High purity PDRN extract derived from microalgae and method for preparing the same

Abstract

The present disclosure relates to a high purity PDRN extract derived from microalgae and a method for producing the same.SOLUTION: The high-purity PDRN extract derived from microalgae according to the present disclosure is easy to produce and has excellent sustainability because it is produced from microalgae that can be mass-produced at any time without external environment or time constraints, is safe because there is no risk of human infection by exogenous viruses, and has an excellent skin absorption rate when applied to products such as cosmetics due to its low molecular weight.SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] The present disclosure relates to a highly purified PDRN extract derived from microalgae and a method for producing the same.

[0002] [Explanation regarding government-supported research and development] This research was conducted under the supervision of Amorepacific Corporation with support from the Ministry of Oceans and Fisheries of Korea's Marine Biomaterial Formulation Technology Development Project (Development of mass production process for microalgae-derived PDRN and standardization of highly effective skin cosmetic materials, Project ID: 1525014981). [Background technology]

[0003] PDRN (Polydeoxyribonucleotide) is a tissue regenerative active substance. It is a polymer with a molecular weight of 50 to 1500 kDa manufactured from DNA that promotes the self-regeneration of damaged cells and tissues. Unlike ordinary DNA, PDRN does not transmit genetic information and exhibits pharmacological activity. It stimulates the A2 receptor, a signaling substance for skin regeneration, promoting the secretion of various growth factors, and has effects such as capillary regeneration through vascular endothelial growth factor (VEGF), improving blood circulation, having anti-inflammatory effects, and preventing capillary leakage.

[0004] PDRN, which resides within human cells, helps maintain optimal health by physiologically stimulating the regeneration and metabolic activity of fibroblasts. PDRN has attracted considerable attention following research results showing its effectiveness in wound treatment, such as burns, by promoting skin regeneration without any particular side effects. It is known to have anti-inflammatory effects by accelerating tissue regeneration, shortening wound healing time, activating cell regeneration, promoting the production of both collagen and noncollagenous proteins, and promoting the differentiation of various cells (stem cells, fibroblasts, osteoblasts, chondrocytes, etc.).

[0005] Because PDRNs are DNA polymers and biological components, they have the advantage of not eliciting allergic reactions or in vivo rejection, which are common with synthetic substances. Most PDRN sources are derived from fish, with DNA extracted from the semen and testes of fish such as salmon, from which PDRNs are mass-purified. However, due to the risk of mass fish deaths and infection, as well as time constraints such as the salmon's migration cycle, there is a need for alternative methods to purify plant-derived PDRNs that can be mass-cultivated and harvested on a continuous basis. In particular, extracting PDRNs from plants rather than animals avoids the risk of zoonotic diseases due to the contamination of viruses from animal tissues and exogenous viruses that pose a risk of human infection. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Korean Patent Publication No. 10-2012-0129693 Summary of the Invention [Problem to be solved by the invention]

[0007] The present disclosure aims to provide a highly pure PDRN extract and a method for producing the same, which is easy to produce, highly sustainable, and safe with no risk of human infection by exogenous viruses, because it is made from microalgae that can be mass-cultivated continuously without being subject to external environmental or time constraints. [Means for solving the problem]

[0008] To achieve the above object, one embodiment of the present invention provides a highly purified polydeoxyribonucleotide (PDRN) extract derived from microalgae, in which the DNA content relative to the total dry weight of the PDRN extract is 49% by weight or more.

[0009] In another embodiment of the present invention, there is provided a cosmetic composition containing the PDRN extract as an active ingredient.

[0010] Furthermore, in another embodiment of the present invention, there is provided a method for producing a highly pure PDRN extract derived from microalgae, comprising the steps of: (1) adding an anionic surfactant and a metal salt to a microalgae lysate and stirring to obtain a cell lysate; (2) centrifuging the cell lysate of step (1), filtering the resulting filtrate, adding a metal salt to the filtrate, and stirring the resulting filtrate; (3) centrifuging the stirred solution of step (2), filtering the resulting filtrate, adding a C1-C4 alcohol to the filtrate, and centrifuging the resulting filtrate to obtain a precipitate; (4) adding a C1-C4 alcohol to the precipitate of step (3), and centrifuging the resulting filtrate to obtain a precipitate; and (5) freeze-drying the precipitate of step (4). [Effects of the Invention]

[0011] The high-purity PDRN extract derived from microalgae disclosed herein is made from microalgae that can be mass-cultivated continuously without being subject to external environmental or time constraints, making it easy to produce and highly sustainable. It is also safe and poses no risk of human infection by foreign viruses, and because it has a low molecular weight, it has excellent skin absorption when used in products such as cosmetics. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 is a diagram showing the PDRN extraction process according to Production Example 1. [Figure 2] FIG. 1 is a graph showing the results of confirming the cell lysis effect depending on the surfactant concentration in Test Example 1. [Figure 3] FIG. 1 shows the results of confirming the PDRN extraction process and cell lysis effect according to Production Example 2. [Figure 4] FIG. 1 shows a process for extracting PDRN according to Production Example 3. [Figure 5] FIG. 1 shows a process for extracting PDRN according to Production Example 4. [Figure 6A]This figure shows the results of confirming the DNA size of PDRNs derived from gold chlorella (Chlorella protothecoides), green chlorella (Chlorella vulgaris), and white chlorella (Chlorella protothecoides). [Figure 6B] This figure shows the results of confirming the DNA size of PDRNs derived from gold chlorella (Chlorella protothecoides), green chlorella (Chlorella vulgaris), and white chlorella (Chlorella protothecoides). [Figure 7] FIG. 1 shows the results of confirming the DNA size of highly purified PDRN derived from Gold Chlorella. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention will be described in detail below.

