A method for simultaneous extraction of plant, algal or fungal pdrn and active ingredients, and products and uses thereof

Abstract

The application belongs to the technical field of substance extraction, and relates to a method for simultaneously extracting PDRN and active ingredients from plants, algae or fungi, products and applications thereof, the method comprising the following steps: mixing a sample with an enzyme-containing aqueous solution to perform enzymolysis, to obtain an enzymolysis solution; performing ultrasonic treatment on the enzymolysis solution, mixing the enzymolysis solution with an extraction buffer to perform extraction, to obtain an extraction solution; performing filtration and centrifugation on the extraction solution, and performing ultrafiltration treatment on the supernatant, to obtain the product; the sample comprises at least one of plants, algae or fungi; the extraction buffer comprises trehalose, L-cysteine, ascorbic acid, sodium citrate, cetyltrimethylammonium bromide, polyvinylpyrrolidone, NaCl, Tris-HCl buffer, EDTA-disodium, and water is used as a solvent. The method can simultaneously extract PDRN and active substances, solves the technical problem that active ingredients cannot be retained when PDRN is extracted, and the obtained extract has excellent anti-inflammatory and soothing effects.

Description

Technical Field

[0001] This invention belongs to the field of material extraction technology, and relates to a method for simultaneously extracting PDRN and active ingredients from plants, algae or fungi, as well as its products and applications. Background Technology

[0002] Polydeoxyribonucleotides (PDRNs), as nucleic acid substances with excellent anti-inflammatory and tissue repair activities, have been widely used in cosmetics, biomedicine, and other fields. Studies have shown that PDRNs can exert significant anti-inflammatory effects by binding to adenosine A2A receptors and promote fibroblast proliferation and angiogenesis, thereby accelerating tissue repair and regeneration. Based on their excellent bioactivity, PDRNs have been widely applied in tissue engineering, wound healing, skin anti-aging, and anti-inflammatory repair in biomedicine and high-end cosmetics.

[0003] Currently, commercial PDRN production primarily relies on extraction from animals (such as salmon testes). However, animal-derived PDRNs present potential risks of viral contamination, interspecies pathogen transmission, and ethical controversies, limiting their widespread application in cosmetics. Therefore, the development of safe, sustainable, and ethically sound plant- or fungal-derived PDRNs has garnered significant attention. However, the complex structures of plant and fungal cells, particularly the dense cell walls and the abundance of interfering components such as polysaccharides, polyphenols, and pigments within the cells, make PDRN extraction and purification challenging. More importantly, existing plant / fungal PDRN extraction processes typically employ harsh chemical or enzymatic conditions, easily leading to the degradation or loss of small-molecule active ingredients such as flavonoids, polyphenols, and polysaccharides within the cells. This prevents the simultaneous retention and co-extraction of nucleic acids and these small-molecule active substances, resulting in a waste of biological resources and limiting the development of products with multiple therapeutic effects.

[0004] Therefore, it is particularly important to provide a method for simultaneously extracting PDRN and active ingredients. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a method for simultaneously extracting PDRN and active ingredients from plants, algae or fungi, as well as its products and applications.

[0006] To achieve this objective, the present invention employs the following technical solution:

[0007] In a first aspect, the present invention provides a method for simultaneously extracting PDRN from plants, algae, or fungi and active ingredients, the method comprising:

[0008] (1) Mix the sample with an enzyme-containing aqueous solution for enzymatic hydrolysis to obtain the enzymatic hydrolysate;

[0009] (2) After sonicating the enzyme hydrolysate, it is mixed with the extraction buffer to extract the solution.

[0010] (3) After filtering the extract, centrifuge it and then ultrafilter the supernatant to obtain the final product;

[0011] The sample includes at least one of plants, algae, or fungi;

[0012] The extraction buffer comprises trehalose, L-cysteine, ascorbic acid, sodium citrate, hexadecyltrimethylammonium bromide, polyvinylpyrrolidone, NaCl, Tris-HCl buffer, and disodium EDTA, with water as the solvent.

[0013] This invention specifically relates to a method for co-extracting PDRN and active ingredients from plants, algae, or fungi with soothing and anti-inflammatory effects, and its application, which solves the technical problem that active ingredients cannot be preserved during existing plant PDRN extraction.

