temperature during the extraction process, does not produce local high temperature, and avoids affecting the quality of PDRN. At the same time, in the zinc salt solution, by ion exchange, the method of adjusting the pH value is directly converted into the precipitation of PDRN zinc salt. The obtained PDRN zinc salt is applied to skin damage repair, realizing barrier repair, scar elimination, acne marks, anti-aging, wrinkle removal, sun protection and regulating the stability of the internal environment of the skin after skin damage.

Description

Technical Field

[0001] The invention relates to a method for preparing a deoxyribonucleotide zinc salt having skin barrier repairing effect, and belongs to the field of biotechnology. Background Art

[0002] PDRN (Polydeoxyribonucleotide) is a natural low molecular weight DNA molecule with a similarity of up to 98% to human DNA. Its natural source is mainly in the testes of fish, and it is composed of a mixture of 50-2000 base pairs combined into chains. PDRN naturally exists in the extracellular matrix (ECM) and is used by cells for metabolism. In addition to participating in cell growth, many preclinical and clinical studies have shown that PDRN can efficiently activate A2A adenosine receptors on the cell surface, promote the production of vascular endothelial growth factor (VEGF) and collagen, the migration of various cells, significantly enhance fibroblast proliferation, reduce inflammatory responses, and activate aging or damaged cells.

[0003] In fact, PDRN has been marketed for its therapeutic properties for various wound healing and inflammation-related diseases. PDRN is licensed as a pharmaceutical product in Europe and other places. It can accelerate the synthesis of DNA. As a natural protector and repair agent for the skin, it has sun protection, anti-inflammatory, cell regeneration, tissue repair, wound healing, scar reduction, pore repair, wrinkle removal, and UV damage resistance. At present, PDRN-related beauty products mainly come from Italy (Mastelli Placentex PDRN salmon water light needle), South Korea (Dermaline PDRN salmon microneedle introduction repair essence, HPCell Pulilan baby needle), etc., and are deeply loved by women.

[0004] Chinese patent document CN 107287186A discloses a method for preparing PDRN, but this method requires 20 hours to digest the cell lysate, separate and centrifuge 3-4 times, the steps are cumbersome and time-consuming; at the same time, ultrasonic decomposition of PDRN is required, and during the ultrasonic process, the PRDN chain will be cleaved, resulting in the Maillard reaction. Chinese patent document CN110747194A discloses a method for preparing PDRN, which requires lysing the testicular tissue at 90-100°C and using a DNA interrupter to break it, which is not easy to operate in large-scale industrial production and will cause the Maillard reaction. Therefore, the existing PDRN extraction technology generally has low extraction yield, cumbersome steps, and will produce local high temperature, affecting product quality.

[0005] Studies have found that zinc is an important factor in promoting the development of immune organs and is of great significance to the development of the human immune organ thymus, which is beneficial to the differentiation of T lymphocytes and promotes the normal physiological function of cellular immune function. At the same time, zinc also helps the healing of skin wounds and traumas. Zinc supplements were first used clinically to treat skin diseases. Zn ions participate in epithelial tissue differentiation, the transport and storage of metallothioneins, reduce ultraviolet-induced cell and gene damage, and improve the tolerance of skin fibroblasts to oxidative stress. Recent studies have confirmed that zinc ions can improve skin elasticity and reduce wrinkles.

[0006] At present, most of the publicly reported ones are polydeoxyribonucleotides. Due to technical defects, there are no reports on PDRN zinc salts, and their application in repairing skin damage has not been found. It is also rare to find raw materials that can quickly repair scars, acne marks, anti-aging, wrinkle removal, sun protection, and regulate the stability of the skin's internal environment to repair skin damage. Summary of the invention

[0007] To solve the above problems, the present invention provides a method for preparing a deoxyribonucleotide zinc salt with skin barrier repair efficacy. The present invention reduces the steps of preparing PDRN while controlling the temperature during the extraction process, does not produce local high temperature, and avoids affecting the quality of PDRN. At the same time, PDRN solid powder is directly converted into the precipitation of PDRN zinc salt by ion exchange in a zinc salt solution to adjust the pH value. The obtained PDRN zinc salt is applied to skin damage repair, and barrier repair, scar elimination, acne marks, anti-aging, wrinkle removal, sun protection and regulating the stability of the internal environment of the skin after skin damage are achieved.

[0008] Terminology Note:

[0009] PDRN zinc salt, also known as deoxyribonucleotide zinc salt.