[0014] In one aspect, the present invention relates to a highly purified polydeoxyribonucleotide (PDRN) extract derived from microalgae, in which the DNA content relative to the total dry weight of the PDRN extract is 49% by weight or more.

[0015] In one embodiment, the PDRN extract has a DNA content of at least 49% by weight, at least 50% by weight, at least 51% by weight, at least 52% by weight, at least 53% by weight, at least 54% by weight, at least 55% by weight, at least 56% by weight, at least 57% by weight, at least 58% by weight, at least 59% by weight, at least 60% by weight, at least 61% by weight, at least 62% by weight, at least 63% by weight, at least 64% by weight, at least 65% by weight, at least 66% by weight, at least 67% by weight, at least 68% by weight, at least 69% by weight, at least 70% by weight, at least 71% by weight, at least 72% by weight, at least 74% by weight, at least 75% by weight, at least 76% by weight, at least 77% by weight, at least 78% by weight, at least 79% by weight, at least 80% by weight, at least 81% by weight, at least 82% by weight, at least 83% by weight, at least 84% by weight, at least 85% by weight, at least 86% by weight, at least 87% by weight, at least 88% by weight, at least 89% by weight, at least 90% by weight, at least 91% by weight, at least 92% by weight, at least 93% by weight, at least 94% by weight, at least 95% by weight, at least 96% by weight, at least 97% by weight, at least 98% by weight, at least 99% by weight, at least 100% by weight, at least 101% by weight, at least 102% by weight, at least 103% by weight, at least 104% % or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more by weight.

[0016] In one embodiment, at least 90% or more of the DNA in the PDRN extract may be low molecular weight DNA having a molecular weight of 20 kDa or less.

[0017] Specifically, at least 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the DNA in the PDRN extract may have a molecular weight of 20 kDa or less, 19 kDa or less, 18 kDa or less, 17 kDa or less, 16 kDa or less, 15 kDa or less, 14 kDa or less, 13 kDa or less, or 12 kDa or less; Alternatively, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the DNA in the PDRN extract may have a molecular weight of 10 kDa or more, 11 kDa or more, 12 kDa or more, 13 kDa or more, 14 kDa or more, 15 kDa or more, 16 kDa or more, 17 kDa or more, 18 kDa or more, or 19 kDa or more.

[0018] In one embodiment, the PDRN extract may be in the form of a white powder.

[0019] In one embodiment, the microalgae may be at least one species selected from Chlorella, Spirulina, Dunaliella, Haematococcus, Euglena, Nannochloropsis, Nostoc, and Schizochytrium.

[0020] The chlorella may be, for example, at least one species selected from gold chlorella (Chlorella protothecoides), green chlorella (Chlorella vulgaris), and white chlorella (Chlorella protothecoides), and in particular, gold chlorella (Chlorella protothecoides).

[0021] In yet another aspect, the present invention relates to a method for producing a highly pure PDRN extract from microalgae.

[0022] In one embodiment, the method may include the steps of: (1) adding an anionic surfactant and a metal salt to a microalgae lysate and stirring the mixture to obtain a cell lysate; (2) centrifuging the cell lysate of step (1), filtering the resulting filtrate, adding a metal salt to the filtrate, and stirring the resulting filtrate; (3) centrifuging the stirred solution of step (2), filtering the resulting filtrate, adding a C1-C4 alcohol to the filtrate, and centrifuging the resulting filtrate to obtain a precipitate; and (4) a second purification step of adding a C1-C4 alcohol to the precipitate of step (3), and centrifuging the resulting filtrate to obtain a precipitate.

[0023] In one embodiment, the microalgae lysate in step (1) is a mixture of microalgae powder and purified water, and the weight of the microalgae lysate may be 20 times or more the weight of the microalgae powder.

[0024] In one embodiment, the microalgae in step (1) may be at least one selected from gold chlorella (Chlorella protothecoides), green chlorella (Chlorella vulgaris), white chlorella (Chlorella protothecoides), Spirulina, Dunaliella, Haematococcus, Euglena, Nannochloropsis, Nostoc, and Schizochytrium.

[0025] In one embodiment, the anionic surfactant in step (1) may be sodium dodecyl sulfate (SDS).

[0026] In one embodiment, the method may further comprise, before step (2), a step of repeatedly applying an ultra-high pressure of 500 to 2,000 bar to the mixture of step (1) three or more times to obtain cell lysates.

[0027] Specifically, the pressure applied to the mixture in step (1) is 500 bar or more, 600 bar or more, 700 bar or more, 800 bar or more, 900 bar or more, 1,000 bar or more, 1,100 bar or more, 1,200 bar or more, 1,300 bar or more, 1,400 bar or more, 1,500 bar or more, 1,600 bar or more, 1,700 bar or more, 1,800 bar or more, or 1,900 bar or more. The pressure applied to the mixture in step (1) may be 2,000 bar or less, 1,900 bar or less, 1,800 bar or less, 1,700 bar or less, 1,600 bar or less, 1,500 bar, 1,400 bar, 1,300 bar, 1,200 bar, 1,100 bar or less, 1,000 bar or less, 900 bar or less, 800 bar or less, 700 bar or less, or 600 bar or less.

