Pdrn, extraction method and application thereof

Abstract

The application provides a PDRN and an extraction method and application thereof, and belongs to the technical field of PDRN extraction. The extraction method of the PDRN comprises the following steps: subjecting plant tissues to stress treatment, so that the cells are in an irreversible damage state; subjecting the plant tissues subjected to the stress treatment to incubation treatment, so that DNA is selectively partially degraded under the action of endogenous deoxyribonuclease; and subjecting the plant tissues subjected to the incubation treatment to crushing treatment and extraction and purification treatment, to obtain the PDRN. The extraction method selectively degrades DNA under mild conditions by using endogenous enzymes (such as DNase) of plants, and has the advantages of high yield, high efficiency, low cost and green environmental protection; meanwhile, the PDRN extracted by the extraction method also has the advantages of high purity, high physiological activity and high safety.

Description

Technical Field

[0001] This application relates to the field of PDRN extraction technology, and more specifically, to a PDRN, its extraction method, and its application. Background Technology

[0002] Polydeoxyribonucleotides (PDRNs) are a class of active polynucleotide mixtures with molecular weights ranging from 50 kDa to 1500 kDa, exhibiting no antigenic properties or systemic toxicity. Due to their anti-inflammatory, anti-apoptotic, and tissue regeneration-promoting biological properties, PDRNs are widely used as bioactive molecules in the field of tissue repair. Currently, commercially available PDRNs are free from interference by active proteins or peptides, resulting in a higher DNA percentage. This allows them to provide more nucleotides in damaged or hypoxic tissues, reactivating cell proliferation and growth. Therefore, PDRNs, as a DNA derivative, have been approved by the Korean Food and Drug Administration.

[0003] However, the available raw materials for PDRN extraction are extremely limited, mainly relying on the reproductive cells of fish such as salmon and trout. These organisms have limited reproductive periods, and harvesting is difficult due to seasonal factors, thus restricting PDRN production and keeping prices high. Furthermore, animal-derived PDRN faces ethical issues and biosafety concerns related to viruses and other organisms. Therefore, researchers have begun to explore extracting PDRN from plants.

[0004] Furthermore, existing methods for PDRN extraction in the market mainly include ultrasonic treatment, acid hydrolysis, exogenous enzyme hydrolysis, and heat treatment. As disclosed in patent document CN 120905189 A: first, an exogenous bio-enzyme composition is extracted; then, a crude PDRN extract from the plant is obtained; next, the crude PDRN extract is hydrolyzed using the exogenous bio-enzyme composition; finally, the hydrolyzed product is purified to extract high-purity PDRN from the plant. However, PDRN extraction using exogenous bio-enzyme compositions is cumbersome, costly, time-consuming, and carries the risk of contamination from residual impurities such as exogenous proteins. This makes industrialization of exogenous bio-enzyme extraction difficult. Moreover, PDRN extracted using this method is difficult to promote and apply in various industries with high safety requirements, such as cosmetics, food, health functional foods, and pharmaceutical biomaterials.

[0005] Therefore, there is an urgent need to develop an endogenous enzyme hydrolysis extraction method to selectively degrade DNA using plant endogenous enzymes (such as deoxyribonuclease, or DNase) under mild conditions, thereby constructing a high-yield, high-efficiency, low-cost, and environmentally friendly extraction process; at the same time, this extraction process can also extract PDRN with high purity, high physiological activity, and high safety. Summary of the Invention

[0006] The purpose of this application is to provide a PDRN, its extraction method, and its application. The extraction method utilizes endogenous plant enzymes (such as DNase enzymes) to selectively degrade DNA under mild conditions, which has the advantages of high yield, high efficiency, low cost, and green environmental protection. At the same time, the PDRN extracted by this method also has the advantages of high purity, high physiological activity, and high safety.

[0007] The embodiments of this application are implemented as follows: In a first aspect, embodiments of this application provide a method for extracting PDRN, comprising the following steps: subjecting plant tissue to stress treatment to induce irreversible cell damage; incubating the stress-treated plant tissue to induce selective partial degradation of DNA under the action of endogenous deoxyribonuclease; and performing fragmentation and extraction purification treatment on the incubated plant tissue to obtain PDRN.

[0008] In the above technical solution, plant tissue is subjected to stress treatment to induce irreversible cell damage. Under this state, the cells' defense functions are essentially lost, while the activity of endogenous DNase enzymes increases and they become more accessible to DNA. Furthermore, the enzyme activity is further increased during subsequent incubation, enabling efficient and selective partial degradation of DNA solely through the action of endogenous enzymes. Then, extraction and purification effectively remove impurities such as proteins and pigments from the extract, resulting in high-purity PDRN. The extraction method provided in this application, through a combination of stress treatment and incubation, can extract PDRN solely through the action of endogenous enzymes, offering advantages such as high yield, high efficiency, low cost, and environmental friendliness. Simultaneously, the PDRN extracted by this method also possesses advantages such as high purity, high physiological activity (the enzymatic hydrolysis conditions are relatively mild, resulting in high physiological activity of PDRN), and high safety (no exogenous proteins or other impurities are introduced).

[0009] In some alternative implementations, the steps of stress treatment on plant tissues include: S11 immersing the plant tissues in a sodium chloride solution with a mass concentration of 0.2% to 0.5% for 20 to 60 minutes; S12 immersing the plant tissues in water for 5 to 20 minutes; S13 repeating steps S11 to S12 multiple times.

[0010] In the above technical solution, the plant tissue is repeatedly soaked in high-osmotic-pressure saline and low-osmotic-pressure pure water, and the parameters at each stage are controlled within the above range. This can effectively bring the cells to an irreversible state of damage. In addition, this stress treatment method has the advantages of simple operation and low cost.

[0011] In some alternative implementations, the surface of the plant tissue is scratched before immersing it in a sodium chloride solution.

[0012] In the above technical solution, scratching the surface of the plant tissue before soaking allows salt water and pure water to enter and exit the cells more quickly and easily, thereby enabling the cells to reach an irreversible state of damage as soon as possible.

[0013] In some alternative implementations, the incubation treatment step includes adding cofactors to the stress-treated plant tissue and incubating it at a pH of 7-7.5 and a temperature of 30°C-40°C.

[0014] In the above technical solution, the addition of cofactors and incubation under the above pH and temperature conditions can effectively improve the activity of endogenous enzymes, so that the enzymatic reaction can proceed fully, thereby enabling DNA to be selectively degraded more efficiently.

[0015] In some alternative embodiments, the incubation treatment lasts for 1 h to 6 h, and / or the cofactor is selected from at least one of calcium ions and magnesium ions.

[0016] In the above technical solution, since the activity of endogenous enzymes changes with the incubation time, limiting the incubation time to the above range ensures that the activity of endogenous enzymes is strong within this time period, allowing the enzymatic reaction to proceed fully, thereby enabling the DNA to be selectively and efficiently degraded. The selection of the above-mentioned cofactors can effectively improve the activity of endogenous enzymes, so that the enzymatic reaction can proceed fully, thereby enabling the DNA to be selectively and efficiently degraded.

[0017] In some alternative implementations, the incubation period is 3 to 5 hours.

[0018] In the above technical solution, limiting the incubation time to the aforementioned range enables precise control of the PDRN molecular weight, ensuring that most of the prepared PDRNs are within the range of 400 bp to 1200 bp. PDRNs within this range exhibit superior effects in promoting cell proliferation and improving cell survival rate, promoting hyaluronic acid synthesis and collagen or elastin expression, inhibiting reactive oxygen species generation and resisting UVB or oxidative stress, anti-inflammatory and soothing effects, anti-wrinkle and skin elasticity improvement effects, promoting cell migration and wound healing effects, regulating the balance of skin microbiota, and anti-glycation and anti-aging effects.

[0019] In some alternative implementations, the plant tissue includes fern tissue.

[0020] In the above technical solutions, fern tissues have the advantage of high DNA content. Using them as extraction raw materials, compared with angiosperm tissues, more PDRN can be extracted with the same amount of raw materials. This can, to some extent, solve the problem that plant-derived PDRN is difficult to fully supply market demand at present.

[0021] In some alternative embodiments, the fern tissue includes at least one of the following: European drynaria tissue, oak fern tissue, and maidenhair fern tissue.