[0010] In one aspect, the present invention provides a method for preparing a zinc salt of deoxyribonucleotide having skin barrier repairing efficacy, comprising the following steps:

[0011] (1) fish testicles were collected, pretreated, and then washed with SSC buffer;

[0012] (2) adding SSC buffer to the washed milt to obtain a homogenate, discarding the supernatant, and taking the precipitate;

[0013] (3) adding SSC buffer to the precipitate of step (2) for homogenization, centrifuging after homogenization, retaining the precipitate, and repeating the steps until the supernatant is clear;

[0014] (4) adding purified water to the precipitate obtained in step (3), homogenizing, then adding urea and proteinase K, adjusting the pH to 7.5-8, obtaining a mixture, and digesting in a water bath;

[0015] (5) After digestion, centrifuge, discard the precipitate, and keep the supernatant;

[0016] (6) adding ethanol to the supernatant obtained in step (5) and slowly stirring to precipitate the DNA;

[0017] (7) The DNA precipitate was washed with anhydrous ethanol 2-3 times, and the DNA precipitate was collected and dried to obtain PDRN sodium salt;

[0018] (8) Adding the PDRN sodium aqueous solution to the zinc salt solution, adjusting the pH, stirring, standing, centrifuging, and drying to obtain the PDRN zinc salt.

[0019] Preferably according to the present invention, in step (1), the fish testis is salmon testis.

[0020] Preferably, in step (1), the pretreatment is to wash and remove blood, drain the water, and then remove the fat tissue debris in the testis.

[0021] Preferably, according to the present invention, in step (1), the SSC buffer solution is a mixture of a NaCl solution and a sodium citrate solution, wherein the NaCl concentration in the mixture is 0.1-0.3 M, and the sodium citrate concentration is 0.005-0.05 M.

[0022] Preferably according to the present invention, in step (2), the amount of SSC buffer added is 1-4 times the volume of milt, and the SSC buffer is the same as the buffer in step (1).

[0023] Preferably, according to the present invention, in step (2), the crushing into a homogenate is to crush the tissue with a tissue crusher at a speed of 7000-9000 rpm / min for 2-6 minutes.

[0024] Preferably, in step (3), the amount of SSC buffer added is 0.5-3 times the volume of milt, and the SSC buffer is the same as the buffer in step (1).

[0025] Preferably, according to the present invention, in step (3), the homogenization is performed by using a tissue crusher at a speed of 7000-9000 rpm / min for 1-4 min, and the centrifugation is performed at a speed of 3000-5000 rpm / min for 10-20 min.

[0026] Preferably according to the present invention, in step (3), the homogenate centrifugation is repeated 2-3 times.

[0027] Preferably according to the present invention, in step (4), the amount of purified water added is 20-40 times the mass of the precipitate.

[0028] Preferably according to the present invention, in step (4), the urea concentration in the mixture is 8-12 mM, and the proteinase K concentration is 0.5-1.5% of the weight of the testis.

[0029] Preferably according to the present invention, in step (4), the digestion temperature is 50-60°C and the digestion time is 1.5-2.5h.

[0030] Preferably according to the present invention, in step (5), the centrifugation is performed at a speed of 7000-9000 rpm / min for 10-20 min.

[0031] Preferably according to the present invention, in step (6), the amount of ethanol added is 1-3 times the volume of the supernatant, and the concentration of ethanol is 70%-100%.

[0032] Preferably, according to the present invention, in step (8), the concentration of the PDRN sodium aqueous solution is 5-15wt%, the concentration of the zinc salt solution is 1wt%-15wt%, and the amount of the zinc salt solution is used to adjust the pH to 5.5-6.5.

[0033] Preferably according to the present invention, in step (8), the zinc salt is a small molecule inorganic or organic zinc salt.

[0034] Preferably according to the present invention, in step (8), the zinc salt is zinc chloride, zinc sulfate or zinc nitrate.

[0035] Preferably, according to the present invention, in step (8), the pH is adjusted to 5.5-6.5, stirred for 6 hours, and allowed to stand for 3 hours.

[0036] The technical effects of the present invention are:

[0037] 1. PDRN is a small molecule DNA from salmon sperm cells, which has a high similarity to human DNA sequence. It can act on adenosine A2A receptors, thereby promoting the production of VEGF, promoting cell regeneration, improving skin quality, and brightening skin tone.