[0028] In one embodiment, the metal salt may be NaCl (sodium chloride).

[0029] In one embodiment, the concentration of the metal salt added in step (2) may be at least twice the concentration of the metal salt added in step (1).

[0030] In one embodiment, in step (3), the volume ratio of the filtrate to the C1-C4 alcohol added to the filtrate may be 1:1 to 3. Specifically, the volume ratio of the filtrate to the C1-C4 alcohol may be 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, or 1:3.

[0031] In one embodiment, in step (2), the metal salt and the cationic surfactant may be added together.

[0032] In one embodiment, the cationic surfactant may be cetyl trimethyl ammonium bromide (CTAB).

[0033] In one embodiment, the step (4) may include the steps of adding purified water to the precipitate from the step (3), stirring and dissolving the precipitate, and adding a C1-C4 alcohol to the dissolved product and centrifuging the mixture to obtain a precipitate.

[0034] In one embodiment, the volume ratio of the lysate to the C1-C4 alcohol added to the lysate may be 1:1 to 3. Specifically, the volume ratio of the lysate to the C1-C4 alcohol added to the lysate may be 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 3:7, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, or 1:3.

[0035] In one embodiment, the C1-C4 alcohol in step (3) and the C1-C4 alcohol in step (4) may be different from each other.

[0036] In one embodiment, the C1 to C4 alcohols may each independently be methanol, ethanol, propyl alcohol, isopropyl alcohol, or butanol.

[0037] In one embodiment, the method may further comprise the step of (5) freeze-drying the precipitate of step (4).

[0038] In one embodiment, in the step (5), purified water may be added to the precipitate from the step (4), followed by stirring and dissolving, and the resulting solution may be freeze-dried.

[0039] In another aspect, the present invention relates to a cosmetic composition containing the PDRN extract as an active ingredient.

[0040] The cosmetic composition according to one embodiment of the present invention may contain a cosmetically or dermatologically acceptable medium or base. This may be any formulation suitable for topical application, such as a solution, gel, solid, anhydrous product, emulsion obtained by dispersing an oil phase in an aqueous phase, suspension, microemulsion, microcapsules, microgranules, or ionic (liposome) and non-ionic vesicular dispersions, or in the form of a cream, lotion, emulsion, powder, ointment, spray, or stick concealer. These compositions may be prepared according to conventional methods in the art. The cosmetic composition may also be used in the form of an aerosol composition further comprising a propellant compressed in the form of a foam.

[0041] The cosmetic composition is not particularly limited in its formulation and can be appropriately selected depending on the purpose. For example, the cosmetic composition may be produced in the form of a skin lotion, a skin softener, a toner, an emulsion, a lotion, a moisturizing lotion, a nutritious lotion, a massage cream, a nutritious cream, a moisturizing cream, a hand cream, a foundation, a beauty serum, a nutritious essence, a pack, a soap, a facial cleanser, a facial cleanser, a facial lotion, a facial cleanser cream, a cleansing water, a powder, a body lotion, a body cream, a body oil, a body cleanser, a body essence, or the like.

[0042] When the cosmetic composition is formulated as a paste, cream, or gel, animal fibers, plant fibers, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. may be used as a carrier component.

[0043] When the cosmetic composition is formulated as a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component, and particularly in the case of a spray, a propellant such as hydrochlorofluorocarbon, propane / butane, or dimethyl ether may further be included.

[0044] When the cosmetic composition is formulated as a solution or emulsion, a solvent, solvating agent, or emulsifier is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerin aliphatic esters, polyethylene glycol, and fatty acid esters of sorbitan.

[0045] When the cosmetic composition is prepared as a suspension, the carrier component may be a liquid diluent such as water, ethanol, or propylene glycol; a suspending agent such as ethoxylated stearyl alcohol, polyoxyethylene sorbitol ester, or polyoxyethylene sorbitan ester; or microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tragacanth.

[0046] When the cosmetic composition is in the form of a surfactant-containing cleanser, examples of the carrier component that may be used include fatty alcohol sulfates, fatty alcohol ether sulfates, sulfosuccinate monoesters, isethionates, imidazolium derivatives, methyl taurates, sarcosinates, fatty acid amide ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, and ethoxylated glycerin fatty acid esters.

[0047] In addition to the green tea peptides, the cosmetic composition may further include functional additives and ingredients commonly found in cosmetic compositions. The functional additives may include ingredients selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, polymeric peptides, polymeric polysaccharides, sphingolipids, and seaweed extracts.

[0048] The composition may further contain, in addition to the functional additives, ingredients commonly contained in cosmetic compositions, as needed, such as oils and fats, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, UV absorbers, preservatives, disinfectants, antioxidants, plant extracts, pH adjusters, alcohols, colorants, fragrances, blood circulation enhancers, cooling agents, antiperspirants, purified water, etc.

[0049] In another aspect, the present invention relates to an injectable composition containing the PDRN extract as an active ingredient.

[0050] In one embodiment, the injectable composition may be an injectable composition for tissue repair.

[0051] In one embodiment, the injectable composition may be injected into the dermal layer of the skin.

[0052] In another aspect, the present invention relates to a food composition, a health functional food composition, or a non-therapeutic oral composition containing the PDRN extract as an active ingredient.

[0053] In one embodiment, the food composition or non-therapeutic oral composition is formulated into, but is not limited to, tablets, granules, pills, powders, liquids such as drinks, caramels, gels, bars, tea bags, etc. In addition to the active ingredient, the composition of each formulation can be formulated by a person of ordinary skill in the art by appropriately selecting and blending ingredients commonly used in the art according to the formulation or intended use, and a synergistic effect can be achieved when other ingredients are used simultaneously.