[0022] In the above-mentioned technical solutions, the above-mentioned types of fern tissues have the advantage of high DNA content. At the same time, there are many types of fern tissues to choose from, which can provide a variety of feasible solutions.

[0023] In some alternative implementations, fern tissue includes leaves.

[0024] In the above technical solution, the leaves of fern tissue have the advantage of high DNA content. Using them as extraction raw materials, compared with other parts of fern tissue, more PDRN can be extracted with the same amount of raw materials, so as to solve the problem that the current plant-derived PDRN is difficult to fully supply the market demand. In addition, when using the leaves of fern tissue as extraction raw materials, the combination of stress treatment and incubation treatment can also effectively improve the yield of PDRN.

[0025] In some alternative implementations, the leaf is selected from spore leaves.

[0026] In the above technical solution, the spore leaves of fern tissue have the advantage of high DNA content. Using them as extraction raw materials, compared with other parts of fern tissue, more PDRN can be extracted with the same amount of raw materials, so as to effectively solve the problem that plant-derived PDRN is difficult to fully supply market demand at this stage. In addition, when using the spore leaves of fern tissue as extraction raw materials, the combination of stress treatment and incubation treatment can more effectively improve the yield of PDRN.

[0027] In some alternative implementations, the plant tissue is fresh plant tissue that has not been dried or heat-treated after harvesting.

[0028] In the above technical solution, fresh plant tissue is selected as the extraction raw material. Its endogenous enzymes have high activity, which can make the enzymatic reaction fully carried out, thereby enabling the DNA to be selectively and efficiently degraded. At the same time, the extracted PDRN also has high physiological activity.

[0029] In some alternative embodiments, the extraction and purification steps include: adding the lysed plant tissue to CTAB buffer and performing thermal extraction at 50°C to 70°C to obtain an extract; adding a mixed solvent of chloroform and isoamyl alcohol to the extract and centrifuging to obtain a precursor solution containing PDRN; adding isopropanol to the precursor solution and centrifuging, washing with alcohol, and drying to obtain PDRN powder.

[0030] The above technical solution uses the CTAB extraction method for PDRN extraction and purification, which has advantages such as good impurity removal effect, mature operation and low cost.

[0031] In some alternative embodiments, the CTAB buffer comprises: 1.5%–2.5% CTAB, 1.3 M–1.5 M NaCl, and 80 mM–120 mM Tris. HCl, 15 mM~25 mM EDTA, 0.5%~2% PVP (mass concentration), and 0.1%~0.3% β-D-methyl (mass concentration) Mercaptoethanol; or / and, the volume ratio of the extract to the mixed solvent is (0.8~1.2):(0.8~1.2), and in the mixed solvent, the volume ratio of chloroform to isoamyl alcohol is (20~30):1.

[0032] In the above technical solution, the CTAB buffer is prepared according to the above formula, which enables cells to be lysed more efficiently and thoroughly, thereby fully releasing PDRN. At the same time, it can effectively protect the structural integrity and physiological activity of PDRN. By limiting the volume ratio of the extract and the mixed solvent within the above range, impurities such as proteins and pigments in the extract can be removed more efficiently and thoroughly, thereby preparing high-purity PDRN.

[0033] Secondly, this application provides a PDRN, which is prepared by the extraction method provided in the first aspect embodiment. The length of some PDRNs is 400 bp to 1200 bp, and the OD260 / OD280 of the PDRNs is 1.8 to 2.1.

[0034] In the above technical solution, PDRN is obtained by the extraction method provided in the first aspect embodiment. Its characteristics are: the length of some PDRN is 400 bp to 1200 bp and the OD260 / OD280 of PDRN is 1.8 to 2.1, that is, the specification of some PDRN is 400 bp to 1200 bp and has high purity. PDRN with this characteristic has excellent effects in promoting cell proliferation and improving cell survival rate, promoting hyaluronic acid synthesis and collagen or elastin expression, inhibiting reactive oxygen generation and anti-UVB or oxidative stress, anti-inflammatory and soothing effects, anti-wrinkle and improving skin elasticity effects, promoting cell migration and wound healing effects, regulating the balance of skin microbiome, and anti-glycation and anti-aging effects.

[0035] Thirdly, the embodiments of this application provide the application of PDRN as provided in the third aspect embodiments in the preparation of products that promote cell proliferation and improve cell survival rate, products that promote hyaluronic acid synthesis and collagen or elastin expression, products that inhibit reactive oxygen species generation and resist UVB or oxidative stress, products that have anti-inflammatory and soothing effects, products that have anti-wrinkle and improve skin elasticity, products that promote cell migration and wound healing, products that regulate the balance of skin microbiota, or products that have anti-glycation and anti-aging effects.

[0036] In some alternative implementations, the product includes at least one of food, pharmaceutical, cosmetic, biomedical material, topical skin preparation, health product, food additive, and cosmetic composition.

[0037] The above technical solutions involve a wide variety of products, providing numerous feasible implementation options, which facilitates the promotion and application of the technical solutions provided in the embodiments of this application. Attached Figure Description

[0038] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1 A process flow diagram of a PDRN extraction method provided in this application embodiment; Figure 2 The distribution results of PDRN molecular weight under different incubation times provided in the embodiments of this application; Figure 3The test results of PDRN promoting cell proliferation and improving cell survival rate under different incubation times provided in the embodiments of this application; Figure 4 Test results of PDRN promoting hyaluronic acid synthesis under different incubation times provided in the embodiments of this application; Figure 5 Test results of PDRN resistance to UVB damage at different incubation times provided in the embodiments of this application; Figure 6 The test results of PDRN inhibiting intracellular ROS generation under different incubation times provided in the embodiments of this application; Figure 7 Test results of PDRN anti-inflammatory and sedative effects under different incubation times provided in the embodiments of this application; Figure 8 Test results of PDRN anti-wrinkle properties under different incubation times provided in the embodiments of this application; Figure 9 Test results of PDRN promoting collagen synthesis under different incubation times provided in the embodiments of this application; Figure 10 The test results of PDRN promoting cell regeneration and wound healing under different incubation times provided in the embodiments of this application; Figure 11 The test results of PDRN regulating the skin microbiome balance under different incubation times provided in the embodiments of this application; Figure 12 The test results of PDRN anti-glycation and anti-aging under different incubation times provided in the embodiments of this application. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0041] It should be noted that the terms "and / or" in this application, such as "feature 1 and / or feature 2", all refer to the three cases of "feature 1" alone, "feature 2" alone, and "feature 1" plus "feature 2".

[0042] In addition, in the description of this application, unless otherwise stated, "one or more" means two or more; the range of "numerical value a to numerical value b" includes the two endpoints "a" and "b"; and "unit of measurement" in "numerical value a to numerical value b + unit of measurement" represents the "unit of measurement" of both "numerical value a" and "numerical value b".

[0043] In existing technologies, although there are studies on extracting PDRN from plants using plant-derived bioenzymes, the extraction of PDRN using exogenous bioenzyme compositions is complicated, costly, time-consuming, and carries the risk of contamination by residual impurities such as exogenous proteins. This makes it difficult to industrialize the exogenous bioenzyme extraction method. Furthermore, the PDRN extracted by this method is difficult to promote and apply in various industrial fields with high safety requirements, such as cosmetics, food, health functional foods, and pharmaceutical biomaterials.

[0044] The inventors discovered that stress treatment of plant tissues increases the activity of endogenous DNase enzymes and makes them more accessible to DNA. Combined with subsequent incubation, this allows for the efficient and selective degradation of DNA using only endogenous enzymes (such as DNase) from plant tissues, resulting in high-purity PDRN and overcoming the limitations of exogenous enzyme extraction methods. Furthermore, the inventors unexpectedly found that stress treatment of plant tissues also effectively improves the yield of PDRN.

[0045] The following is a detailed description of a PDRN, its extraction method, and its application according to an embodiment of this application.

[0046] In a first aspect, embodiments of this application provide a method for extracting PDRN, comprising the following steps: subjecting plant tissue to stress treatment to induce irreversible cell damage; incubating the stress-treated plant tissue to induce selective partial degradation of DNA under the action of endogenous deoxyribonuclease; and performing fragmentation and extraction purification treatment on the incubated plant tissue to obtain PDRN.