[0014] The extraction buffer used in this invention does not contain toxic reagents such as β-mercaptoethanol, chloroform, and phenol, and meets the safety requirements for cosmetic raw materials. This buffer is also suitable for the flowers, stems, leaves, roots of plants and large fungi, and can simultaneously extract PDRN and other plant active ingredients such as polysaccharides, polyphenols, and flavonoids. During the extraction process, browning and oxidation are inhibited, resulting in a clear and highly stable cosmetic raw material solution.

[0015] The addition of Tris-HCl buffer helps maintain the stability of the extraction system, ensures sufficient cell lysis, and prevents the degradation of nucleic acid structures; the addition of EDTA-disodium helps chelate metal ions, inhibit nucleases, and reduce metal-catalyzed polyphenol oxidative browning; the high-salt environment promotes nucleic acid dissolution, protein and polysaccharide precipitation, and reduces interference; hexadecyltrimethylammonium bromide (CTAB), as a cationic surfactant, efficiently lyses plant / fungal cell walls and solubilizes polyphenols, flavonoids, and lipid-soluble components; polyvinylpyrrolidone (PVP) strongly complexes polyphenols, flavonoids, and tannins. The extract contains pigments; it removes polysaccharide adhesion and prevents irreversible binding of polyphenols to nucleic acids, while chelating but not precipitating polyphenols, thus retaining them in the extract; trehalose acts as a bioprotective agent, stabilizing nucleic acids, proteins, and polyphenols; L-cysteine ​​is a strong reducing agent, inhibiting polyphenol oxidative browning and protecting flavonoids and anthocyanins from destruction; ascorbic acid has antioxidant and anti-browning effects, assisting in color protection and brightening, and can form a dual antioxidant system with L-cysteine; sodium citrate assists in chelating metal ions, regulating the osmotic pressure of the system, enhancing the stability of the system, and ensuring that the extract does not change color during long-term storage.

[0016] Preferably, the extraction buffer comprises, by concentration, trehalose 2-20 g / L, L-cysteine ​​0.1-2 g / L, ascorbic acid 0.3-2 g / L, sodium citrate 0.5-5 g / L, hexadecyltrimethylammonium bromide 5-30 g / L, polyvinylpyrrolidone 10-40 g / L, and NaCl 2-20 g / L, and by molar concentration, Tris-HCl buffer 0.01-0.5 mol / L and EDTA-disodium 0.01-0.05 mol / L, with water as the solvent.

[0017] The concentrations of trehalose can be selected from 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, and 20 g / L, etc. The concentrations of L-cysteine ​​can be selected from 0.1 g / L, 0.2 g / L, 0.5 g / L, 0.8 g / L, 1 g / L, 1.2 g / L, 1.5 g / L, 1.8 g / L, and 2 g / L, etc. The concentrations of ascorbic acid can be selected from 0.3 g / L, 0.5 g / L, 0.8 g / L, 1 g / L, 1.2 g / L, 1.5 g / L, 1.8 g / L, and 2 g / L, etc. For sodium citrate, concentrations can be selected from 0.5 g / L, 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, 5 g / L, etc. For hexadecyltrimethylammonium bromide, concentrations can be selected from 5 g / L, 8 g / L, 10 g / L, 12 g / L, 15 g / L, 18 g / L, 20 g / L, 22 g / L, 25 g / L, 28 g / L, 30 g / L, etc. For polyvinylpyrrolidone, concentrations can be selected from 10 g / L, 12 g / L, 15 g / L, 18 g / L, 20 g / L, 22 g / L, 25 g / L, 28 g / L, 30 g / L, 32 g / L, 35 g / L, 38 g / L, 40 g / L, etc. For NaCl, concentrations can be selected from 2 g / L, 5 g / L, etc. The molar concentrations of Tris-HCl buffer can be selected from 0.01 mol / L, 0.02 mol / L, 0.05 mol / L, 0.08 mol / L, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, and 0.5 mol / L. The molar concentrations of EDTA-disodium can be selected from 0.01 mol / L, 0.02 mol / L, 0.03 mol / L, 0.04 mol / L, and 0.05 mol / L. Other specific values ​​within the above ranges can also be selected, and will not be elaborated further here.