[0054] In one embodiment, the food composition or non-therapeutic oral composition can be administered in a variety of ways, such as by simple ingestion, drinking, injection, spraying, squeezing, etc.

[0055] In one embodiment, the food composition or non-therapeutic oral composition may be, for example, various foods such as chewing gum, chocolate, caramel products, candies, ice creams, and confectioneries; beverages such as soft drinks, mineral water, and alcoholic beverages; or functional foods containing vitamins, minerals, and the like.

[0056] In one embodiment, the food composition or non-therapeutic oral composition may be ingested as is or may be used together with other foods or food ingredients, and may be used appropriately in a conventional manner. The food composition or non-therapeutic oral composition may contain, in addition to the PDRN extract, a phytologically acceptable food supplement additive, and the amount of the active ingredient to be mixed is determined appropriately depending on the purpose of use.

[0057] In this specification, the term "supplementary food additive" refers to a component added to food as a supplement, which is added when producing functional foods of each formulation, and can be appropriately selected and used by those skilled in the art. Examples of supplementary food additives include various supplements, vitamins, minerals (electrolytes), flavorings such as synthetic flavorings and natural flavorings, colorants and fillers, pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloids, thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonation agents used in carbonated beverages, but the types of supplementary food additives of the present invention are not limited to these examples.

[0058] As used herein, the term "functional food" refers to food manufactured and processed into the form of tablets, capsules, powders, granules, liquids, pills, etc. using raw materials or ingredients that have beneficial functions for the human body. Here, "functional" refers to regulating nutrients for the structure and function of the human body or providing beneficial effects for health purposes, such as physiological effects. The functional food of the present invention can be manufactured by methods commonly used in the art, and can be manufactured by adding raw materials and ingredients commonly added in the art. Furthermore, the functional food formulation may be any formulation recognized as a functional food. The food composition can be manufactured into various forms of formulations.

[0059] The present invention can provide the following embodiment as one embodiment.

[0060] The first embodiment can provide a highly pure polydeoxyribonucleotide (PDRN) extract derived from microalgae, in which the DNA content relative to the total dry weight of the PDRN extract is 49% by weight or more.

[0061] The second embodiment can provide a PDRN extract in the first embodiment, in which the DNA content relative to the total dry weight of the PDRN extract is 70% by weight or more.

[0062] The third embodiment can provide a PDRN extract in at least one of the first and second embodiments, in which at least 90% or more of the DNA in the PDRN extract is low-molecular-weight DNA having a molecular weight of 20 kDa or less.

[0063] A fourth embodiment can provide a PDRN extract in at least one of the first to third embodiments, wherein the PDRN extract is in the form of a white powder.

[0064] A fifth embodiment is a PDRN extract according to at least one of the first to fourth embodiments, wherein the microalgae is at least one selected from gold chlorella (Chlorella protothecoides), green chlorella (Chlorella vulgaris), white chlorella (Chlorella protothecoides), Spirulina, Dunaliella, Haematococcus, Euglena, Nannochloropsis, Nostoc, and Schizochytrium.

[0065] The sixth embodiment includes the steps of: (1) adding an anionic surfactant and a metal salt to a microalgae lysate and stirring the mixture to obtain a cell lysate; (2) centrifuging the cell lysate from step (1), filtering the filtrate, adding a metal salt to the filtrate, and stirring the filtrate; (3) a first purification step in which the stirred solution of step (2) is centrifuged, filtered, and a C1-C4 alcohol is added to the filtrate obtained by centrifugation, followed by centrifugation to obtain a precipitate; (4) A method for producing a highly pure PDRN extract derived from microalgae can be provided, which comprises a second purification step of adding a C1-C4 alcohol to the precipitate from step (3) and centrifuging the mixture to obtain a precipitate.

[0066] The seventh embodiment can provide a manufacturing method in which, in the sixth embodiment, the microalgae dissolution solution in step (1) is a mixture of microalgae powder and purified water, and the weight of the microalgae dissolution solution is 20 times or more the weight of the microalgae powder.

[0067] An eighth embodiment can provide a production method according to at least one of the sixth to seventh embodiments, wherein the microalgae in the step (1) is at least one selected from gold chlorella (Chlorella protothecoides), green chlorella (Chlorella vulgaris), white chlorella (Chlorella protothecoides), Spirulina, Dunaliella, Haematococcus, Euglena, Nannochloropsis, Nostoc, and Schizochytrium.

[0068] A ninth embodiment can provide a production method according to at least one of the sixth to eighth embodiments, wherein the anionic surfactant in step (1) is sodium dodecyl sulfate (SDS).

[0069] The tenth embodiment can provide a manufacturing method in at least one of the sixth to ninth embodiments, further comprising, before step (2), a step of repeatedly applying an ultrahigh pressure of 500 to 2,000 bar to the mixture of step (1) three or more times to obtain cell lysates.

[0070] An eleventh embodiment can provide the production method according to at least one of the sixth to tenth embodiments, wherein the metal salt is NaCl.

[0071] The twelfth embodiment can provide a manufacturing method in which, in at least one of the sixth to eleventh embodiments, the concentration of the metal salt added in step (2) is at least twice the concentration of the metal salt added in step (1).

[0072] A thirteenth embodiment can provide a production method in at least one of the sixth to twelfth embodiments, wherein in step (2), the metal salt and the cationic surfactant are added together.