[0047] In this application, plant tissues are subjected to stress treatment to induce irreversible cell damage, resulting in the near loss of cellular defense functions. This leads to increased activity of endogenous DNase enzymes, which become more readily accessible to DNA. Furthermore, the enzyme activity is further enhanced during subsequent incubation, enabling efficient and selective partial degradation of DNA solely through the action of endogenous enzymes. Extraction and purification effectively remove impurities such as proteins and pigments from the extract, yielding high-purity PDRN. The extraction method provided in this application, through a combination of stress and incubation treatments, allows for the extraction of PDRN solely through the action of endogenous enzymes, offering advantages such as high yield, high efficiency, low cost, and environmental friendliness. Simultaneously, the PDRN extracted using this method also exhibits high purity, high physiological activity (due to the relatively mild conditions of the enzymatic hydrolysis method), and high safety (without the introduction of exogenous proteins or other impurities).

[0048] As an example, plant tissue refers to fresh plant tissue that has not been dried or heat-treated after harvesting.

[0049] In this embodiment, fresh plant tissue is selected as the extraction raw material. Its endogenous enzymes have high activity, which can make the enzymatic reaction fully carried out, thereby enabling the DNA to be selectively and efficiently degraded. At the same time, the extracted PDRN also has high physiological activity.

[0050] As an example, the steps for stress treatment of plant tissue include: S11 Immersing the plant tissue in a sodium chloride solution with a mass concentration of 0.2% to 0.5% (e.g., but not limited to any one of the concentrations of 0.2%, 0.3%, 0.4%, and 0.5%, or any range between any two) for 20 to 60 minutes (e.g., but not limited to any one of the durations of 20, 30, 40, 50, and 60 minutes, or any range between any two); S12 Immersing the plant tissue in water for 5 to 20 minutes (e.g., but not limited to any one of the durations of 5, 10, 15, and 20 minutes, or any range between any two); S13 Repeating steps S11 to S12 multiple times.

[0051] In this embodiment, the plant tissue is repeatedly soaked in high-osmotic-pressure saline and low-osmotic-pressure pure water, and the parameters at each stage are controlled within the above-mentioned range. This can efficiently induce irreversible damage to the cells (specifically, repeated osmotic stress will gradually reduce the function of the cell membrane and vacuolar membrane, causing irreversible loss of the cell's physiological activity, while the cell nucleus structure and high molecular weight DNA structure will remain relatively stable without being affected). This stress treatment method also has the advantages of being simple to operate and low in cost.

[0052] As an example, repeat steps S11 to S12 2 to 5 times.

[0053] In this embodiment, by repeating the operation as described above a certain number of times, most plant tissues can be brought to a state of irreversible damage.

[0054] It should be noted that, in order to determine the specific state of the cells more accurately, a small amount of plant tissue can be taken in pure water and observed under a microscope after each cycle. When it is observed that the protoplasmic structure of the cells is no longer recovering, it proves that the plant tissue is in a state of irreversible damage, and thus it can be prepared for incubation treatment.

[0055] As an example, the process of scrambling the surface of the plant tissue before immersing it in a sodium chloride solution also includes the treatment of the plant tissue surface.

[0056] In this embodiment, scratching the surface of the plant tissue before soaking allows the salt water and pure water to enter and exit the cells more quickly and easily, thereby enabling the cells to reach an irreversible state of damage as soon as possible.

[0057] It should be noted that there are no restrictions on the tools used in the scratch treatment steps; for example, a knife or tweezers can be used.

[0058] As an example, the incubation treatment involves adding a cofactor to the stress-treated plant tissue and incubating it at a pH of 7–7.5 (e.g., but not limited to any one of pH values ​​7, 7.1, 7.2, 7.3, 7.4, and 7.5, or a range between any two) and a temperature of 30–40°C (e.g., but not limited to any one of temperature values ​​30°C, 32°C, 34°C, 36°C, 38°C, and 40°C, or a range between any two).

[0059] In this embodiment, the addition of a cofactor and incubation under the aforementioned pH and temperature conditions can effectively enhance the activity of endogenous enzymes, allowing the enzymatic reaction to proceed fully and thus enabling more efficient and selective partial degradation of DNA.

[0060] It should be noted that the inventors discovered that the activity of endogenous enzymes during incubation does not exhibit a simple linear relationship with time, but rather increases first and then decreases with incubation time. Therefore, the incubation duration needs to be optimized based on the actual activity of endogenous enzymes at different incubation times.

[0061] As an example, the incubation treatment duration is 1 h to 6 h, for example, but not limited to any one of the incubation durations of 1 h, 2 h, 3 h, 4 h, 5 h and 6 h or any range between two.

[0062] In this embodiment, since the activity of endogenous enzymes changes with incubation time, the incubation time is limited to the above range. During this time period, the activity of endogenous enzymes is stronger, which allows the enzymatic reaction to proceed fully, thereby enabling the DNA to be selectively and efficiently degraded.

[0063] As an example, the cofactor is selected from at least one of calcium ions and magnesium ions.

[0064] In this embodiment, selecting the aforementioned types of cofactors can effectively enhance the activity of endogenous enzymes, allowing the enzymatic reaction to proceed fully, thereby enabling the DNA to be selectively and efficiently degraded.

[0065] It should be noted that the inventors' research found that the specifications of the extracted PDRN are closely related to the incubation length. Specifically, the longer the incubation time, the smaller the length of the obtained PDRN (i.e., the smaller the molecular weight), and the shorter the incubation time, the larger the length of the obtained PDRN (i.e., the larger the molecular weight). At the same time, the efficacy of PDRN is also closely related to its specifications. Therefore, the incubation time can be optimized based on the actual efficacy of PDRN to obtain PDRN of suitable specifications.

[0066] As an example, the incubation treatment duration is 3 h to 5 h, for example, but not limited to any one of the durations of 3 h, 3.5 h, 4 h, 4.5 h and 5 h, or any range between two of them.

[0067] In this embodiment, limiting the incubation time to the above-mentioned range enables precise control of the molecular weight of PDRN, so that most of the prepared PDRNs are in the range of 400 bp to 1200 bp. PDRNs in this range have excellent effects in promoting cell proliferation and improving cell survival rate, promoting hyaluronic acid synthesis and collagen or elastin expression, inhibiting reactive oxygen species generation and anti-UVB or oxidative stress, anti-inflammatory and soothing effects, anti-wrinkle and skin elasticity improvement effects, promoting cell migration and wound healing effects, regulating the balance of skin microbiota, and anti-glycation and anti-aging effects.

[0068] Understandably, enzyme inactivation is necessary after the incubation process to terminate the enzymatic hydrolysis in a timely manner.

[0069] It should be noted that the method of enzyme inactivation is not limited and can be carried out in accordance with conventional processes in this field. For example, it can be to add EDTA or citric acid to the incubated plant tissue, or to subject the incubated plant tissue to instantaneous cooling or heating treatment.

[0070] As an example, plant tissues include fern tissues.

[0071] It should be noted that, to the inventor's knowledge, this application is the first time that fern tissue has been used as a raw material for PDRN extraction.

[0072] In this embodiment, fern tissue has the advantage of high DNA content. Using it as an extraction raw material, compared with angiosperm tissue, more PDRN can be extracted with the same amount of raw material. This can, to some extent, solve the problem that plant-derived PDRN is difficult to fully supply market demand at present.

[0073] As an example, fern tissues include at least one of the following: European hairy fern tissue, oak fern tissue, and maidenhair fern tissue.

[0074] In this embodiment, the above-mentioned fern tissues have the advantage of high DNA content. At the same time, there are many types of fern tissues to choose from, which can provide a variety of feasible implementation schemes.

[0075] As an example, fern tissue includes leaves.

[0076] In this embodiment, the leaves of fern tissue have the advantage of high DNA content. Using them as extraction raw materials, compared with other parts of fern tissue, more PDRN can be extracted with the same amount of raw materials, thus solving the problem that plant-derived PDRN is difficult to fully supply market demand at present. In addition, when using the leaves of fern tissue as extraction raw materials, the combination of stress treatment and incubation treatment can also effectively improve the yield of PDRN.

[0077] As an example, the leaf is selected from spore leaves (i.e., leaves containing spores).

