A plant-derived PDRN microcrystalline inclusion complex, its preparation method and application

Abstract

This invention relates to a plant-derived PDRN microcrystalline inclusion complex, its preparation method, and its application. The raw materials for preparing the microcrystalline inclusion complex, by weight, include 8-12 parts of plant-derived PDRN, 12-18 parts of arginine, 0.1-1 parts of pH adjuster, 0.1-2 parts of preservative, and 5-20 parts of stabilizer. The PDRN and arginine form a nanoscale microcrystalline inclusion complex through electrostatic self-assembly. The molecular weight of the plant-derived PDRN is 30kD-160kD. The stabilizer includes glycerol, trehalose, and sorbitol. The plant-derived PDRN microcrystalline inclusion complex provided by this invention solves the technical problems of high cost, high residue, low transdermal delivery efficiency, poor stability, low encapsulation rate, and limited raw material sources in existing PDRN extraction processes.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a plant-derived PDRN microcrystalline inclusion complex, its preparation method, and its application. Background Technology

[0002] Polydeoxyribonucleotides (PDRNs) are bioactive mixtures composed of deoxyribonucleotide chains, with molecular weights typically ranging from 50 to 1500 kDa. Studies have shown that PDRNs can provide DNA synthesis raw materials to damaged cells through technological means, enhance the proliferation and growth of fibroblasts, regulate the expression of vascular endothelial growth factor, promote angiogenesis, and improve microcirculation, demonstrating broad application prospects in skin regeneration and anti-aging.

[0003] However, the application of PDRN in cosmetics faces several technical bottlenecks: First, PDRN has a large molecular weight and strong negative charge, making it difficult to penetrate the stratum corneum of the skin, resulting in low transdermal delivery efficiency; second, PDRN requires various solvents during extraction and purification, causing residues, affecting quality, and increasing purification process costs; third, PDRN is easily degraded by nucleases in vivo, resulting in low bioavailability; and fourth, PDRN has poor stability and is easily inactivated in cosmetic formulations.

[0004] To address these issues, researchers have explored various transdermal delivery technologies. Liposome encapsulation can encapsulate PDRN within nanoliposomes, but the encapsulation rate is typically less than 70%, and liposomes suffer from poor stability and high cost. Existing technologies also disclose methods for forming nanoparticles through electrostatic self-assembly of nonamericarginine or polycationic polymers with nucleic acids (application number CN201480072258.0, patent title: Nanoscale carriers for delivery or co-delivery of chemotherapeutic agents, nucleic acids, and photosensitizers). However, the preparation process for nonamericarginine is complex and costly, and polycationic polymers pose biosafety risks. In recent years, a technical solution has emerged utilizing the self-assembly of PDRN with mussel protein to form supramolecular nanoparticles (application number CN202511801065.0, patent title: Polydeoxyribonucleotide self-assembled supramolecular nanoparticle composition and its preparation method, uses, and cosmetics). However, the source of mussel protein is limited, extraction costs are high, and it is not suitable for large-scale application. In addition, existing PDRN raw materials mainly rely on extraction from animal tissues such as fish testes, which poses problems such as unstable raw material supply, allergy risk, and animal ethics.

[0005] Therefore, developing a plant-derived PDRN delivery system that is stable in origin, has a mild process, low residue, low cost, high transdermal efficiency, and high encapsulation rate has significant industrial value and innovative significance. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a plant-derived PDRN microcrystalline inclusion complex, its preparation method, and its application, thereby solving the technical problems of high cost, high residue, low transdermal delivery efficiency, poor stability, low encapsulation rate, and limited raw material sources in existing PDRN extraction processes.

[0007] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a plant-derived PDRN microcrystalline inclusion complex, wherein the raw materials for preparing the microcrystalline inclusion complex include, by weight, 8-12 parts of plant-derived PDRN, 12-18 parts of arginine, 0.1-1 parts of pH adjuster, 0.1-2 parts of preservative and 5-20 parts of stabilizer; The PDRN and arginine form a nanoscale microcrystalline inclusion complex through electrostatic self-assembly. The molecular weight of the plant-derived PDRN is 30kD-160kD, for example, it can be 30kD, 40kD, 50kD, 60kD, 80kD, 100kD, 120kD, 130kD, 150kD or 160kD, etc. The stabilizers include glycerol, trehalose, and sorbitol.

[0008] The weight percentage of plant-derived PDRN in the raw materials for preparing the microcrystalline inclusion complex of the present invention can be 8 parts, 8.2 parts, 8.5 parts, 9 parts, 9.5 parts, 10 parts, 10.5 parts, 11 parts, 11.8 parts, or 12 parts.

[0009] The weight percentage of arginine can be 12, 12.5, 13, 14, 15, 15.5, 16, 17, 17.5 or 18 parts, etc.

[0010] The weight percentage of the pH adjuster can be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 part, etc.

[0011] The weight percentage of the preservative can be 0.1 parts, 0.3 parts, 0.5 parts, 0.8 parts, 1 part, 1.2 parts, 1.4 parts, 1.6 parts, 1.8 parts, or 2 parts, etc.

[0012] The weight percentage of the stabilizer can be 5, 6, 8, 10, 12, 14, 16, 18, 19, or 20 parts, etc.