[0073] A fourteenth embodiment can provide a production method according to at least one of the sixth to thirteenth embodiments, wherein the cationic surfactant is cetyltrimethylammonium bromide (CTAB).

[0074] The fifteenth embodiment can provide a production method in which, in at least one of the sixth to fourteenth embodiments, the C1-C4 alcohol in step (3) and the C1-C4 alcohol in step (4) are different from each other.

[0075] The sixteenth embodiment can provide a cosmetic composition comprising, as an active ingredient, the PDRN extract of any one of the first to fifth embodiments, or the PDRN extract produced according to the method of any one of the sixth to fifteenth embodiments.

[0076] The seventeenth embodiment can provide a food composition comprising, as an active ingredient, the PDRN extract of any one of the first to fifth embodiments, or the PDRN extract produced according to the method of any one of the sixth to fifteenth embodiments.

[0077] The present invention will be described in more detail below with reference to embodiments and test examples. However, these embodiments and test examples are merely provided to aid in understanding the present invention, and do not limit the scope of the present invention. Modifications, substitutions, and insertions commonly known in the art may be made, and these are also included within the scope of the present invention.

[0078] [Production Example 1] Production of PDRN extract derived from Gold Chlorella Figure 1 outlines the procedure for producing PDRN extract from Gold Chlorella. Specifically, 900 g of purified water was added to 100 g of dried Gold Chlorella powder (manufactured by Daesang Co., Ltd.), and the mixture was homogenized in a mixer at 25–30°C for 30 minutes. Sodium dodecyl sulfate (SDS) and sodium chloride (NaCl) were added to the homogenate to a concentration of 1% (w / w), and the mixture was then lysed at 60°C for 2 hours with stirring. The lysate was centrifuged at 7000 rpm for 30 minutes to obtain the supernatant. The supernatant was then filtered through a 1 μm, 0.45 μm, and 0.2 μm particle size range. Ethanol was added to the filtrate at a volumetric ratio of 30:70, and the mixture was allowed to precipitate overnight at 0–4°C. The precipitate was then centrifuged at 5000 rpm for 10 minutes to obtain the precipitate. 100 g of purified water was added to this precipitate, which was then redissolved by stirring at room temperature for 30 minutes. Ethanol was added to this solution so that the volume ratio of solution to ethanol was 30:70, and the solution was allowed to precipitate overnight at 0-4°C. The precipitate was then centrifuged (5000 rpm, 10 minutes). The precipitate was then dried with hot air at room temperature for 3 hours to obtain a PDRN extract derived from Gold Chlorella.

[0079] The resulting PDRN extract was dissolved in purified water to a concentration of 10 mg / mL, and then the concentration and purity of DNA in the PDRN extract were measured using a Nanodrop-1000 instrument (Thermo-Fisher, Wilmington, DE, USA). DNA purity was assessed by the ratio of absorbance at 260 nm to 280 nm and the ratio of absorbance at 260 nm to 230 nm. Generally, a 260 / 280 ratio of 1.8 or higher indicates high-purity DNA with little protein contamination, while a 260 / 230 ratio of 1.8 or higher indicates high-purity DNA with little polysaccharide or polyphenol contamination. The results are shown in Table 1.

[0080] [Table 1]

[0081] From the results in Table 1, the 260 / 280 ratio was 1.99, indicating that proteins were sufficiently removed, but the 260 / 230 ratio was low at 1.21, indicating that polysaccharides and other substances were not removed and that the purity was low. The DNA content in the PDRN extract was approximately 10.7%, indicating that the purity of the DNA in the PDRN extract was also low.

[0082] [Test Example 1] Evaluation of cell lysis effect depending on anionic surfactant concentration 900 g of purified water was added to 100 g of dried gold chlorella powder (Daiso Co., Ltd.), and then homogenized in a mixer at 25–30°C for 30 minutes. Sodium dodecyl sulfate (SDS) and sodium chloride (NaCl) were added to the homogenate at concentrations of 1% (w / w) or 5% (w / w) and 1% (w / w), respectively, and the mixture was stirred at 60°C for 2 hours to lyse the cells. Cell morphology was then observed using an optical microscope (SZX16, Olympus Corporation, Japan). The results are shown in Figure 2.

[0083] The results in Figure 2 confirm that increasing the amount of anionic surfactant (SDS) used does not increase the chlorella cell lysis effect.

[0084] [Production Example 2] Production of PDRN extract derived from Gold Chlorella using an ultra-high pressure disperser Figure 3 shows a schematic diagram of the procedure for producing PDRN extract from gold chlorella using an ultra-high-pressure disperser. Specifically, 900 g of purified water was added to 100 g of dried gold chlorella powder (manufactured by Daesang Co., Ltd.), and the mixture was then homogenized in a mixer at 25–30°C for 30 minutes. SDS and NaCl were added to the homogenate to a concentration of 1% each, and the mixture was then stirred at 60°C for 2 hours to carry out cell lysis. The cell lysate was then disrupted three times using an ultra-high-pressure disperser (M110EH Microfluidizer, Microfluidics International Corporation, USA) at 1500 bar pressure. As shown in Figure 3, the cell disruption was confirmed to have a creamy texture. The morphology of the Chlorella cells after the first and second extractions was observed under an optical microscope, confirming complete lysis, as shown in Figure 3. The cell disruption was then centrifuged at 7000 rpm for 20 minutes to obtain the supernatant. The resulting supernatant was filtered through 1 μm, 0.45 μm, and 0.2 μm stages. Ethanol was added to the filtrate at a volumetric ratio of 30:70 filtrate:ethanol. The mixture was allowed to settle at 0-4°C for 1 hour, and then centrifuged at 5000 rpm for 10 minutes to obtain a precipitate. 100 g of purified water was added to the precipitate, which was then redissolved with stirring at room temperature for 30 minutes. 100 g of purified water was added to the lysate at a volumetric ratio of 30:70 lysate:ethanol. The mixture was allowed to settle at 0-4°C for 1 hour, and then centrifuged at 5000 rpm for 10 minutes to obtain a precipitate. 100 g of purified water was then added to the precipitate, which was then redissolved with stirring at room temperature for 30 minutes. The solution was then dried in a freeze dryer for 2 days, and a PDRN extract derived from Gold Chlorella was obtained using an ultra-high-pressure disperser.