[0078] In this embodiment, the spore leaves of fern tissue have the advantage of high DNA content. Using them as extraction raw materials, more PDRN can be extracted compared to other parts of fern tissue with the same amount of extraction raw materials, so as to effectively solve the problem that plant-derived PDRN is difficult to fully supply market demand at this stage. In addition, when using the spore leaves of fern tissue as extraction raw materials, the combination of stress treatment and incubation treatment can more effectively improve the yield of PDRN.

[0079] It should also be noted that the spores of fern tissues have a single-cell structure. After removing the relatively thick cell wall, the content of impurities such as protein, polysaccharide and pigment is low, which is conducive to obtaining high-purity PDRN.

[0080] As an example, the crushing process includes freezing the incubated plant tissue in liquid nitrogen and then grinding it into powder.

[0081] In this embodiment, the above-described fragmentation method can efficiently break the cell wall while maximally protecting the structural integrity of the DNA.

[0082] As an example, the extraction and purification process includes: adding the lysed plant tissue to CTAB buffer and performing thermal extraction at 50°C to 70°C (e.g., but not limited to processing temperatures of 50°C, 60°C, and 70°C) to obtain an extract; adding a mixed solvent of chloroform and isoamyl alcohol to the extract and centrifuging to obtain a precursor solution containing PDRN; adding isopropanol to the precursor solution and centrifuging, washing with alcohol, and drying to obtain PDRN powder.

[0083] In this embodiment, the CTAB extraction method is used for the extraction and purification of PDRN, which has advantages such as good impurity removal effect, mature operation and low cost.

[0084] It should be noted that there is no limit to the hot extraction time, which can be adjusted according to actual needs, for example, 20 min to 50 min.

[0085] It should be noted that the steps of adding a mixed solvent of chloroform and isoamyl alcohol to the extract and centrifuging can be repeated multiple times to improve the impurity removal effect.

[0086] As an example, CTAB buffer comprises: CTAB at a mass concentration of 1.5% to 2.5% (e.g., but not limited to any one of 1.5%, 1.7%, 2.0%, 2.2%, and 2.5%, or a range between any two); NaCl at a mass concentration of 1.3 M to 1.5 M (e.g., but not limited to any one of 1.3 M, 1.4 M, and 1.5 M, or a range between any two); and Tris at a mass concentration of 80 mM to 120 mM (e.g., but not limited to any one of 80 mM, 90 mM, 100 mM, 110 mM, and 120 mM, or a range between any two). HCl, EDTA at concentrations of 15 mM to 25 mM (e.g., but not limited to any one of 15 mM, 17 mM, 20 mM, 22 mM, and 25 mM, or any range between them), PVP at concentrations of 0.5% to 2% (e.g., but not limited to any one of 0.5%, 1%, 1.5%, and 2%, or any range between them), and β-D at concentrations of 0.1% to 0.3% (e.g., but not limited to any one of 0.1%, 0.2%, and 0.3%, or any range between them). Mercaptoethanol.

[0087] In this embodiment, the CTAB buffer is prepared according to the above formula, which enables cells to lyse more efficiently and thoroughly, thereby fully releasing PDRN. At the same time, it can effectively protect the structural integrity and physiological activity of PDRN.

[0088] As an example, the volume ratio of the extract to the mixed solvent is (0.8~1.2):(0.8~1.2), for example, but not limited to any one of the volume ratios of 0.8:0.8, 0.8:1.0, 0.8:1.2, 1.0:0.8, 1.0:1.2, 1.2:0.8, and 1.2:1.0, or any range between the two, and in the mixed solvent, the volume ratio of chloroform to isoamyl alcohol is (20~30):1, for example, but not limited to any one of the volume ratios of 20:1, 22:1, 24:1, 26:1, 28:1, and 30:1, or any range between the two.

[0089] In this embodiment, by limiting the volume ratio of the extract to the mixed solvent within the above-mentioned range, impurities such as proteins and pigments in the extract can be removed more efficiently and thoroughly, thereby preparing high-purity PDRN.

[0090] As an example, the steps of adding isopropanol to the precursor solution and centrifuging include: adding isopropanol to the precursor solution and letting it stand at -20°C for at least 1 h, and then centrifuging at 4°C and 12000 g to 15000 g for 10 min to 20 min.

[0091] As an example, the steps of the alcohol washing treatment include: repeatedly washing the precipitate after centrifugation with a 70% ethanol solution 2 to 5 times.

[0092] As an example, after drying, the process also includes a step of redissolving the PDRN powder in TE buffer (composed of 10 mM Tris-HCl and 1 mM EDTA).

[0093] In this embodiment, PDRN is stored in TE buffer, which can better maintain its physiological activity.

[0094] It should be noted that for any process or step in the PDRN extraction process that is not specifically described or limited, it can be carried out in accordance with conventional processes in this field.

[0095] As an example, a process flow diagram of the PDRN extraction method is exemplarily shown below. Figure 1 .

[0096] Secondly, this application provides a PDRN, which is prepared by the extraction method provided in the first aspect embodiment. The length of some PDRNs is 400 bp to 1200 bp, and the OD260 / OD280 of the PDRNs is 1.8 to 2.1.

[0097] In this application, PDRN is prepared using the extraction method provided in the first aspect embodiment. Its characteristics are: the length of some PDRN is 400 bp to 1200 bp and the OD260 / OD280 of PDRN is 1.8 to 2.1, that is, the specification of some PDRN is 400 bp to 1200 bp and has high purity. PDRN with these characteristics has excellent effects in promoting cell proliferation and improving cell survival rate, promoting hyaluronic acid synthesis and collagen or elastin expression, inhibiting reactive oxygen species generation and anti-UVB or oxidative stress, anti-inflammatory and soothing effects, anti-wrinkle and skin elasticity improvement effects, promoting cell migration and wound healing effects, regulating the balance of skin microbiota, and anti-glycation and anti-aging effects.

[0098] Thirdly, the embodiments of this application provide the application of PDRN as provided in the third aspect embodiments in the preparation of products that promote cell proliferation and improve cell survival rate, products that promote hyaluronic acid synthesis and collagen or elastin expression, products that inhibit reactive oxygen species generation and resist UVB or oxidative stress, products that have anti-inflammatory and soothing effects, products that have anti-wrinkle and improve skin elasticity, products that promote cell migration and wound healing, products that regulate the balance of skin microbiota, or products that have anti-glycation and anti-aging effects.

[0099] As an example, the product includes at least one of food, pharmaceuticals, cosmetics, biomedical materials, topical skin preparations, health products, food additives, and cosmetic compositions.

[0100] In this embodiment, there are many types of products, which can provide a variety of feasible solutions, thereby facilitating the promotion and application of the technical solutions provided in the embodiments of this application.

[0101] The features and performance of this application will be further described in detail below with reference to the embodiments.

[0102] Example 1 This application provides a method for extracting PDRN, including the following steps: Stress treatment: Fresh leaves of the European dry bark fern (without spores) were soaked in a 0.5% saline solution for 30 minutes, then the saline solution was drained and pure water was added and soaked for 10 minutes. After the treatment, a portion of the plant tissue in the pure water was taken and observed under a microscope to see if the protoplasm of the cells could be restored. After repeating the treatment 4 times, it was found that the protoplasm layer of the cells did not recover, indicating that the cells were in an irreversible state of damage.

[0103] Incubation treatment: Calcium and magnesium ions were added to the stress-treated plant tissues and incubated at pH 7.1 and 35°C for 1 h to allow DNA to be selectively partially degraded by endogenous deoxyribonuclease; then EDTA was immediately added to the plant tissues to inhibit enzyme activity.

[0104] Crushing process: The incubated plant tissues are frozen in liquid nitrogen and then ground into powder.

[0105] Extraction and purification: 1 g of the lysed plant tissue was added to CTAB buffer (composition: 2% CTAB, 1.4 M NaCl, 100 mM Tris). HCl, 20 mM EDTA, 1% PVP (by mass), and 0.2% β-D-glucan (by mass) were used. Extraction was performed in mercaptoethanol at 60°C for 30 min to obtain an extract. The extract was added to an equal volume of a mixed solvent (composed of chloroform and isoamyl alcohol in a volume ratio of 24:1) and centrifuged three times to remove impurities such as proteins and pigments, resulting in a precursor solution containing PDRN. Isopropanol was added to the precursor solution and the mixture was allowed to stand at -20°C for 2 h, followed by centrifugation at 13000 g for 15 min at 4°C. The PDRN precipitate was then washed three times with a 70% ethanol solution and air-dried to obtain dried PDRN. Finally, the dried PDRN was redissolved in TE buffer for storage.