[0013] The stabilizers include glycerin, trehalose, and sorbitol. The three work synergistically to significantly improve the stability of the microcrystalline inclusion complex, prevent the degradation of plant-derived PDRN, and also provide some moisturizing effect, thus synergizing with the repairing effects of PDRN.

[0014] Preferably, the plant comprises oat germ.

[0015] Preferably, the pH adjuster includes citric acid.

[0016] Preferably, the preservative includes pentylene glycol and / or hexanediol.

[0017] Preferably, the mass ratio of glycerol, trehalose and sorbitol is (2-6):(1-3):(1-2).

[0018] Where “2-6” can be 2.1, 2.3, 2.5, 2.8, 3, 3.2, 3.5, 3.8, 4, 4.2, 4.5, 4.8, 5, 5.2, 5.5 or 5.8, etc.; "1-3" can be 1.1, 1.3, 1.5, 1.7, 1.9, 2, 2.1, 2.3, 2.5, 2.7, or 2.9, etc.; “1-2” can be 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 or 1.9, etc.

[0019] Preferably, the particle size of the plant-derived PDRN microcrystalline inclusion complex is 200-800 nm (e.g., 200 nm, 300 nm, 400 nm, 500 nm, 550 nm, 600 nm, 700 nm, 750 nm, 780 nm, or 800 nm, etc.), the encapsulation efficiency is greater than 80% (e.g., 81%, 83%, 85%, 88%, or 90%, etc.), and the zeta potential is -10 to -30 mV (e.g., -10 mV, -12 mV, -15 mV, -18 mV, -20 mV, -22 mV, -25 mV, -27 mV, -29 mV, or -30 mV, etc.).

[0020] Preferably, the plant-derived PDRN is prepared using the following method, the method comprising: (1) Crush the plant raw materials, then add them to n-hexane and stir to defatt them, then filter and collect the residue; (2) Mix the filter residue with water, disodium EDTA and TE buffer, and homogenize to obtain a homogenate; (3) The homogenate was mixed with Tris buffer, proteinase K and sodium dodecyl sulfate, and then lysed in a water bath to obtain the lysate; (4) After centrifuging the lysate, take the supernatant, add sodium acetate solution and anhydrous ethanol to the supernatant, let it stand, centrifuge, collect the precipitate, and purify the precipitate to obtain the plant-derived PDRN.

[0021] Preferably, the pulverized material in step (1) is passed through a 40-80 mesh sieve. For example, the sieve mesh can be 40 mesh, 45 mesh, 50 mesh, 55 mesh, 60 mesh, 65 mesh, 70 mesh, 75 mesh, 78 mesh, or 80 mesh.

[0022] Preferably, the amount of n-hexane added in step (1) is 2-3 times the mass of the plant raw material, for example, it can be 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 2.6 times, 2.7 times or 2.9 times.

[0023] Preferably, the degreasing treatment in step (1) is performed 2-3 times, each time for 2-3 hours, for example, 2.0h, 2.1h, 2.2h, 2.3h, 2.4h, 2.5h, 2.6h, 2.7h, 2.9h or 3.0h.

[0024] Preferably, the mass ratio of filter residue, water, disodium EDTA and TE buffer in step (2) is 1:(5-20):(0.1-1):(0.1-0.5).

[0025] "5-20" can be 5, 7, 9, 10, 12, 14, 16, 18, 19 or 20, etc.

[0026] "0.1-1" can be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1, etc.

[0027] "0.1-0.5" can be 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.48 or 0.5, etc.

[0028] Preferably, the homogenization process in step (2) is carried out in a homogenizer.

[0029] Preferably, the homogenization time in step (2) is 3-10 min, for example, it can be 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 8.5 min, 9 min, 9.5 min or 10 min.

[0030] Preferably, the mass ratio of the homogenate in step (3) to Tris buffer, proteinase K and sodium dodecyl sulfate is 1:(0.1-1):(0.05-0.5):(0.2-1).

[0031] "0.1-1" can be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1, etc.

[0032] "0.05-0.5" can be 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45 or 0.5, etc.

[0033] "0.2-1" can be 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1, etc.

[0034] Preferably, the temperature of the water bath in step (3) is 50-60℃, for example, it can be 51℃, 52℃, 53℃, 54℃, 55℃, 56℃, 57℃, 59℃ or 60℃, etc.

[0035] Preferably, the pyrolysis treatment in step (3) is carried out under stirring, the stirring speed is 100-200 rpm (for example, it can be 100 rpm, 120 rpm, 140 rpm, 150 rpm, 160 rpm, 170 rpm, 180 rpm, 190 rpm, 195 rpm or 200 rpm, etc.), and the time is 2-4 h (for example, it can be 2.2 h, 2.4 h, 2.5 h, 2.6 h, 2.8 h, 3.0 h, 3.2 h or 3.8 h, etc.).

[0036] Preferably, the centrifugation speed in step (4) is 8000-10000 rpm (e.g., 8200 rpm, 8500 rpm, 8800 rpm, 9000 rpm, 9200 rpm, 9500 rpm, 9800 rpm or 9900 rpm, etc.), and the time is 12-20 min (e.g., 12.5 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min or 19.5 min, etc.).