[0038] 2. The raw materials PDRN and zinc ions used in the present invention are essential to the human body, safe and non-toxic to the human body, will not cause irritation, allergy and drug resistance, and have extremely high biosafety.

[0039] 3. The PDRN zinc of the present invention can also effectively repair damaged skin and restore the skin barrier function. At the same time, it can eliminate scars, acne marks, anti-aging, wrinkle removal, sun protection and regulate the stability of the internal environment of the skin. It is suitable for skin repair after medical beauty projects such as laser, micro plastic surgery and photorejuvenation on the face or other parts. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a bar graph showing the effects of different formulas on cell activity. DETAILED DESCRIPTION

[0041] The technical scheme of the present invention is described below in conjunction with specific embodiments and test examples. The following description is only for explaining the present invention, but does not limit the present invention in any way. The method of the present invention is conventional in the art unless otherwise specified. The reagents of the present invention are commercially available reagents unless otherwise specified.

[0042] Example 1

[0043] A method for preparing a deoxyribonucleotide zinc salt having skin barrier repairing effect, comprising the following steps:

[0044] (1) Cleaning salmon testicles

[0045] Selectively collect testes from mature salmon, take 100g of salmon testes, soak and thaw in purified water, wash to remove blood, drain the water, remove fat tissue and other debris in the testes, and wash with SSC buffer; SSC buffer is a mixture of NaCl solution and sodium citrate solution, the NaCl concentration in the mixture is 0.2M, and the sodium citrate concentration is 0.01M;

[0046] (2) Centrifugal cleaning

[0047] Add 2 volumes of SSC buffer to the milt, crush the tissue with a tissue crusher, homogenize at 8000rpm / min for 3min to make a homogenate, pour out the homogenate, centrifuge the homogenate at 4000rpm / min for 15min, discard the supernatant, retain the precipitate, add 1.5 volumes of SSC buffer to the precipitate, crush the tissue with a tissue crusher, homogenize at 8000rpm / min for 3min to make a homogenate, pour out the homogenate, centrifuge the slurry at 4000rpm / min for 15min, discard the supernatant, retain the precipitate, repeat the above steps 2 times until the centrifugal supernatant is clarified;

[0048] (3) Cell lysis

[0049] Add 3 L of purified water to the precipitate obtained by centrifugation in step (2), homogenize with a tissue grinder, add urea and proteinase K to make the urea concentration 10 mM and the amount of proteinase K 0.5% of the weight of the testis proteinase K, adjust the pH to 7.5, and digest in a water bath at 55°C for 1.5 h.

[0050] (4) Centrifugation

[0051] The lysate was centrifuged at 8000 rpm for 15 min and the supernatant was taken.

[0052] (5) Alcohol precipitation

[0053] Add pure ethanol to the supernatant obtained in step (4) at a volume ratio of 1:1, stir slowly, collect the precipitate, and wash twice with anhydrous ethanol;

[0054] (6) Drying

[0055] The precipitate obtained in step (5) is dried to obtain PDRN sodium salt.

[0056] (7) Zinc salt replacement

[0057] Dissolve 1 g of PDRN sodium salt in 500 mL of water, add 15% zinc sulfate solution, adjust the pH to 5.5, stir for 6 h, let stand for 3 h, centrifuge at 8000 rpm / min for 15 min, and dry to obtain PDRN zinc salt.

[0058] Example 2

[0059] A method for preparing a deoxyribonucleotide zinc salt having skin barrier repairing effect, comprising the following steps:

[0060] (1) Cleaning salmon testicles

[0061] Selectively collect testes from mature salmon, take 100g of salmon testes, soak and thaw in purified water, wash to remove blood, drain the water, remove fat tissue and other debris in the testes, and wash with SSC buffer, which is a mixture of NaCl solution and sodium citrate solution, with a NaCl concentration of 0.2M and a sodium citrate concentration of 0.01M in the mixture;

[0062] (2) Centrifugal cleaning

[0063] Add 2 volumes of SSC buffer to the milt, crush the tissue with a tissue crusher, homogenize at 8000rpm / min for 3 minutes to make a homogenate, pour out the homogenate, centrifuge the homogenate at 4000rpm / min for 15 minutes, discard the supernatant, and keep the precipitate. Add 1.5 volumes of SSC buffer to the precipitate, crush the tissue with a tissue crusher, homogenize at 8000rpm / min for 3 minutes to make a homogenate, pour out the homogenate, centrifuge the slurry at 4000rpm / min for 15 minutes, discard the supernatant, and keep the precipitate. Repeat the above steps twice until the centrifuged supernatant is clear.