[0106] Example 2 This application provides a method for extracting PDRN, which differs from Example 1 only in that the plant tissue is the spore leaf of the European drynose fern.

[0107] Example 3 This application provides a method for extracting PDRN, which differs from Example 1 only in that the plant tissue is the leaf of a fern.

[0108] Example 4 This application provides a method for extracting PDRN, which differs from Example 1 only in that the plant tissue is the spore leaf of the oak fern.

[0109] Example 5 This application provides a method for extracting PDRN, which differs from Example 1 only in that the plant tissue is the leaf of Adiantum capillus-veneris.

[0110] Example 6 This application provides a method for extracting PDRN, which differs from Example 1 only in that the plant tissue is the spore leaf of Adiantum capillus-veneris.

[0111] Example 7 This application provides a method for extracting PDRN, which differs from Example 1 only in that the plant tissue is green tea leaves.

[0112] Example 8 This application provides a method for extracting PDRN, which differs from Example 4 only in that the incubation time is 2 hours.

[0113] Example 9 This application provides a method for extracting PDRN, which differs from Example 4 only in that the incubation time is 3 hours.

[0114] Example 10 This application provides a method for extracting PDRN, which differs from Example 4 only in that the incubation time is 4 hours.

[0115] Example 11 This application provides a method for extracting PDRN, which differs from Example 4 only in that the incubation time is 5 hours.

[0116] Example 12 This application provides a method for extracting PDRN, which differs from Example 4 only in that the incubation time is 6 hours.

[0117] Comparative Example 1 This application provides a comparative method for extracting PDRN, which differs from Example 1 only in that no stress treatment is performed.

[0118] Comparative Example 2 This application provides a comparative method for extracting PDRN, which differs from Example 2 only in that no stress treatment is performed.

[0119] Comparative Example 3 This application provides a comparative method for extracting PDRN, which differs from Example 3 only in that no stress treatment is performed.

[0120] Comparative Example 4 This application provides a comparative method for extracting PDRN, which differs from Example 4 only in that no stress treatment is performed.

[0121] Comparative Example 5 This application provides a comparative method for extracting PDRN, which differs from Example 5 only in that no stress treatment is performed.

[0122] Comparative Example 6 This application provides a comparative method for extracting PDRN, which differs from Example 6 only in that no stress treatment is performed.

[0123] Comparative Example 7 This application provides a comparative method for extracting PDRN, which differs from Example 7 only in that no stress treatment is performed.

[0124] Comparative Example 8 This application provides a comparative method for extracting PDRN, which differs from Example 1 only in that no stress treatment is performed and the plant tissue is corn leaf.

[0125] Comparative Example 9 This application provides a comparative method for extracting PDRN, which differs from Example 1 only in that no stress treatment is performed and the plant tissue is lettuce leaf.

[0126] Comparative Example 10 This application provides a comparative method for extracting PDRN, which differs from Example 1 only in that no stress treatment is performed and the plant tissue is bamboo leaves.

[0127] Comparative Example 11 This application provides a comparative method for extracting PDRN, which differs from Example 4 only in that no incubation treatment is performed.

[0128] Comparative Example 12 This application provides a comparative method for extracting PDRN, which differs from Example 8 only in that no stress treatment or incubation treatment is performed.

[0129] Comparative Example 13 This application provides a comparative method for extracting PDRN, which differs from Example 8 only in that no stress treatment is performed.

[0130] Comparative Example 14 This application provides a comparative method for extracting PDRN, which differs from Example 9 only in that no stress treatment is performed.

[0131] Comparative Example 15 This application provides a comparative method for extracting PDRN, which differs from Example 10 only in that no stress treatment is performed.

[0132] Comparative Example 16 This application provides a comparative method for extracting PDRN, which differs from Example 11 only in that no stress treatment is performed.

[0133] Comparative Example 17 This application provides a comparative method for extracting PDRN, which differs from Example 12 only in that no stress treatment is performed.

[0134] To better understand the process differences between the various embodiments and comparative examples, a summary is provided here in tabular form, as shown in Table 1.

[0135] Table 1

[0136] Experimental Example 1 DNA extraction volume and concentration testing: Test method: The products extracted from Examples 1-7 and Comparative Examples 1-10 were used as samples. The concentration of each sample was tested and the extraction yield per unit weight and purity were calculated. Specifically, the absorbance of each sample at 260 nm and 280 nm was measured using a spectrophotometer. The concentration of DNA and the extraction yield per unit weight were calculated based on the absorbance at 260 nm. At the same time, the purity was calculated based on the absorbance at 260 nm and 280 nm. Finally, the results are summarized in Table 2.

[0137] Table 2

[0138] Referring to Table 2, the test results of Examples 1-6 show that using spore leaves of fern tissue as the raw material for DNA extraction results in a significantly higher extraction rate (i.e., extraction amount per unit weight) compared to using leaves of fern tissue without spores as the raw material for DNA extraction, and the extracted DNA has a higher purity.

[0139] The test results of Examples 1-7 and Comparative Examples 1-7 show that stress treatment of plant tissues can effectively improve the DNA extraction rate (i.e., the amount extracted per unit weight) compared to no stress treatment. Moreover, the improvement effect is more significant for the leaves of fern tissues with high DNA content, especially the spore leaves.

[0140] The test results of Comparative Examples 1 to 10 show that using fern tissue as the raw material for DNA extraction results in a significantly higher extraction rate (i.e., extraction amount per unit weight) compared to using other types of plant tissue, and the extracted DNA has a higher purity.

[0141] Experimental Example 2 Endogenous enzyme activity test Test Method: The incubation solutions of Examples 4, 8-12, and Comparative Examples 4 and 11-17 after incubation but before enzyme inactivation were used as samples. A fluorescence DNase activity assay kit (Abcam DNase I Activity Assay Kit, product number ab234056) was used, following standard operating procedures. Each sample was mixed with a DNase-specific fluorescent substrate and reacted for a certain time. The fluorescence signal generated after substrate degradation was then measured using a spectrophotometer. The measured fluorescence signal was then plotted against a standard curve using standard DNase solutions to calculate the enzyme activity value. Note: The DNase activity unit is defined as RAU (Relative Activity Unit), which is the enzyme activity corresponding to a 0.1 change in absorbance (or fluorescence intensity) under the test conditions. All measurements were repeated under the same conditions and expressed as mean ± standard deviation. The results are summarized in Table 3.

[0142] Table 3

[0143] Referring to Table 3, the test results of Examples 2-12 and Comparative Example 11 show that after stress treatment of plant tissues, the activity of endogenous enzymes initially increased and then decreased with increasing incubation time. Specifically, the increase in endogenous enzyme activity was most significant when the incubation time was between 2 and 5 hours. The test results of Comparative Examples 12, 4, and 13-17 show that without stress treatment, the activity of endogenous enzymes also initially increased and then decreased with increasing incubation time. However, the activity level of endogenous enzymes was lower at all time points than after stress treatment, especially when the incubation time was between 2 and 5 hours. This indicates that stress treatment can effectively increase the activity of endogenous enzymes, which is consistent with the extraction results in Table 2.

[0144] Experimental Example 3 PDRN molecular weight test Test method: The products extracted from Comparative Example 11, Example 4, and Examples 8-12 were used as samples, with Comparative Example 11 corresponding to 0 h, Example 4 to 1 h, and Examples 8-12 to 2 h, 3 h, 4 h, 5 h, and 6 h, respectively. Electrophoresis analysis was then performed on each sample. The specific steps for electrophoresis analysis were as follows: 1.0% agarose gel was dissolved in TAE buffer, and EtBr was added to prepare the gel; 2-5 μL of the extracted product was mixed with 6 times the amount of loading buffer and added to the sample wells, along with a DNA gradient standard (DNA ladder). Electrophoresis was performed at 100 V for 30-40 minutes, and the electrophoresis results were photographed.