[0037] Preferably, the sodium acetate solution in step (4) is a 3M sodium acetate solution.

[0038] Preferably, the volume ratio of the supernatant in step (4) to sodium acetate and anhydrous ethanol is 1:(0.05-0.5):(2-3).

[0039] The "0.05-0.5" can be 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45 or 0.5, etc.

[0040] “2-3” can be 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7 or 2.9, etc.

[0041] Preferably, the settling time in step (4) is 6-12 hours (e.g., 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 10.5 hours, 11 hours, 11.5 hours, 11.8 hours, or 12 hours), and the temperature is -10 to 20°C (e.g., -8°C, -5°C, 0°C, 5°C, 10°C, 12°C, 15°C, or 18°C).

[0042] Preferably, the purification step (4) is as follows: the precipitate is washed 2-3 times with 65-75% (e.g., 66%, 67%, 68%, 69%, 70%, 71%, 72%, or 74%) ethanol solution, then purified using an ion exchange chromatography column, the eluent is collected after elution, the precipitate is dialyzed to remove salts and then precipitated with alcohol, the precipitate is collected after centrifugation, and the precipitate is dried to obtain the plant-derived PDRN.

[0043] In a second aspect, the present invention provides a method for preparing plant-derived PDRN microcrystalline inclusion complexes as described in the first aspect, the method comprising: (a) Add plant-derived PDRN to a pH adjuster to adjust the pH to 5.5-6.5; Arginine was dissolved in deionized water to prepare an arginine solution; (b) Arginine solution was added dropwise to plant-derived PDRN, the pH was adjusted to 6-7 with a pH adjuster, and then some stabilizer was added. After stirring, the crude microcrystalline inclusion complex was obtained. (c) The crude microcrystalline inclusion complex is filtered and the permeate is collected. Preservatives and residual stabilizers are added to the permeate and stirred evenly to obtain the plant-derived PDRN microcrystalline inclusion complex.

[0044] Preferably, the pH adjustment in step (a) is to 5.5-6.5, for example, it can be 5.6, 5.8, 6, 6.2 or 6.4.

[0045] Preferably, the concentration of the arginine solution in step (a) is 0.05-0.5%, for example, it can be 0.06%, 0.08%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4% or 0.45%, etc.

[0046] Preferably, step (b) involves adjusting the pH to 6-7, for example, to 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, or 6.9.

[0047] Preferably, the stabilizer in step (b) is glycerol and sorbitol.

[0048] Preferably, the stirring speed in step (b) is 100-200 rpm (e.g., 120 rpm, 140 rpm, 150 rpm, 160 rpm, 170 rpm, 180 rpm, 190 rpm or 195 rpm, etc.), and the time is 30-60 min (e.g., 35 min, 40 min, 45 min, 50 min, 52 min, 55 min, 58 min or 59 min, etc.).

[0049] Preferably, the pore size of the membrane used for filtration in step (c) is 0.1-0.3 μm, for example, it can be 0.12 μm, 0.15 μm, 0.18 μm, 0.2 μm, 0.22 μm, 0.25 μm, 0.28 μm or 0.29 μm, etc.

[0050] Preferably, the remaining stabilizer in step (c) is trehalose.

[0051] Thirdly, the present invention provides the application of plant-derived PDRN microcrystalline inclusion complexes as described in the first aspect in the preparation of cosmetics. All the specific point values ​​within the above range can be selected, and will not be elaborated on here.

[0052] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention uses plant-derived PDRN, which solves the problems of allergenicity risk, animal ethics, unstable raw material supply and high cost of traditional animal-derived PDRN. It is in line with the trend of pure cosmetics and sustainable development. Moreover, the molecular weight of plant-derived PDRN (30-160kDa) is significantly lower than that of traditional salmon-derived PDRN (50-1500kDa), which is more conducive to transdermal absorption and enhances the efficacy of cosmetics.

[0053] (2) The raw materials used in this invention are highly safe. Arginine, which is listed in the "Catalogue of Used Cosmetic Raw Materials", is used as the encapsulation material and can be used directly as a cosmetic raw material. It is mild, safe and moisturizing, and avoids the compliance risks of spermidine and other raw materials not listed in the catalogue of used cosmetic raw materials.

[0054] (3) The preparation process of the PDRN microcrystalline inclusion complex provided by this invention is mild, requiring only a few steps of mixing, pH adjustment, and standing. It does not require high-pressure homogenization or chemical cross-linking agents, thus avoiding damage to the PDRN structure. By optimizing the mass ratio of arginine to PDRN and the pH conditions, the method yields a high encapsulation rate of PDRN microcrystalline inclusion complex, reaching over 80%, which is significantly better than existing technologies. The nanocomposite particles formed by electrostatic self-assembly have a particle size between 200-800 nm, which can significantly improve transdermal absorption capacity and greatly enhance product efficacy. At the same time, the process cost is low, far lower than that of nonamericarginine or liposome materials. Attached Figure Description

[0055] Figure 1 To prepare ultraviolet gel imaging of the sample obtained in Example 1; Figure 2 To prepare the ultraviolet gel imaging of the sample prepared in Example 2. Detailed Implementation

[0056] To further illustrate the technical means and effects of the present invention, the following describes the technical solution of the present invention in conjunction with preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0057] The following detailed description of the features and advantages of the present invention is sufficient to enable those skilled in the art to understand the technical content of the present invention and to implement it accordingly. Furthermore, based on this specification, claims, and drawings, those skilled in the art can easily understand the related objectives and advantages of the present invention.