[0064] (3) Cell lysis

[0065] Add 3 L of purified water to the precipitate obtained by centrifugation in step (2), homogenize with a tissue grinder, add 10 mM urea and 1.5% proteinase K by weight of the testis, adjust the pH to 7.5, and digest in a water bath at 60° C. for 2 h.

[0066] (4) Centrifugation

[0067] The lysate was centrifuged at 8000 rpm for 15 min and the supernatant was taken.

[0068] (5) Alcohol precipitation

[0069] Add pure ethanol to the supernatant obtained in step (4) at a volume ratio of 1:1, stir slowly, collect the precipitate, and wash twice with anhydrous ethanol.

[0070] (6) Drying

[0071] The precipitate obtained in step (5) is dried to obtain PDRN sodium salt.

[0072] (7) Zinc salt replacement

[0073] 1 g of the dried DNA sodium salt was dissolved in 500 mL of water, 15% zinc sulfate solution was added, the pH was adjusted to 5.5, stirred for 6 h, allowed to stand for 3 h, centrifuged at 8000 rpm / min for 15 min, and dried to obtain PDRN zinc salt.

[0074] Comparative Example 1

[0075] The same preparation method as described in Example 2, except that:

[0076] In step (7), 5% zinc sulfate was used to adjust the pH to 5.5, and the remaining steps (1) to (6) were the same as in Example 2.

[0077] Comparative Example 2

[0078] The same preparation method as described in Example 2, except that:

[0079] In step (7), 5% zinc sulfate was used to adjust the pH to 5.5, and the remaining steps (1) to (6) were the same as in Example 2.

[0080] Comparative Example 3

[0081] The same preparation method as described in Example 2, except that:

[0082] In step (7), 10% zinc sulfate solution was used to adjust the pH to 6. The remaining steps (1) to (6) were the same as those in Example 2.

[0083] Comparative Example 4

[0084] The same preparation method as described in Example 2, except that:

[0085] In step (7), 10% zinc sulfate solution was used to adjust the pH to 6. The remaining steps (1) to (6) were the same as those in Example 2.

[0086] Comparative Example 5

[0087] The same preparation method as described in Example 2, except that:

[0088] In step (7), 15% zinc sulfate solution was used to adjust the pH to 6. The remaining steps (1) to (6) were the same as those in Example 2.

[0089] Comparative Example 6

[0090] The same preparation method as described in Example 2, except that:

[0091] In step (7), 5% zinc sulfate solution was used to adjust the pH to 6.5. The remaining steps (1) to (6) were the same as those in Example 2.

[0092] Comparative Example 7

[0093] The same preparation method as described in Example 2, except that:

[0094] In step (7), 10% zinc sulfate solution was used to adjust the pH to 6.5. The remaining steps (1) to (6) were the same as those in Example 2.

[0095] Comparative Example 8

[0096] The same preparation method as described in Example 2, except that:

[0097] In step (7), 15% zinc sulfate solution was used to adjust the pH to 6.5. The remaining steps (1) to (6) were the same as those in Example 2.

[0098] Test Example 1: Purity Detection Calculation

[0099] After the product is dissolved in pure water, the absorbance at 260 nm and 280 nm is measured using a spectrophotometer. The purity of the polydeoxyribonucleotide in the product is calculated according to the following formula:

[0100] Purity = OD260 / OD280.

[0101] The purity of the polydeoxyribonucleotide finally obtained in Example 1 was calculated to be 1.72, and the purity of the polydeoxyribonucleotide finally obtained in Example 1 was calculated to be 1.92. Protein and PDRN cannot be fully separated, the purity of PDRN is reduced, and the protein content is increased.

[0102] Test Example 2: Determination of zinc content

[0103] In the present invention, the method for determining the zinc content of the obtained high molecular weight zinc hyaluronate is flame atomic absorption spectrometry. The specific determination method is as follows.

[0104] Standard curve preparation:

[0105] Accurately weigh 1 mL of zinc single element solution standard substance (1000 μg / mL) into a 100 mL volumetric flask, dilute to volume with 1% nitric acid to obtain a standard stock solution (10 μg / mL). Measure 0, 0.2, 0.4, 0.8, and 1.6 mL of the standard stock solution into 5 clean volumetric flasks, and dilute to volume with 1% nitric acid. The zinc standard solutions with zinc concentrations of 0, 0.2, 0.4, 0.8, and 1.6 μg / mL are obtained.