[0145] See Figure 2 It can be seen that in the sample incubated for 0 h, the electrophoresis results showed that there were high molecular weight PDRNs of 5000 bp to 10000 bp at the top. As the incubation time increased, low molecular weight PDRNs gradually appeared. When the incubation time was 4 h to 5 h, the proportion of PDRNs in the range of 400 bp to 1200 bp was the highest. As the incubation time continued to increase, the molecular weight of PDRNs further decreased, and PDRNs below 400 bp began to appear.

[0146] Test Example 4 Tests to demonstrate that PDRN promotes cell proliferation and improves cell viability Test method: The products extracted from Comparative Example 11, Example 4, and Examples 8-12 were used as samples, with Comparative Example 11 corresponding to 0 h, Example 4 corresponding to 1 h, and Examples 8-12 corresponding to 2 h, 3 h, 4 h, 5 h, and 6 h, respectively. The effect of each sample on cell viability was then tested.

[0147] Test procedure: Human skin-derived fibroblasts (HDF) were seeded in 96-well plates, with 1 × 10⁶ cells per well. 4 Cells were cultured for 24 hours to stabilize their state, and then PDRN samples corresponding to each incubation time were added to the culture medium. PDRN treatment concentrations were set at three gradients: 10, 50, and 100 μg / mL. The untreated control group (control) received no PDRN under the same conditions. Cells were cultured for another 48 hours. After culture, MTT [3] was added to each well. (4,5 dimethylthiazol 2 yl) 2,5 The diphenyltetrazolium bromide reagent was prepared to a final concentration of 0.5 mg / mL and reacted at 37°C for 3 hours. The supernatant was then discarded, and DMSO was added to each well to fully dissolve the formed formazan crystals. The absorbance was measured at 570 nm. The measured absorbance values ​​were converted to cell viability (%) relative to the untreated control group for evaluation.

[0148] See Figure 3It is known that the PDRN prepared according to the extraction process provided in the embodiments of this application has the effect of promoting cell proliferation and improving cell survival rate. Among them, when the incubation time is 5 h, the PDRN with the highest proportion of 400 bp to 1200 bp has a more stable and obvious effect.

[0149] Experimental Example 5 Tests to demonstrate PDRN's promotion of hyaluronic acid synthesis Test method: The products extracted from Comparative Example 11, Example 4, and Examples 8-12 were used as samples, with Comparative Example 11 corresponding to 0 h, Example 4 corresponding to 1 h, and Examples 8-12 corresponding to 2 h, 3 h, 4 h, 5 h, and 6 h, respectively. The effect of each sample on hyaluronic acid synthesis was then tested.

[0150] Test Procedure: Human keratinocytes were aliquoted into 96-well plates and allowed to adhere stably. Then, PDRN samples at a final concentration of 100 μg / mL were added to each well, and the plates were treated for 24 hours. Experimental groups consisted of PDRN samples prepared from the same pretreated raw material at different incubation times (0, 1, 2, 3, 4, 5, and 6 hours). The untreated control group received culture medium without any sample, and the positive control group received culture medium supplemented with 5 ng / mL TGF. β1 treatment group. After culture, cells were washed twice with PBS, and proteins were extracted using RIPA buffer. Total protein content was quantified using the BCA method. Hyaluronic acid content was quantified using a commercial Hyaluronic Acid ELISA kit. Absorbance was measured at 450 nm, and the HA production in each group was calculated using the standard curve.

[0151] See Figure 4 It is known that the PDRN prepared according to the extraction process provided in the embodiments of this application has the effect of promoting hyaluronic acid synthesis. Among them, when the incubation time is 4 h to 5 h, the PDRN with the highest proportion of 400bp to 1200bp has a more obvious effect.

[0152] Experimental Example 6 PDRN UVB damage test Test method: The products extracted from Comparative Example 11, Example 4, and Examples 8-12 were used as samples, with Comparative Example 11 corresponding to 0 h, Example 4 corresponding to 1 h, and Examples 8-12 corresponding to 2 h, 3 h, 4 h, 5 h, and 6 h, respectively. The UVB damage resistance efficacy of each sample was then tested.

[0153] Test Procedure: Human Dermal Fibroblasts (HDF) were seeded in 96-well plates and cultured until sufficient cell density was reached. Each sample was then treated with a concentration of 100 μg / mL for 2 hours (pretreatment). After pretreatment, UVB at 60 mJ / cm² was applied to each well. 2 The cells were then exposed to UVB radiation and cultured for 24 hours. Cell viability was evaluated using the MTT assay, and the measured absorbance was converted to relative survival (%) compared to the unexposed UVB control group.

[0154] See Figure 5 It is known that the PDRN prepared according to the extraction process provided in the embodiments of this application has the effect of resisting UVB damage. Among them, when the incubation time is 4 h to 5 h, the PDRN with the highest proportion of 400 bp to 1200 bp has a more obvious effect.

[0155] Experimental Example 7 Tests to inhibit intracellular ROS production by PDRN Test method: The products extracted from Comparative Example 11, Example 4, and Examples 8-12 were used as samples, with Comparative Example 11 corresponding to 0 h, Example 4 corresponding to 1 h, and Examples 8-12 corresponding to 2 h, 3 h, 4 h, 5 h, and 6 h, respectively. The effect of each sample on inhibiting intracellular ROS generation was then tested.

[0156] Test Procedure: After culturing human skin fibroblasts (HDF) to an appropriate state, different samples were pretreated with the cells at a concentration of 100 μg / mL for 2 hours. Subsequently, 500 μM hydrogen peroxide was added to induce oxidative stress. After a certain reaction time, the cells were analyzed using the fluorescent probe CM. Cells were stained with H2DCFDA (10 μM) to detect ROS generation. After staining, the fluorescence intensity of the cells was measured using a microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 528 nm to quantitatively analyze the ROS level.

[0157] In this experiment, the inhibition effect of intracellular ROS generation was compared with the normal control group without peroxidation treatment as the biological reference. However, to more clearly compare the relative protective and recovery effects of each treatment group under oxidative stress, the ROS fluorescence value of the peroxidation treatment control group was set to 100 in the graphs and quantitative analysis. The results are expressed as "relative change rate (% of oxidative stress control group)". "Stress control" is indicated.

[0158] See Figure 6It is known that the PDRN prepared according to the extraction process provided in the embodiments of this application has the effect of inhibiting the generation of intracellular ROS. Among them, when the incubation time is 4 h to 5 h, the PDRN with the highest proportion of 400 bp to 1200 bp has a more obvious effect.

[0159] Experimental Example 8 PDRN anti-inflammatory and sedative test Test method: The products extracted from Comparative Example 11, Example 4, and Examples 8-12 were used as samples, with Comparative Example 11 corresponding to 0 h, Example 4 corresponding to 1 h, and Examples 8-12 corresponding to 2 h, 3 h, 4 h, 5 h, and 6 h, respectively. The anti-inflammatory and sedative effects of each sample were then tested.

[0160] Test Procedure: The test was conducted using the mouse macrophage line RAW264.7 in a lipopolysaccharide (LPS)-induced nitric oxide production model. RAW264.7 cells were seeded into 96-well plates and cultured stably. Samples were added to each well at a concentration of 100 μg / mL. LPS was then added to a final concentration of 1 μg / mL to induce an inflammatory response, and the cells were cultured for 48 hours. After culture, the supernatant from each well was collected, reacted with Griess reagent, and the absorbance was measured at 540 nm to quantify NO production. NO concentration was calculated using a nitrite standard curve.

[0161] In this experiment, the NO inhibition rate was calculated using the LPS-treated inflammation-induced group as the control group, with its NO production level set at 100. The reduction in NO production in each PDRN-treated group relative to this value was converted into the relative inhibition rate (% of control). The NO inhibition rate in the untreated control group was set to 0. All experiments were repeated twice under the same conditions, and the results are expressed as mean and standard deviation.

[0162] See Figure 7 It is known that the PDRN prepared according to the extraction process provided in the embodiments of this application has anti-inflammatory and sedative effects. Among them, when the incubation time is 4 h to 5 h, the PDRN with the highest proportion of 400 bp to 1200 bp has a more obvious effect.