[0058] The terminology and expressions used herein are for descriptive purposes only, and the invention should not be limited to these terms and expressions. The use of these terms and expressions does not imply the exclusion of any illustrative and descriptive equivalents (or parts thereof), and it should be recognized that various modifications that may exist should also be included within the scope of the claims. Other modifications, variations, and substitutions may also exist. Accordingly, the claims should be considered to cover all such equivalents.

[0059] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0060] The weight parts mentioned in the following examples and comparative examples are based on the actual active ingredients contained in commercially available raw materials. These commercially available raw materials may also selectively contain any one or a combination of at least two of the following: solvents, fillers, diluents, stabilizers, pH adjusters, antibacterial agents, antioxidants, or impurities within permissible limits.

[0061] The sources of some of the raw materials used in the embodiments and comparative examples of this invention are shown in Table 1 below: Table 1 All other raw materials can be used as long as they are purchased from authorized distributors.

[0062] Preparation Example 1 This preparation example provides an oat germ PDRN, the specific preparation method of which is as follows: (1) Raw material pretreatment: Take oat germ, wash, dry, crush and pass through a 60-mesh sieve to obtain oat germ powder; (2) Degreasing treatment: Add the crushed oat germ powder to 2.5 times the mass of n-hexane, stir at 25°C for 2.5 hours to degrease, filter, collect the filter residue, repeat the degreasing twice and then air dry; (3) Cell wall breaking treatment: Add pure water, disodium EDTA and TE buffer to a homogenizer and stir evenly. Then add the dried filter residue. The mass ratio of filter residue, water, disodium EDTA and TE buffer is 1:12:0.5:0.3. Start high speed for homogenization. Homogenize for 2 minutes each time and repeat twice to obtain homogenate. (4) Cyclic extraction: Add the homogenate to the extraction pot, add Tris buffer, proteinase K and SDS (sodium dodecyl sulfate). The mass ratio of the homogenate, Tris buffer, proteinase K and SDS is 1:0.5:0.25:0.6. Under the conditions of water bath at 55°C, lyse for 3 hours, stirring at 150 rpm, with intermittent stirring (stop for 10 minutes after every 30 minutes of stirring). (5) Centrifugation to remove impurities: After cooling the lysate to 25°C, centrifuge it in a centrifuge at 9000 rpm for 15 minutes and take the supernatant. (6) Precipitation and purification: Add 3M sodium acetate solution and anhydrous ethanol to the supernatant. The volume ratio of the supernatant, sodium acetate and anhydrous ethanol is 1:0.3:2.5. Let it stand at 5°C for 9 hours to precipitate. Centrifuge to collect the precipitate, wash it twice with 70% ethanol, and dry it. Then purify it using an ion exchange chromatography column. Elute with buffer containing 0.3M sodium chloride at a volume of 5 BV. Collect the eluent, dialyze the eluent to remove salt, and precipitate it again with 70% ethanol after desalting. Centrifuge and dry the precipitate at 5°C to obtain PDRN powder, which is the oat germ PDRN product.

[0063] Preparation Example 2 This preparation example provides an oat germ PDRN, the specific preparation method of which is as follows: (1) Raw material pretreatment: Take oat germ, wash, dry, crush and pass through a 40-mesh sieve to obtain oat germ powder; (2) Degreasing treatment: Add the crushed oat germ powder to 2 times the mass of n-hexane, stir at 26°C for 3 hours to degrease, filter, collect the filter residue, repeat the degreasing twice and then air dry; (3) Cell wall breaking treatment: Add pure water, disodium EDTA and TE buffer to a homogenizer and stir evenly. Then add the dried filter residue. The mass ratio of filter residue, water, disodium EDTA and TE buffer is 1:5:1:0.1. Start high speed for homogenization. Homogenize for 1 minute each time and repeat 3 times. (4) Lysis and extraction: Add the homogenate to the extraction pot, add Tris buffer, proteinase K and SDS. The mass ratio of the homogenate, Tris buffer, proteinase K and SDS is 1:0.1:0.5:1. Under the conditions of a water bath at 50°C, lyse for 4 hours with a stirring speed of 100 rpm and intermittent stirring (stop for 10 minutes after stirring for 20 minutes). (5) Centrifugation to remove impurities: After the lysate is cooled to 25°C, it is placed in a centrifuge for centrifugation at 8000 rpm for 15 minutes, and the supernatant is taken. (6) Precipitation and purification: Add 3M sodium acetate solution and anhydrous ethanol to the supernatant. The volume ratio of the supernatant, sodium acetate and anhydrous ethanol is 1:0.5:3. Let it stand at -10℃ for 12 hours to precipitate. Centrifuge to collect the precipitate, wash it twice with 65% ethanol, and dry it. Then purify it using an ion exchange chromatography column. Elute with buffer containing 0.1M sodium chloride. The elution volume is 6 BV. Collect the eluent. Dialyze the eluent to remove salt. After desalting, precipitate it again with 65% ethanol. Centrifuge and dry the precipitate at 8℃ to obtain PDRN powder, which is the oat germ PDRN product.