[0085] The resulting PDRN extract was dissolved in purified water to a concentration of 10 mg / mL, and the DNA concentration and purity in the PDRN extract were then measured using a Nanodrop-1000 instrument (Thermo-Fisher, Wilmington, DE, USA). The results are shown in Table 2 below.

[0086] [Table 2]

[0087] The results in Table 2 show that, even when the ultra-high pressure disperser was used, the 260 / 280 ratio was 1.87, indicating that proteins were sufficiently removed, as in Production Example 1. However, the 260 / 230 ratio was low at 1.14, indicating that polysaccharides and the like were not removed and the purity was low. The DNA content in the PDRN extract was approximately 16.7%, indicating that although the purity of DNA in the PDRN extract had increased compared to Production Example 1, it was still at a low level.

[0088] [Production Example 3] Production of PDRN extract derived from Gold Chlorella using high-concentration metal salts Figure 4 shows a schematic diagram of the procedure for producing PDRN extract from gold chlorella using a high concentration of metal salts. Specifically, 900 g of purified water was added to 100 g of dried gold chlorella powder (manufactured by Daesang Co., Ltd.), and the mixture was then homogenized in a mixer at 25–30°C for 30 minutes. SDS and NaCl were added to the homogenate to a concentration of 1%, and the mixture was then stirred at 60°C for 2 hours to carry out cell lysis. The cell lysate was then disrupted three times using an ultra-high-pressure disperser at 1500 bar. The disrupted cell was then centrifuged at 7000 rpm for 20 minutes to obtain the supernatant. The resulting supernatant was filtered through a 1 μm, 0.45 μm, and 0.2 μm particle size range. NaCl was added to the filtrate to concentrations of 0.5 M, 1 M, 1.5 M, 2 M, and 2.5 M, respectively, and the mixture was then stirred at room temperature for 1 hour. The supernatant was then centrifuged at 7000 rpm for 20 minutes to obtain the supernatant. The resulting supernatant was filtered at 1 μm, 0.45 μm, and 0.2 μm pores. Isopropyl alcohol (IPA) was added to the filtrate at a volumetric ratio of 50:50 filtrate:IPA. The filtrate was allowed to settle for 1 hour at 0-4°C, and then centrifuged at 5000 rpm for 10 minutes to obtain a precipitate. 100 g of purified water was added to the filtrate and redissolved with stirring at room temperature for 30 minutes. Ethanol was added to the filtrate at a volumetric ratio of 30:70 filtrate:ethanol. The filtrate was allowed to settle for 1 hour at 0-4°C, and then centrifuged at 5000 rpm for 10 minutes to obtain a precipitate. 100 g of purified water was added to the filtrate and redissolved with stirring at room temperature for 30 minutes. The lysate was then dried in a freeze dryer for 2 days to obtain a PDRN extract derived from Gold Chlorella.

[0089] The resulting PDRN extract was dissolved in purified water to a concentration of 1 mg / mL or 10 mg / mL, and the DNA concentration and purity in the PDRN extract were then measured using a Nanodrop-1000 instrument (Thermo-Fisher, Wilmington, DE, USA). The results are shown in Table 3.

[0090] [Table 3]

[0091] The results in Table 3 show that treatment with NaCl at concentrations of 1.5M, 2M, and 2.5M resulted in a 260 / 230 ratio of 1.8 or greater, confirming the removal of contaminants such as polysaccharides. Furthermore, at high NaCl treatment concentrations of 2M and 2.5M, the DNA content in the Chlorella PDRN extract was approximately 50%, confirming a significant increase in the purity of DNA in the PDRN extract. Furthermore, as the purity of DNA in the PDRN extract increased, a white powdery PDRN extract was obtained, unlike the yellow powder obtained in Preparation Examples 1 and 2, and it was confirmed that unpleasant odors such as fishy odors were significantly reduced.