[0163] Experimental Example 9 PDRN anti-wrinkle test Test method: The products extracted from Comparative Example 11, Example 4, and Examples 8-12 were used as samples, with Comparative Example 11 corresponding to 0 h, Example 4 corresponding to 1 h, and Examples 8-12 corresponding to 2 h, 3 h, 4 h, 5 h, and 6 h, respectively. The anti-wrinkle effect of each sample was then tested.

[0164] Test Procedure: Human dermal fibroblasts (HDF) were used. After cell culture, oxidative stress was induced by adding 200 μM hydrogen peroxide, followed by the addition of PDRN samples at a concentration of 100 μg / mL, and the cells were cultured for 48 hours. The control group was the oxidative stress group treated with H2O2 only, and the positive control group was the group treated with 50 μM epigallocatechin gallate (EGCG), a potent antioxidant. After culture, the culture supernatant was collected and analyzed using a commercial MMP. 1. Quantitative analysis of MMP using an ELISA kit 1. Protein expression level.

[0165] In this experiment, the MMP of the H2O2-treated group was... The expression level was set at 100 as the baseline for relative expression (% of control). Results showed that the MMP levels in the EGCG-treated group were... 1. The expression level decreased to below 5%, indicating that it can significantly block oxidative stress-induced MMPs. 1. Upward adjustment.

[0166] See Figure 8 It is known that the PDRN prepared according to the extraction process provided in the embodiments of this application has anti-wrinkle effect. Among them, when the incubation time is 4 h to 5 h, the PDRN with the highest proportion of 400 bp to 1200 bp has a more obvious effect.

[0167] Experimental Example 10 Tests to demonstrate PDRN's role in promoting collagen synthesis Test method: The products extracted from Comparative Example 11, Example 4, and Examples 8-12 were used as samples, with Comparative Example 11 corresponding to 0 h, Example 4 corresponding to 1 h, and Examples 8-12 corresponding to 2 h, 3 h, 4 h, 5 h, and 6 h, respectively. The efficacy of each sample in promoting collagen synthesis was then tested.

[0168] Test procedure: HDF cells were prepared at a rate of 2 × 10⁻⁶. 5 Inoculate the cells at a density of 1 cell / well in 6-well plates and incubate for 24 hours to allow them to adhere. The culture medium contains 10% FBS and 1% penicillin. DMEM containing streptomycin was used. The culture medium was then changed, and cells were treated with PDRN samples at a final concentration of 100 μg / mL for 72 hours. The control group consisted of untreated cells supplemented with an equal volume of distilled water (approximately 200 μg), while the positive control group consisted of cells supplemented with 5 ng / mL of TGF, known to promote collagen synthesis. β1 treatment group. After incubation, the culture supernatant from each well was collected, and PIP was measured using a commercial Procollagen Type I ELISA kit according to the manufacturer's instructions. 1. Protein content. The measured PIP... 1. The numerical values ​​were standardized using the untreated control group (0) as a baseline and converted to relative expression level (% of control) for comparison and analysis.

[0169] See Figure 9 It is known that the PDRN prepared according to the extraction process provided in the embodiments of this application has the effect of promoting collagen synthesis. Among them, when the incubation time is 4 h to 5 h, the PDRN with the largest proportion of 400 bp to 1200 bp has a more obvious effect.

[0170] Experimental Example 11 Tests to demonstrate PDRN's role in promoting cell regeneration and wound healing Test method: The products extracted from Comparative Example 11, Example 4, and Examples 8-12 were used as samples, with Comparative Example 11 corresponding to 0 h, Example 4 corresponding to 1 h, and Examples 8-12 corresponding to 2 h, 3 h, 4 h, 5 h, and 6 h, respectively. The cell regeneration and wound healing effects of each sample were then tested.

[0171] Test Procedure: Human skin fibroblasts (HDF) were seeded into 6-well plates and cultured to form a monolayer. A linear artificial wound was then created in the center of the cell layer using a sterile scratcher. Cells were subsequently washed with PBS to remove detached cells, and PDRN sample was added to each well to a final concentration of 100 μg / mL. The untreated control group received only culture medium. At 0 hours after wound formation and at the end of 24 hours of culture, the wound area was photographed using an optical microscope. Image analysis software was used to calculate the ratio of the initial wound area to the wound closure area after 24 hours. The wound repair rate of each sample was converted to a relative value (%) based on the 100% repair rate of the untreated control group for comparison.

[0172] See Figure 10It is known that the PDRN prepared according to the extraction process provided in the embodiments of this application has the effect of promoting cell regeneration and wound healing. Among them, when the incubation time is 4 h to 5 h, the PDRN with the highest proportion of 400 bp to 1200 bp has a more obvious effect.

[0173] Experimental Example 12 Tests on the regulation of skin microbiome balance by PDRN Test method: The products extracted from Comparative Example 11, Example 4, and Examples 8-12 were used as samples, with Comparative Example 11 corresponding to 0 h, Example 4 corresponding to 1 h, and Examples 8-12 corresponding to 2 h, 3 h, 4 h, 5 h, and 6 h, respectively. The efficacy of each sample in regulating the balance of skin microbiota was then tested.

[0174] Test Procedure: This study aimed to evaluate the effects of PDRN incubation times on the growth and community balance of representative skin microbiota, including probiotics (Staphylococcus epidermidis ATCC 12228), harmful bacteria (Staphylococcus aureus ATCC 6538), and Propionibacterium acnes ATCC 6919.

[0175] The microbial strains used in the experiment were all prepared to 1×10⁻⁶ using their respective suitable methods. 6 Inoculum solution of cfu / mL. Single colonies of *S. epidermidis* and *S. aureus* were picked from TSA plates and inoculated into TSB liquid medium, and cultured at 37°C, aerobic conditions, and 130 rpm for 48 hours. To prepare the inoculum solution, the culture was centrifuged (6000×g, 20 min), the supernatant was discarded, and the culture was resuspended and washed with 0.85% NaCl; this process was repeated three times. Single colonies of *P. acnes* were picked from RCM plates and cultured in RCM liquid medium at 37°C, anaerobic conditions (using an anaerobic tank / gas bag), and 130 rpm for 72 hours. The inoculum solution was then prepared by washing with NaCl as described above. The bacterial suspension concentration was adjusted uniformly according to the OD600 value before culturing and counting all strains.

[0176] The minimal culture medium used in this experiment was formulated as follows: 10 g glucose (1%), 5 g NaCl, 0.05 g MgSO4·7H2O, and 0.01 g CaCl2 (optional) per 1 L. It contained no nitrogen or phosphorus sources (no added ammonium salts, phosphates, peptones, yeast extracts, etc.), and the final pH was adjusted to approximately 7.0. In this medium, nitrogen and phosphorus were provided solely by the added PDRN samples. Each PDRN sample was added to the minimal culture medium at a concentration of 100 μg / mL, serving as the treatment group; the control group consisted of only the culture medium without any extracts.

[0177] The microbial mixing and cultivation process was as follows: 250 mL of minimal culture medium was dispensed into sterile 500 mL Erlenmeyer flasks, and then equal amounts of *S. epidermidis* ATCC 12228, *S. aureus* ATCC 6538, *S. aureus* ATCC 6539, and *P. acnes* ATCC 6919 were inoculated. The flasks were tightly sealed with screw caps and wrapped with 1-2 layers of sealing film and aluminum foil to prevent outside air from entering. This maintained a relatively aerobic environment in the early stages of cultivation, and as time progressed, the oxygen in the flasks was gradually depleted, naturally forming microaerobic-anaerobic conditions. All flasks under these conditions were incubated at 37°C and 130 rpm on a constant-temperature shaker for 48 hours. *S. epidermidis* and *S. aureus* mainly proliferated in the aerobic environment in the early stages of cultivation, while *P. acnes* proliferated more favorably in the later stages of cultivation under the subsequently formed microaerobic / anaerobic conditions. After 48 hours of incubation, each sample was inoculated onto TSA agar (for *S. epidermidis* and *S. aureus*) plates using the dilution plating method and incubated at 37°C under aerobic conditions. For *P. acnes*, samples were inoculated onto RCM agar plates and incubated at 37°C under anaerobic conditions. Bacterial species were distinguished by colony morphology: *S. epidermidis* were white to milky white, small, glossy colonies (1–2 mm); *S. aureus* were golden yellow to pale yellow, glossy, round colonies; and *P. acnes* ATCC 6919 were milky white to pale white, very small, and flat colonies. The colony counts (CFU) obtained under each condition were calculated using the initial inoculum concentration of 1×10⁻⁶ for the Control (0 h) group. 6 The number of colonies corresponding to cfu / mL is 100%, which is expressed as a relative growth rate (%).