[0064] Preparation Example 3 This preparation example provides an oat germ PDRN, the specific preparation method of which is as follows: (1) Raw material pretreatment: Take oat germ, wash, dry, crush and pass through an 80-mesh sieve to obtain oat germ powder; (2) Degreasing treatment: Add the crushed oat germ powder to 3 times the volume of n-hexane, stir at 26°C for 2 hours to degrease, filter, collect the filter residue, repeat the degreasing 3 times and then dry. (3) Cell wall breaking treatment: Add pure water, disodium EDTA and TE buffer to a homogenizer and stir evenly. Then add the dried filter residue. The mass ratio of filter residue, water, disodium EDTA and TE buffer is 1:20:0.1:0.5. Start high speed for homogenization. Each homogenization takes 3 minutes and is repeated once. (4) Lysis and extraction: Add the homogenate to the extraction pot, add Tris buffer, proteinase K and SDS. The mass ratio of the homogenate, Tris buffer, proteinase K and SDS is 1:1:0.05:0.2. Under the conditions of water bath at 60℃, lyse for 2 hours, stir at 200 rpm, and stir intermittently (stop for 10 minutes after stirring for 40 minutes). (5) Centrifugation to remove impurities: After the lysate is cooled to 26°C, it is placed in a centrifuge for centrifugation at 10,000 rpm for 10 minutes, and the supernatant is taken. (6) Precipitation and purification: Add 3M sodium acetate solution and anhydrous ethanol to the supernatant. The volume ratio of the supernatant, sodium acetate and anhydrous ethanol is 1:0.1:2. Let it stand at 20°C for 6 hours to precipitate. Centrifuge to collect the precipitate, wash it 3 times with 75% ethanol, and dry it. Then purify it using an ion exchange chromatography column. Elute with buffer containing 0.5M sodium chloride. The elution volume is 5 BV. Collect the eluent. Dialyze the eluent to remove salt. After desalting, precipitate it again with 75% ethanol. Centrifuge and dry the precipitate at 3°C ​​to obtain PDRN powder, which is the oat germ PDRN product.

[0065] Preparation Example 4 This preparation example provides an oat germ PDRN, the only difference between which is the preparation method and that of Preparation Example 1 is the absence of the defatting step (2). The oat germ powder is directly subjected to the cell wall breaking treatment in step (3). The remaining steps and parameters are the same as those of Preparation Example 1.

[0066] Preparation Example 5 This preparation example provides an oat germ PDRN, the only difference between its preparation method and that of Preparation Example 1 is the absence of the cell wall breaking treatment step (3). The filter residue from step (2) is mixed with water (mass ratio of 1:12) and then directly subjected to the pyrolysis treatment in step (4). The remaining steps and parameters are the same as those in Preparation Example 1.

[0067] Preparation Example 6 This preparation example provides an oat germ PDRN, the only difference between its preparation method and that of Preparation Example 1 is the absence of the pyrolysis treatment step (4), and the homogenate from step (3) is directly centrifuged to remove impurities in step (5), while the remaining steps and parameters are the same as those of Preparation Example 1.

[0068] Preparation Example 7 This preparation example provides an oat germ PDRN, the preparation method of which differs from that of Preparation Example 1 only in that the Tris buffer is not used in the lysis treatment step (4), and the mass ratio of homogenate, proteinase K and SDS is kept unchanged. The remaining steps and parameters are the same as those of Preparation Example 1.

[0069] Preparation Example 8 This preparation example provides an oat germ PDRN, the only difference between which is the preparation method and that of Preparation Example 1 is that proteinase K is not used in the lysis treatment step (4), and the mass ratio of homogenate, Tris buffer and SDS is kept unchanged. The remaining steps and parameters are the same as those of Preparation Example 1.

[0070] Preparation Example 9 This preparation example provides an oat germ PDRN, the preparation method of which differs from that of Preparation Example 1 only in that SDS is not used in the lysis treatment step (4), and the mass ratio of homogenate, Tris buffer and proteinase K is kept unchanged. The remaining steps and parameters are the same as those of Preparation Example 1.

[0071] Example 1 This embodiment provides a plant-derived PDRN microcrystalline inclusion complex. The raw materials for preparing the PDRN microcrystalline inclusion complex include, by weight, 10 parts of PDRN from Preparation Example 1, 15 parts of arginine, 0.8 parts of citric acid, 1 part of preservative, and 18 parts of stabilizer. The stabilizer is glycerol, trehalose and sorbitol in a mass ratio of 4:3:2; The preservative is pentanediol and hexanediol in a mass ratio of 4:3; Its preparation method is as follows: (1) Take PDRN and adjust the pH to 6.0 with citric acid; (2) Dissolve arginine in deionized water to prepare an arginine solution with a concentration of 0.275%; (3) Under stirring conditions, arginine solution was slowly added dropwise to PDRN solution to obtain a mixture, and the addition time was 20 minutes; (4) After the addition is complete, adjust the pH of the mixture to 6.5 with citric acid, add glycerol and sorbitol, and stir at 24°C for 45 minutes to allow PDRN and arginine to fully form crude microcrystalline inclusion complex through electrostatic self-assembly. (5) The crude microcrystalline inclusion complex was filtered through a 0.2 μm filter membrane, and then preservatives and trehalose were added and stirred evenly to obtain the plant-derived PDRN microcrystalline inclusion complex.