[0092] [Production Example 4] Production of PDRN extract derived from Gold Chlorella using a cationic surfactant Figure 5 shows a schematic diagram of the procedure for producing PDRN extract from gold chlorella using a cationic surfactant. Specifically, 900 g of purified water was added to 100 g of dried gold chlorella powder (manufactured by Daezo Co., Ltd.) (Production Example 4-1), or 950 g of purified water was added to 50 g of dried gold chlorella powder (manufactured by Daezo Co., Ltd.) (Production Example 4-2). The mixture was then placed in a stirrer and homogenized at 25–30°C for 30 minutes. SDS and NaCl were added to the homogenate to a concentration of 1%, and the mixture was then lysed at 60°C for 2 hours with stirring. The cell lysate was disrupted by treating it three times at 1500 bar pressure using an ultra-high-pressure disperser. The disrupted cell mixture was then centrifuged at 7000 rpm for 20 minutes to obtain the supernatant. The resulting supernatant was filtered through a 1 μm, 0.45 μm, and 0.2 μm particle size range. Cetyltrimethylammonium bromide (CTAB) and NaCl were added to the filtrate to concentrations of 1% (w / w) and 2.5M, respectively, and the mixture was stirred at room temperature for 1 hour. The mixture was then centrifuged at 7000 rpm for 20 minutes to obtain the supernatant. The resulting supernatant was filtered through a 1 μm, 0.45 μm, and 0.2 μm filtration step. Isopropyl alcohol (IPA) was added to the filtrate at a volumetric ratio of 50:50 filtrate:IPA, and the mixture was allowed to precipitate at 0-4°C for 1 hour. The precipitate was then centrifuged at 5000 rpm for 10 minutes to obtain a precipitate. 100 g of purified water was added to the precipitate, which was then redissolved with stirring at room temperature for 30 minutes. Ethanol was added to the solution at a volumetric ratio of 30:70 filtrate:ethanol, and the mixture was allowed to precipitate at 0-4°C for 1 hour. The precipitate was then centrifuged at 5000 rpm for 10 minutes to obtain a precipitate. After that, 100 g of purified water was added to the precipitate and redissolved by stirring at room temperature for 30 minutes. The solution was dried in a freeze dryer for 2 days to obtain a PDRN extract derived from Gold Chlorella.

[0093] The resulting PDRN extract was dissolved in purified water to a concentration of 1 mg / mL, and then the concentration and purity of DNA in the PDRN extract were measured using a Nanodrop-1000 instrument (Thermo-Fisher, Wilmington, DE, USA). The results are shown in Table 4 below.

[0094] [Table 4]

[0095] The results in Table 4 confirmed that, compared to Production Example 3, Production Example 4, which also used a cationic surfactant, resulted in a higher DNA content in the PDRN extract. In particular, Production Example 4-2, in which the weight of the cell lysate (dried chlorella powder + purified water) was 20 times that of the dried chlorella powder, confirmed that DNA extraction with significantly higher purity was possible. As a result, when a cell lysate in an amount 20 times that of the raw material (dried chlorella powder) was treated with a cationic surfactant and a high concentration of metal salt, chlorella PDRN with a high DNA content of approximately 80% or more could be obtained.

[0096] [Test Example 2] Comparison of PDRN extraction yield and DNA purity using an ultra-high pressure disperser In order to confirm whether or not the presence or absence of a cell disruption process using an ultra-high pressure disperser affects the extraction yield and DNA purity of Chlorella PDRN, Chlorella PDRN was obtained in the same manner as in Production Example 4-2, except that the cell disruption process using an ultra-high pressure disperser was not performed (Production Example 4-3), and the DNA purity and PDRN extraction yield were measured. The results are shown in Table 5 below.

[0097] [Table 5]

[0098] From the results in Table 5, it was confirmed that in Production Example 4-3, which did not undergo the ultra-high pressure dispersion process, the DNA purity in the PDRN extract was about 79%, similar to Production Example 4-2. However, the yield of PDRN extract relative to the raw material was very low at about 1%, confirming that not using an ultra-high pressure disperser is disadvantageous in terms of raw material yield.

[0099] [Production Example 5] Extraction of PDRN from Green Chlorella and White Chlorella PDRN extracts were obtained in the same manner as in Production Example 4-2, except that dried green chlorella powder (Production Example 5-1) and dried white chlorella powder (Production Example 5-2) were used instead of dried gold chlorella powder.

[0100] The resulting PDRN extract was dissolved in purified water to a concentration of 1 mg / mL, and then the concentration and purity of DNA in the PDRN extract were measured using a Nanodrop-1000 instrument (Thermo-Fisher, Wilmington, DE, USA). The results are shown in Table 6 below.

[0101] [Table 6]

[0102] The results in Table 6 show that the PDRN extracts extracted from the three types of chlorella (gold, green, and white) all had 260 / 280 and 260 / 230 ratios of 2 or greater, confirming that contaminants such as proteins and polysaccharides had been almost completely removed and that they were extremely pure. Furthermore, the DNA content in the PDRN extracts was confirmed to be extremely high, at approximately 81% and 82% for the PDRNs derived from gold and green chlorella, respectively.

[0103] [Test Example 3] Comparison of size and purity of three types of chlorella-derived PDRN Electrophoresis was performed to measure the size of each PDRN from Production Example 4-2 (Gold Chlorella-derived PDRN), Production Example 5-1 (Green Chlorella-derived PDRN), and Production Example 5-2 (White Chlorella-derived PDRN). Specifically, each PDRN extract was dissolved in DNA gel loading buffer (ThermoScientific, USA) at a concentration of 10 mg / mL and then electrophoresed using a 1% agarose gel, which is a level that allows separation of DNA fragments ranging from 200 bp to 50 kb. For comparison, Chlorella PDRN raw material extracted with a DNA content of 10-20% was dissolved at the same concentration (10 mg / mL) and then electrophoresed. The results are shown in Figure 6A. As shown in Figure 6A, the PDRNs from the highly purified Green and Gold Chlorella were less than 100 bp, while the PDRN from White Chlorella was a mixture of DNA fragments corresponding to the 100-200 bp level. On the other hand, the three Chlorella PDRNs with a purity of 10-20% were confirmed in a wide range of 100-700 bp, and showed very low band intensity compared to high-purity PDRNs. This is presumed to be due to the DNA forming complexes with contaminating components such as proteins and polysaccharides during the PDRN extraction process, which is believed to be the cause of the large DNA size, low band intensity, and band dragging phenomenon during electrophoresis.