[0106] Test solution: Accurately weigh 50 mg of the test sample into a 100 mL volumetric flask, add 1 mL of nitric acid, heat in a water bath until completely dissolved, make up to volume with water, take 1 mL of the above solution into a 50 mL volumetric flask, and make up to volume with 1% nitric acid.

[0107] Determination: After adjusting to zero with blank solution, take appropriate amount of each solution and measure the absorbance by flame atomic absorption spectrometry with detection wavelength of 213.9nm. Perform linear regression on the concentration of zinc (X, μg / mL) with the absorbance of the test solution (Y), draw a standard curve, and calculate the zinc concentration in the test solution using the following formula.

[0108]

[0109] Where, Ci—Zinc concentration in the test solution, μg / mL;

[0110] W—test sample weight, mg;

[0111] h%—loss on drying of the test sample.

[0112] The effect is explained below with reference to test examples.

[0113] Table 1 Zinc content of each example sample (%)

[0114]

[0115]

[0116] Example 2 is carried out according to the established preparation parameters. In the preparation of the PDRN zinc of the present invention, by adjusting the preparation conditions such as pH and zinc salt solution concentration, the zinc content of the obtained PDRN zinc is greater than 6.0%.

[0117] Compared with Example 1 and Example 2, the higher the PDRN purity, the higher the zinc content. Compared with Comparative Examples 1 and 2, Example 2 uses a higher zinc ion concentration for replacement, and the zinc content of the obtained PDRN zinc increases accordingly.

[0118] Compared with Comparative Examples 3 and 6, Example 2 has different degrees of changes in pH conditions. The change in pH increases the instability of the product, resulting in a significant degradation of the molecular weight of the product, reducing the molecular weight of the final product, and causing the obtained zinc ion content to be less than 7.0%.

[0119] Experimental Example 3: Test for promoting fibroblast proliferation

[0120] In order to verify that the composition of the present invention has the effect of promoting the growth of fibroblasts, the stock solutions of Examples 1-2 and Comparative Examples 1-8 were subjected to an evaluation test on promoting the proliferation of fibroblasts.

[0121] The MTT cell proliferation evaluation method was adopted, CTCC-197-Hum fibroblasts were selected as experimental cells, 10% fetal bovine serum culture medium was selected as experimental culture medium, and the experiment set up sample group, positive control group, negative control group and zero adjustment group.

[0122] The specific experimental steps are as follows:

[0123] 1. Prepare fibroblasts into 2×10 4 The cell suspension of 100 cells / mL is ready for use;

[0124] 2. 2×10 4 The cells / mL cell suspension was inoculated into a 96-well cell culture plate (200 μL / well) and incubated in a 37°C, 5% CO2 incubator;

[0125] 3. When the cells in the well plate are attached and the plating rate reaches 30%, grouping and drug administration are carried out, with 5 replicate wells (200 μL / well) for each group:

[0126] (1) Sample group, a total of 10 groups, respectively added with cell culture medium containing the stock solution of Example 1, Example 2, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5, Comparative Example 6, Comparative Example 7, and Comparative Example 8, and the amount of the tested stock solution added was 1 g / mL;

[0127] (2) Positive control group, 1 group, added with cell culture medium containing TGF-β1 protein, the added amount of TGF-β1 protein was 100 ng / mL;

[0128] (3) Negative control pair, 1 group in total, added with cell culture medium;

[0129] (4) Zero adjustment group: 1 group, all of which are wells without inoculated cells and added with cell culture medium.

[0130] After the administration, the well plate was placed in a 37° C., 5% CO 2 incubator for 48 h.

[0131] 4. After incubation, discard the supernatant, add 20 μL of MTT solution to each well, and then continue incubation in a 37°C, 5% CO2 incubator for 4 hours. Discard the MTT solution, add 150 μL of DMSO solution to each well, shake the plate, place it on a microtiter plate photometer to measure the absorbance at 490 nm, and calculate the cell proliferation rate (RGR, %) according to the following formula.