[0072] Example 2 This embodiment provides a plant-derived PDRN microcrystalline inclusion complex. The raw materials for preparing the PDRN microcrystalline inclusion complex include, by weight, 10 parts of PDRN from Preparation Example 2, 12 parts of arginine, 0.6 parts of citric acid, 1.2 parts of preservative, and 15 parts of stabilizer. The stabilizer is glycerol, trehalose and sorbitol in a mass ratio of 2:3:1; The preservative is pentanediol and hexanediol in a mass ratio of 1:1; Its preparation method is as follows: (1) Take oat germ PDRN and adjust the pH to 5.5 with citric acid; (2) Dissolve arginine in deionized water to prepare an arginine solution with a concentration of 0.5%; (3) Under stirring conditions, arginine solution was slowly added dropwise to PDRN solution to obtain a mixture, and the addition time was 30 minutes; (4) After the addition is complete, adjust the pH of the mixture to 6.0 with citric acid, add glycerol and sorbitol, and stir at 25°C for 30 minutes to allow PDRN and arginine to fully form crude microcrystalline inclusion complex through electrostatic self-assembly. (5) The crude microcrystalline inclusion complex was filtered through a 0.1 μm filter membrane, and then preservatives and trehalose were added and stirred evenly to obtain the plant-derived PDRN microcrystalline inclusion complex.

[0073] Example 3 This embodiment provides a plant-derived PDRN microcrystalline inclusion complex. The raw materials for preparing the PDRN microcrystalline inclusion complex include, by weight, 10 parts of PDRN from Preparation Example 3, 18 parts of arginine, 0.9 parts of citric acid, 0.8 parts of preservative, and 20 parts of stabilizer. The stabilizer is glycerol, trehalose and sorbitol in a mass ratio of 6:1:2; The preservative is pentanediol and hexanediol in a mass ratio of 1:2; Its preparation method is as follows: (1) Take PDRN and adjust the pH to 6.5 with citric acid; (2) Dissolve arginine in deionized water to prepare an arginine solution with a concentration of 0.1%; (3) Under stirring conditions, arginine solution was slowly added dropwise to PDRN solution to obtain a mixture, and the addition time was 15 minutes; (4) After the addition is complete, adjust the pH of the mixture to 7.0 with citric acid, add glycerol and sorbitol, and continue stirring at 25°C for 60 minutes to allow PDRN and arginine to fully form crude microcrystalline inclusion complex through electrostatic self-assembly. (5) The obtained microcrystalline inclusion complex was filtered through a 0.3 μm filter membrane, and then preservatives and trehalose were added and stirred evenly to obtain the plant-derived PDRN microcrystalline inclusion complex.

[0074] Examples 4-9 The embodiments provide six plant-derived PDRN microcrystalline inclusion complexes. The only difference between the PDRN microcrystalline inclusion complexes and those of Example 1 is that the PDRN in Preparation Example 1 is replaced with the PDRN in Preparation Examples 4-9 in equal amounts. The other components and amounts remain unchanged, and the preparation method is the same as in Example 1.

[0075] Example 10 This embodiment provides a plant-derived PDRN microcrystalline inclusion complex. The only difference between the PDRN microcrystalline inclusion complex and that in Example 1 is that the pH is adjusted to 8.5 in step (4) of the preparation method, and the amount of citric acid is reduced accordingly. The other components and their amounts remain unchanged. The preparation method is the same as in Example 1.

[0076] Example 11 This embodiment provides a plant-derived PDRN microcrystalline inclusion complex. The only difference between the PDRN microcrystalline inclusion complex and that in Example 1 is that the pH is adjusted to 4.5 in step (4) of the preparation method, and the amount of citric acid is increased accordingly. The other components and their amounts remain unchanged. The preparation method is the same as in Example 1.

[0077] Comparative Example 1 This comparative example provides a plant-derived PDRN microcrystalline inclusion complex. The only difference between the PDRN microcrystalline inclusion complex and Example 1 is that arginine is replaced with an equal amount of nonapolyarginine, while the other components and amounts remain unchanged. The preparation method is the same as in Example 1.

[0078] Comparative Example 2 This comparative example provides a plant-derived PDRN microcrystalline inclusion complex. The only difference between the PDRN microcrystalline inclusion complex and Example 1 is that the amount of arginine added is 22 parts, while the other components and amounts remain unchanged. The preparation method is the same as in Example 1.

[0079] Comparative Example 3 This comparative example provides a plant-derived PDRN microcrystalline inclusion complex. The only difference between the PDRN microcrystalline inclusion complex and Example 1 is that the amount of arginine added is 8 parts, while the other components and amounts remain unchanged. The preparation method is the same as in Example 1.