[0018] In this invention, the term "Tris-HCl buffer" refers to a buffer solution prepared from tris(hydroxymethyl)aminomethane (Tris) and hydrochloric acid, wherein the concentration refers to the concentration of Tris(hydroxymethyl)aminomethane. Those skilled in the art will understand that the pH of this buffer solution is adjusted by the amount of hydrochloric acid added, and that the total concentration of Tris(hydroxymethyl)aminomethane in the buffer solution is fixed, while the amount of hydrochloric acid used is determined according to the target pH.

[0019] Preferably, the pH of the Tris-HCl buffer solution is 7-8.5, such as 7.5, 7.6, 7.7, 7.8, 7.9, 8, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0020] Preferably, the volume ratio of the enzymatic hydrolysate after ultrasonic treatment to the extraction buffer is (0.5-2):(0.5-2).

[0021] The specific point values ​​in (0.5-2) can be selected from 0.5, 0.8, 1, 1.2, 1.5, 1.8, 2, etc. Other specific point values ​​within the above range can also be selected, which will not be elaborated here.

[0022] Preferably, the plant includes any one or a combination of at least two of the following: rose, camellia, peony, honeysuckle, peony, chamomile, gentian, gentian, astragalus, rhodiola, ginseng, dendrobium, tea, artemisia, aloe, or mint.

[0023] Preferably, the algae include any one or a combination of at least two of Spirulina, kelp, Chlorella, or Ulva.

[0024] Preferably, the fungus includes black truffle.

[0025] Preferably, the sample is a combination of rose petals, astragalus root, tea leaves, and black truffle.

[0026] Preferably, the enzyme is a combination of cellulase, pectinase and neutral protease or a combination of β-glucanase, chitinase and neutral protease.

[0027] Preferably, the mass ratio of cellulase, pectinase and neutral protease is (1-1.5):(0.5-1):(0.2-0.5).

[0028] Preferably, the mass ratio of β-glucanase, chitinase and neutral protease is (1-1.5):(0.5-1):(0.2-0.5).

[0029] The specific point values ​​in (1-1.5) can all be selected from 1, 1.1, 1.2, 1.3, 1.4, 1.5, etc.; the specific point values ​​in (0.5-1) can all be selected from 0.5, 0.6, 0.7, 0.8, 0.9, 1, etc.; the specific point values ​​in (0.2-0.5) can all be selected from 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, etc. Other specific point values ​​within the above range can also be selected, which will not be elaborated here.

[0030] When the sample is a plant or algae, the enzyme is a combination of cellulase, pectinase and neutral protease.

[0031] When the sample is a fungus, the enzyme is a combination of β-glucanase, chitinase, and neutral protease.

[0032] Preferably, the ratio of the sample to the enzyme-containing aqueous solution is (1-2) g:(10-20) mL.

[0033] The specific point values ​​in (1-2) can all be selected from 1, 1.2, 1.4, 1.6, 1.8, 2, etc., and the specific point values ​​in (10-20) can all be selected from 10, 12, 14, 16, 18, 20, etc. Other specific point values ​​within the above range can also be selected, which will not be elaborated here.

[0034] Preferably, the enzyme content in the enzyme-containing aqueous solution is 1.5-4% by mass, such as 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0035] Preferably, the enzymatic hydrolysis is performed at a temperature of 37-50°C for 3-5 hours.

[0036] Temperatures can be selected from 37℃, 40℃, 42℃, 45℃, 48℃, 50℃, etc., and time can be selected from 3 h, 3.2 h, 3.5 h, 3.8 h, 4 h, 4.2 h, 4.5 h, 4.8 h, 5 h, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0037] Preferably, the ultrasonic treatment has a power of 200-300W, a temperature of 50-60℃, a working time of 2-5 seconds, an interval of 2-5 seconds, and a total ultrasonic treatment time of 10-40 minutes.