[0132]

[0133] The cell proliferation rate after 48 h of culture is shown in Table 1

[0134] Table 1 Cell proliferation rate (%) changes:

[0135] Group Cell proliferation rate% Example 1 187.56±2.83 Example 2 240.18±3.61 Comparative Example 1 225.61±3.13 Comparative Example 2 212.29±3.01 Comparative Example 3 199.73±2.97 Comparative Example 4 191.34±2.42 Comparative Example 5 183.48±2.67 Comparative Example 6 174.74±2.76 Comparative Example 7 156.81±2.37 Comparative Example 8 149.76±2.59 Negative control group 108.25±2.13 Positive control group 297.64±3.42

[0136] Compared with the negative control group, the cell proliferation rate of the positive control group increased significantly, indicating that the fibroblast proliferation evaluation test system of the present invention is effective and can verify whether the stock solution of the present invention has the effect of promoting fibroblast proliferation.

[0137] Compared with the negative control group, Examples 1-2 and Comparative Examples 1-8 groups all had a significant effect of promoting fibroblast proliferation at 48h, and the higher the zinc content, the more obvious the cell proliferation effect. The PDRN zinc salt prepared in the embodiment of the present invention significantly promotes fibroblast proliferation.

[0138] Experimental Example 4: Oxidative stress test

[0139] Human epidermal cells (HaCaT) were cultured and divided into a normal group, a model group, and experimental groups 1-10 (respectively, Example 1, Example 2, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5, Comparative Example 6, Comparative Example 7, and Comparative Example 8, a total of 11 groups. Except for the normal group, 150 nM H2O2 was added to the other groups to cause oxidative stress damage, and then the prepared sample solution was added to a final concentration of 100 μg / mL, and the cells were cultured at 37°C and 5% CO2 for 48 hours. The activity of each group of cells was determined by the MTT method. The effects of different formulations on cell activity are shown in Figure 1 .

[0140] Depend on Figure 1As shown, oxidative stress can significantly reduce the activity of HaCaT cells. After different prescriptions are given, cell activity is significantly improved. It can be seen from the graph that the zinc content increases cell activity and reduces oxidative stress damage. A large number of studies have shown that PDRN has the effects of increasing cell growth factors, improving collagen / non-collagen synthesis, inducing cell regeneration, and promoting wound healing; Zn ions participate in epithelial tissue differentiation, reduce ultraviolet-induced cell and gene damage, and improve the tolerance of skin fibroblasts to oxidative stress response. This study shows that the prescription of Example 2 has the best effect in reducing oxidative stress damage and improving cell activity expression.

Claims

1. A method for preparing a deoxyribonucleotide zinc salt having skin barrier repairing effect, comprising the following steps: (1) Cleaning salmon testicles Selectively collect testes from mature salmon. Take 100 g of salmon testes, soak and thaw in purified water, wash to remove blood, drain the water, remove fat tissue and other debris in the testes, and wash with SSC buffer. SSC buffer is a mixture of NaCl solution and sodium citrate solution. The NaCl concentration in the mixture is 0.2 M and the sodium citrate concentration is 0.01 M. (2) Centrifugal cleaning Add 2 times the volume of SSC buffer to the milt, crush the tissue with a tissue crusher, homogenize at 8000rpm / min for 3min to make a homogenate, pour out the homogenate, centrifuge the homogenate at 4000rpm / min for 15min, discard the supernatant, and retain the precipitate; add 1.5 times the volume of SSC buffer to the precipitate, crush the tissue with a tissue crusher, homogenize at 8000rpm / min for 3min to make a homogenate, pour out the homogenate, centrifuge the slurry at 4000rpm / min for 15min, discard the supernatant, and retain the precipitate; repeat the above steps 2 times until the centrifugal supernatant is clarified; (3) Cell lysis Add 3 L of purified water to the precipitate obtained by centrifugation in step (2), homogenize with a tissue grinder, add 10 mM urea and 1.5% proteinase K by weight of the testis, adjust the pH to 7.5, and digest in a water bath at 60°C for 2 h; (4) Centrifugation The lysate was centrifuged at 8000 rpm for 15 min and the supernatant was taken; (5) Alcohol precipitation Add pure ethanol to the supernatant obtained in step (4) at a volume ratio of 1:1, stir slowly, collect the precipitate, and wash it twice with anhydrous ethanol; (6) Drying The precipitate obtained in step (5) is dried to obtain PDRN sodium salt; (7) Zinc salt replacement 1 g of the PDRN sodium salt was dissolved in 500 mL of water, 15% zinc sulfate solution was added, the pH was adjusted to 5.5, stirred for 6 h, allowed to stand for 3 h, centrifuged at 8000 rpm / min for 15 min, and dried to obtain PDRN zinc salt.

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