[0080] Test Example 1: Molecular Weight and Purity Test (1) The molecular weight was determined using agarose gel electrophoresis. The specific method is as follows: Prepare a 1.2%-1.5% agarose gel, add an appropriate amount of nucleic acid dye to the agarose gel, mix well, pour into a gel casting mold, and cool at 25°C for at least 30 minutes to obtain a solidified agarose gel; Take an appropriate amount of solidified agarose gel and place it in the electrophoresis tank. Add an appropriate amount of TAE electrophoresis buffer until the gel surface is submerged by 2-3 mm to obtain the sample loading mixture. Samples from Preparation Examples 1-9, commercially available salmon-derived PDRN, and the loading buffer were mixed at a volume ratio of (3-7):1 and then loaded. A PDRN standard (2000 bp molecular weight) was added as a reference. The electrophoresis voltage was set to 80-120V, and the electrophoresis time was 30-60 minutes. After electrophoresis, the samples were observed under a UV gel imaging system, and then the molecular weight was determined according to "T-SHRH079-2026 Cosmetic Raw Materials Polydeoxyribonucleic Acid (PDRN)" (results were rounded to integers).

[0081] (2) Purity test The samples from Preparation Examples 1-9 were prepared into 1% solutions with water, and then the purity was tested using the method GB / T34796-2017 "Determination of Concentration and Purity of Nucleic Acids in Aqueous Solutions - Ultraviolet Spectrophotometry".

[0082] The results are shown in Table 2.

[0083] Table 2 As shown in Table 2, the molecular weight of PDRN obtained using the specific raw materials and methods of this invention is between 30-160 kDa, which is significantly lower than that of commercially available animal-derived PDRN.

[0084] As can be seen from preparation examples 4-6, the degreasing, cell wall disruption and pyrolysis treatments in the preparation method provided by the present invention are combined to obtain PDRN with higher purity.

[0085] As can be seen from preparation examples 7-9, the Tris buffer, proteinase K and SDS used in the lysis step also affect the purity of PDRN.

[0086] Test Example 2: Particle Size Distribution and Zeta Potential Test The mean particle size, polydispersity index (PDI), and zeta potential of the plant-derived PDRN microcrystalline inclusion complexes of Examples 1-11 and Comparative Examples 1-3 were measured using a dynamic light scattering (DLS) instrument.

[0087] The results are shown in Table 3.

[0088] Table 3 Test Example 3: Encapsulation Ratio Test Test subjects: PDRN microcrystalline inclusion complexes of Examples 1-3, 10-11 and Comparative Examples 1-3.

[0089] Test method: The PDRN microcrystalline inclusion complex was completely lysed with a lysis buffer to release the internal PDRN. The PDRN in the lysis buffer was quantified using a micro spectrophotometer to calculate the amount of PDRN loaded into the microcrystalline inclusion complex. The encapsulation efficiency was calculated using the following formula: Encapsulation efficiency = (Amount of PDRN loaded into the microcrystalline inclusion complex / Total amount of PDRN initially added) x 100%.

[0090] The results are shown in Table 4.

[0091] Table 4 Test Example 4: In vitro transdermal absorption test Test subjects: PDRN microcrystalline inclusion complexes of Examples 1-11 and Comparative Examples 1-3.

[0092] Test Method: Pigskin was used to simulate human epidermal skin. The pigskin was cut into 5cm x 5cm pieces and then soaked in physiological saline for 30 minutes to serve as a transdermal barrier. A Franz diffusion cell was used as the static diffusion cell. The test sample was used as the transdermal release solution, and 30% physiological saline was used as the transdermal absorption solution. The transdermal absorption test was conducted at a constant temperature of 35°C and a constant flow rate of 800 rpm. After 24 hours, 5 mL of the permeate was collected from each sampling point using a clean syringe, and then 5 mL of 30% physiological saline was added using the same syringe. The PDRN content in the sample was determined using ultraviolet spectrophotometry. PDRN transmittance = (Amount of PDRN in the receiving cell / Amount of PDRN in the sample) × 100%.

[0093] The results are shown in Table 5.

[0094] Table 5 As shown in Tables 3-5, the PDRN microcrystalline inclusion complex prepared by a specific method and using specific raw materials in this invention not only has low process cost and high encapsulation rate, but also has nanocomposite particles with a diameter between 200-800 nm formed by electrostatic self-assembly, which have the potential for transdermal absorption and greatly improve product efficacy.

[0095] Test Example 5: Security Test The specific method was as follows: Forty women aged 18-35 years with no history of allergies were selected. Each subject used samples prepared according to Examples 1-3. After cleaning the subject's arm, approximately 0.020 mL-0.025 mL of test sample was added to the patch. The patch containing the sample was then applied to the selected location on the arm using non-irritating adhesive tape. After application, the patch was gently pressed with a finger to ensure even adhesion to the skin, and left for 48 hours. During these 48 hours, the patch area was kept dry, and subjects were advised to avoid strenuous exercise, scratching, and prolonged sun exposure. After 48 hours, the patch was removed and marked. The results were assessed under sufficient light after 30 minutes, once the pressure marks disappeared.