[0104] Furthermore, to compare the degree of protein contamination in the three types of Chlorella PDRN extracted with high and low purity, electrophoresis was performed in the same manner as above, except that a 12% acrylamide gel was used. The results are shown in Figure 6B. As shown in Figure 6B, the three types of high-purity Chlorella PDRN were confirmed to have almost no protein contamination. On the other hand, Gold and White Chlorella PDRN, which had purities of 10-20%, showed a high level of protein contamination, most of which was confirmed to be 10 kDa or less in size.

[0105] In conclusion, it was confirmed that the PDRNs of Production Example 4-2 (PDRN derived from Gold Chlorella), Production Example 5-1 (PDRN derived from Green Chlorella), and Production Example 5-2 (PDRN derived from White Chlorella) are mixtures of low molecular weight DNA fragments with minimal protein contamination.

[0106] [Test Example 4] Comparison of size and purity of PDRN derived from high-purity Gold Chlorella To more accurately confirm the DNA size of the PDRN derived from Gold Chlorella in Production Example 4-2, the PDRN extract derived from Gold Chlorella in Production Example 4-2 was dissolved in DNA gel loading buffer at a concentration of 10 mg / mL, and then electrophoresis was performed using a 5% agarose gel. The results are shown in Figure 7.

[0107] As shown in Figure 7, the size of the DNA in the PDRN extract derived from Gold Chlorella in Production Example 4-2 was confirmed to be 20 to 30 bp. In calculating the molecular weight of DNA, the average molecular weight of one base pair is 650 daltons. Therefore, it was confirmed that the molecular weight of the DNA in the highly purified PDRN derived from Gold Chlorella according to one embodiment of the present invention is approximately 12 to 18 kDa, which is significantly lower than the molecular weight range of DNA in conventional salmon PDRN (50 to 1500 kDa).

Claims

1. A highly purified PDRN (Polydeoxyribonucleotide) extract derived from microalgae, having a DNA content of 49% by weight or more relative to the total dry weight of the PDRN extract.

2. The PDRN extract of claim 1, wherein the DNA content of the PDRN extract is 70% by weight or more based on the total dry weight of the PDRN extract.

3. 2. The PDRN extract of claim 1, wherein at least 90% or more of the DNA in the PDRN extract is low-molecular-weight DNA having a molecular weight of 20 kDa or less.

4. 2. The PDRN extract of claim 1, wherein the PDRN extract is in the form of a white powder.

5. 2. The PDRN extract according to claim 1, wherein the microalgae is at least one selected from gold chlorella (Chlorella protothecoides), green chlorella (Chlorella vulgaris), white chlorella (Chlorella protothecoides), spirulina, Dunaliella, Haematococcus, Euglena, Nannochloropsis, Nostoc, and Schizochytrium.

6. (1) Adding an anionic surfactant and a metal salt to a microalgae lysate and stirring the mixture to obtain a cell lysate; (2) centrifuging the cell lysate of step (1), filtering the filtrate, adding a metal salt to the filtrate, and stirring the filtrate; (3) a first purification step of centrifuging the stirred solution of step (2), filtering the filtrate, adding a C1-C4 alcohol to the filtrate, and centrifuging the filtrate to obtain a precipitate; (4) A method for producing a highly pure PDRN extract derived from microalgae, comprising a second purification step of adding a C1-C4 alcohol to the precipitate from step (3) and centrifuging the mixture to obtain a precipitate.

7. 7. The method according to claim 6, wherein the microalgae dissolution solution in step (1) is a mixture of microalgae powder and purified water, and the weight of the microalgae dissolution solution is 20 times or more the weight of the microalgae powder.

8. 7. The method according to claim 6, wherein the microalgae in step (1) is at least one selected from the group consisting of gold chlorella (Chlorella protothecoides), green chlorella (Chlorella vulgaris), white chlorella (Chlorella protothecoides), Spirulina, Dunaliella, Haematococcus, Euglena, Nannochloropsis, Nostoc, and Schizochytrium.

9. The method according to claim 6, wherein the anionic surfactant in step (1) is sodium dodecyl sulfate (SDS).

10. The method according to claim 6, further comprising, before step (2), applying ultra-high pressure of 500 to 2,000 bar to the mixture of step (1) three or more times to obtain cell lysates.

11. The method of claim 6, wherein the metal salt is NaCl.

12. 7. The method according to claim 6, wherein the concentration of the metal salt added in step (2) is at least twice the concentration of the metal salt added in step (1).

13. The method according to claim 6, wherein in step (2), the metal salt and the cationic surfactant are added together.

14. The method of claim 13, wherein the cationic surfactant is cetyltrimethylammonium bromide (CTAB).

15. The method according to claim 6, wherein the C1-C4 alcohol in step (3) and the C1-C4 alcohol in step (4) are different from each other.

16. A cosmetic composition comprising, as an active ingredient, the PDRN extract according to any one of claims 1 to 5 or the PDRN extract produced according to the method according to any one of claims 6 to 15.

17. A food composition comprising, as an active ingredient, the PDRN extract according to any one of claims 1 to 5 or the PDRN extract produced according to the method according to any one of claims 6 to 15.

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