[0178] See Figure 11It is evident that the PDRN prepared according to the extraction process provided in this application all exhibit the effect of regulating the balance of the skin microbiome. Specifically, it can promote the growth of beneficial skin bacteria such as *S. epidermidis*, while having a relatively smaller effect on the growth of harmful bacteria and acne-causing bacteria. Among these, the effect is more pronounced when the incubation time is 4-5 hours, as the proportion of PDRN in the 400-1200 bp range is the highest. This is presumably due to the combined effect of multiple factors, including the nitrogen and phosphorus supply capacity of PDRN, its molecular weight distribution, pH regulation, and the resource competition structure among microorganisms, under optimal incubation conditions, which leads to the dominance of beneficial bacteria and the effective regulation of the proliferation of harmful bacteria and acne-causing bacteria in the skin microbiome environment.

[0179] Experimental Example 13 Tests on PDRN's anti-glycation and anti-aging effects Test method: The products extracted from Comparative Example 11, Example 4, and Examples 8-12 were used as samples, with Comparative Example 11 corresponding to 0 h, Example 4 corresponding to 1 h, and Examples 8-12 corresponding to 2 h, 3 h, 4 h, 5 h, and 6 h, respectively. The anti-glycation and anti-aging effects of each sample were then tested.

[0180] Test Procedure: This study aims to evaluate the inhibitory effect of PDRN with different incubation times on the production of advanced glycation end products (AGEs) induced by high concentration of glucose (500 mM) stimulation in human skin fibroblasts (HDF).

[0181] HDF cells used in the experiment were pre-differentiated and stabilized before being aliquoted into 6-well plates. Each sample group was treated with 100 μg / mL of PDRN prepared at different incubation times for 24 hours. Separate control groups were included: an untreated control group, a stimulation group with only high-concentration glucose (500 mM), and a positive control group treated with aminoguanidine (500 μg / mL), a standard for inhibiting AGEs. In all groups, high-glucose stimulation was applied under conditions containing PDRN or the control substance for a total of 24 hours. Cell supernatants were then collected, and the amount of AGEs (advanced glycosylation end products) generated was quantitatively analyzed using a commercial ELISA kit. The AGEs generation in the untreated control group was set as 100%, and the AGEs levels under each condition were converted to relative values.

[0182] See Figure 12It was found that in the high glucose treatment group, the amount of AGEs generated was significantly higher than that in the untreated control group, reaching more than twice that of the control group. In the positive control group (aminoguanidine 500 μg / mL), the amount of AGEs generated was inhibited by more than 70% to 75% compared with the high glucose treatment group, showing a strong AGEs inhibition effect. In each PDRN sample group, the inhibitory effect on AGEs generation gradually increased with the extension of incubation time: especially at 4 hours and 5 hours, the level of AGEs generation was reduced by more than 40% compared with the high glucose stimulation group; a considerable degree of inhibition was also shown at 3 hours and 6 hours of maturation; and the generation of AGEs was also significantly reduced in the other maturation time groups (0-2 hours) compared with the high glucose control group.

[0183] This significant AGEs-inhibiting effect is believed to stem not only from the direct inhibition of glycation by the nucleotides and their combinations contained in PDRN, but also from its comprehensive effects through multiple indirect pathways, including alleviating intracellular oxidative stress (antioxidant), regulating inflammatory signals (anti-inflammatory), improving the cellular metabolic environment, interfering with the RAGE (AGEs receptor) signaling pathway, and activating anti-aging and antioxidant signaling molecules such as sirtuin and PPARG. It is thus hypothesized that PDRN maintains the metabolic homeostasis of cell membranes, DNA, and proteins, inhibits reactive oxygen species generation, promotes damage repair, and enhances anti-aging signals, enabling cells to maintain normal metabolic activity even in an environment of AGEs accumulation. In summary, the fern spore extract of this invention exhibits excellent effects in inhibiting intracellular AGEs production, possessing not only anti-glycation functions but also antioxidant and anti-aging functions. This verifies that this material can serve as a high-functionality new material for preventing skin aging, reducing glycation stress, and for use in health-functional cosmetics.

[0184] The embodiments described above are some, but not all, of the embodiments of this application. The detailed description of the embodiments of this application is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

Claims

1. A method for extracting PDRN, characterized in that, Includes the following steps: Stress treatment is applied to plant tissues to induce irreversible cell damage. The plant tissues subjected to stress treatment were incubated to allow DNA to be selectively and partially degraded by endogenous deoxyribonuclease. The plant tissue after incubation was broken up and purified to obtain PDRN.

2. The PDRN extraction method according to claim 1, characterized in that, The steps of stress treatment of plant tissues include: S11 Immerse the plant tissue in a sodium chloride solution with a mass concentration of 0.2%~0.5% for 20 min~60 min; S12 Immerse the plant tissue in water for 5 min to 20 min; S13 Repeat steps S11~S12 multiple times; Optionally, before immersing the plant tissue in the sodium chloride solution, the surface of the plant tissue may be scratched.

3. The PDRN extraction method according to claim 1, characterized in that, The incubation treatment steps include: adding cofactors to the stress-treated plant tissue and incubating it under conditions of pH 7-7.5 and temperature 30℃-40℃; Optionally, the incubation treatment lasts for 1 h to 6 h, or / and the cofactor is selected from at least one of calcium ions and magnesium ions; Optionally, the incubation treatment can last for 3 to 5 hours.

4. The method for extracting PDRN according to any one of claims 1 to 3, characterized in that, The plant tissues include fern tissues; Optionally, the fern tissue includes leaves; Optionally, the leaf is selected from spore leaves.

5. The PDRN extraction method according to claim 4, characterized in that, The plant tissue is fresh plant tissue that has not been dried or heat-treated after harvesting, and / or the fern tissue includes at least one of the following: European hairy fern tissue, oak fern tissue, and maidenhair fern tissue.

6. The method for extracting PDRN according to any one of claims 1 to 3, characterized in that, The extraction and purification process includes the following steps: The shredded plant tissue was added to CTAB buffer and subjected to thermal extraction at 50℃~70℃ to obtain the extract. A mixed solvent of chloroform and isoamyl alcohol was added to the extract and centrifuged to obtain a precursor solution containing PDRN. Isopropanol was added to the precursor solution and the mixture was centrifuged, washed with alcohol, and dried to obtain PDRN powder.

7. The PDRN extraction method according to claim 6, characterized in that, The CTAB buffer solution comprises: 1.5%–2.5% CTAB (w / w), 1.3 M–1.5 M NaCl, and 80 mM–120 mM Tris. HCl, 15 mM~25 mM EDTA, 0.5%~2% PVP (mass concentration), and 0.1%~0.3% β-D-methyl (mass concentration) Mercaptoethanol; Or / and, the volume ratio of the extract to the mixed solvent is (0.8~1.2):(0.8~1.2), and the volume ratio of chloroform to isoamyl alcohol in the mixed solvent is (20~30):

1.

8. A PDRN, characterized in that, The PDRN is prepared by the extraction method according to any one of claims 1 to 7, wherein the length of a portion of the PDRN is 400 bp to 1200 bp, and the OD260 / OD280 of the PDRN is 1.8 to 2.

1.

9. The use of the PDRN as described in claim 8 in the preparation of products that promote cell proliferation and improve cell survival rate, products that promote hyaluronic acid synthesis and collagen or elastin expression, products that inhibit reactive oxygen species generation and resist UVB or oxidative stress, products that have anti-inflammatory and soothing effects, products that have anti-wrinkle and improve skin elasticity, products that promote cell migration and wound healing, products that regulate the balance of skin microbiota, or products that have anti-glycation and anti-aging effects.

10. The application according to claim 9, characterized in that, The products include at least one of the following: food, pharmaceuticals, cosmetics, biomedical materials, topical skin preparations, health products, food additives, and cosmetic compositions.

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