[0096] The grading criteria for adverse skin reactions are shown in Table 6: Table 6 The results are shown in Table 7: Table 7 As shown in Table 7, no adverse skin reactions were observed in the volunteers after the spot tester was removed. Therefore, the product described in this invention is safe and non-irritating, has no adverse reactions on the human body, and is highly safe.

[0097] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0098] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0099] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A plant-derived PDRN microcrystalline inclusion complex, characterized in that, The raw materials for preparing the PDRN microcrystalline inclusion complex include, by weight, 8-12 parts of plant-derived PDRN, 12-18 parts of arginine, 0.1-1 parts of pH adjuster, 0.1-2 parts of preservative, and 5-20 parts of stabilizer; The PDRN and arginine form a nanoscale microcrystalline inclusion complex through electrostatic self-assembly. The molecular weight of the plant-derived PDRN is 30kD-160kD; The stabilizers include glycerol, trehalose, and sorbitol.

2. The plant-derived PDRN microcrystalline inclusion complex as described in claim 1, characterized in that, The plant includes oat germ; The pH adjuster includes citric acid; The preservatives include pentylene glycol and / or hexanediol.

3. The plant-derived PDRN microcrystalline inclusion complex as described in claim 1 or 2, characterized in that, The plant-derived PDRN microcrystalline inclusion complex has a particle size of 200-800 nm, an encapsulation efficiency of greater than 80%, and a zeta potential of -10 to -30 mV.

4. The plant-derived PDRN microcrystalline inclusion complex as described in claim 1, characterized in that, The plant-derived PDRN is prepared using the following method, which includes: (1) Crush the plant raw materials, then add them to n-hexane and stir to defatt them, then filter and collect the residue; (2) Mix the filter residue with water, disodium EDTA and TE buffer, and homogenize to obtain a homogenate; (3) The homogenate was mixed with Tris buffer, proteinase K and sodium dodecyl sulfate, and then lysed in a water bath to obtain the lysate; (4) After centrifuging the lysate, take the supernatant, add sodium acetate solution and anhydrous ethanol to the supernatant, let it stand, centrifuge, collect the precipitate, and purify the precipitate to obtain the plant-derived PDRN.

5. The plant-derived PDRN microcrystalline inclusion complex as described in claim 4, characterized in that, The material described in step (1) is pulverized and then passed through a 40-80 mesh sieve; The amount of n-hexane added in step (1) is 2-3 times the mass of the plant raw material; The degreasing treatment in step (1) is performed 2-3 times, each time for 2-3 hours; In step (2), the mass ratio of filter residue, water, disodium EDTA, and TE buffer is 1:(5-20):(0.1-1):(0.1-0.5). The homogenization process described in step (2) is carried out in a homogenizer; The homogenization process in step (2) takes 3-10 minutes.

6. The plant-derived PDRN microcrystalline inclusion complex as described in claim 4, characterized in that, The mass ratio of the homogenate to Tris buffer, proteinase K, and sodium dodecyl sulfate in step (3) is 1: (0.1-1): (0.05-0.5): (0.2-1). The temperature of the water bath in step (3) is 50-60℃; The pyrolysis process described in step (3) is carried out under stirring, with a stirring speed of 100-200 rpm and a time of 2-4 hours; In step (4), the centrifugation speed is 8000-10000 rpm and the time is 12-20 min; The sodium acetate solution mentioned in step (4) is a 3M sodium acetate solution; The volume ratio of the supernatant to 3M sodium acetate solution and anhydrous ethanol in step (4) is 1: (0.05-0.5): (2-3). The standing time in step (4) is 6-12 hours, and the temperature is -10~20℃; The purification steps in step (4) are as follows: wash the precipitate 2-3 times with 65-75% ethanol solution, then purify it using an ion exchange chromatography column, collect the eluent after elution, dialyze to remove salt, precipitate with alcohol, collect the precipitate after centrifugation, and dry the precipitate to obtain the plant-derived PDRN.

7. A method for preparing plant-derived PDRN microcrystalline inclusion complexes as described in any one of claims 1-6, characterized in that, The preparation method includes: (a) Add plant-derived PDRN to a pH adjuster to adjust the pH to 5.5-6.5; Arginine was dissolved in deionized water to prepare an arginine solution; (b) Arginine solution was added dropwise to plant-derived PDRN, pH was adjusted to 6-7 with pH adjuster, then some stabilizer was added, and after stirring, crude microcrystalline inclusion complex was obtained; (c) The crude microcrystalline inclusion complex is filtered and the permeate is collected. Preservatives and residual stabilizers are added to the permeate and stirred evenly to obtain the plant-derived PDRN microcrystalline inclusion complex.

8. The preparation method according to claim 7, characterized in that, The concentration of the arginine solution in step (a) is 0.05-0.5%; The stabilizers mentioned in step (b) are glycerol and sorbitol; The stirring speed in step (b) is 100-200 rpm, and the stirring time is 30-60 min.

9. The preparation method according to claim 7, characterized in that, The pore size of the membrane used for filtration in step (c) is 0.1-0.3 μm; The remaining stabilizer in step (c) is trehalose.

10. The use of a plant-derived PDRN microcrystalline inclusion complex as described in any one of claims 1-6 in the preparation of cosmetics.

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