[0038] Specific values ​​within the range of 2-5 seconds can be selected from 2, 3, 4, 5, etc.; power can be selected from 200 W, 220 W, 240 W, 260 W, 280 W, 300 W, etc.; temperature can be selected from 50℃, 52℃, 54℃, 56℃, 58℃, 60℃, etc.; and total duration can be selected from 10 min, 12 min, 15 min, 18 min, 20 min, 22 min, 25 min, 28 min, 30 min, 32 min, 35 min, 38 min, 40 min, etc. Other specific values ​​within the above range can also be selected, which will not be elaborated here.

[0039] Preferably, the extraction temperature is 50-60℃ and the extraction time is 10-30 min.

[0040] Temperatures can be selected from 50℃, 52℃, 54℃, 56℃, 58℃, 60℃, etc., and time can be selected from 10 min, 12 min, 15 min, 18 min, 20 min, 22 min, 25 min, 28 min, 30 min, etc. Other specific values ​​within the above range can also be selected, which will not be elaborated here.

[0041] Preferably, before filtration, the extract is further treated at 60-70°C for 10-20 minutes.

[0042] Temperatures can be selected from 60℃, 62℃, 64℃, 66℃, 68℃, 70℃, etc., and time can be selected from 10 min, 12 min, 14 min, 16 min, 18 min, 20 min, etc. Other specific values ​​within the above range can also be selected, which will not be elaborated here.

[0043] Preferably, the centrifugation speed is 8000-12000 rpm, the temperature is 0-30℃, and the time is 2-10 min.

[0044] The rotation speed can be selected from 8000 rpm, 8500 rpm, 9000 rpm, 9500 rpm, 10000 rpm, 10500 rpm, 11000 rpm, 11500 rpm, 12000 rpm, etc. The temperature can be selected from 0℃, 5℃, 10℃, 15℃, 20℃, 25℃, 30℃, etc. The time can be selected from 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, etc. Other specific values ​​within the above range can be selected, which will not be described in detail here.

[0045] Preferably, the ultrafiltration membrane used in the ultrafiltration process has a molecular weight cutoff of 3-10 kDa, such as 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0046] Preferably, the ultrafiltration process specifically includes: concentrating the extract using an ultrafiltration membrane to obtain a concentrate, adding water to the concentrate to the original volume, and repeating the above operation 2-3 times to obtain the final product.

[0047] Preferably, the volume ratio of the concentrate to the extract is 1:(3-4), and the specific values ​​in (3-4) can be selected from 3, 3.2, 3.4, 3.6, 3.8, 4, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0048] In a second aspect, the present invention provides an extract prepared by the method described in the first aspect for simultaneously extracting PDRN and active ingredients from plants, algae or fungi.

[0049] Preferably, the extract includes a single extract or a mixture of extracts.

[0050] Preferably, the mixed extracts include rose extract, astragalus extract, tea extract, and black truffle extract.

[0051] This invention discovers that the target points of plant-derived extracts and fungal-derived extracts are complementary. When extracted separately and then combined, they can achieve a synergistic effect of anti-inflammatory and soothing properties. Rose extract, astragalus extract, tea extract, and black truffle extract have certain synergistic effects in soothing and anti-inflammatory aspects.

[0052] Preferably, the mass ratio of the rose extract, astragalus extract, tea extract and black truffle extract is (0.5-2):(0.5-2):(0.5-2):(0.2-1).

[0053] The specific point values ​​in (0.5-2) can all be selected from 0.5, 0.8, 1, 1.2, 1.5, 1.8, 2, etc., and the specific point values ​​in (0.2-1) can all be selected from 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, etc. Other specific point values ​​within the above range can also be selected, which will not be elaborated here.

[0054] Thirdly, the present invention provides the use of the extract according to the second aspect in the preparation of products having soothing and anti-inflammatory effects.

[0055] Preferably, the product includes cosmetics or skincare products.

[0056] Compared with the prior art, the present invention has the following beneficial effects:

[0057] (1) This invention provides a non-toxic and non-irritating co-extraction buffer solution that does not contain toxic reagents such as β-mercaptoethanol, chloroform, and phenol, and meets the safety requirements for cosmetic raw materials;

[0058] (2) The extraction method of the present invention is simple and has a wide range of applications. It can be applied to the flowers, stems, leaves, and roots of plants, algae, and large fungi, and can simultaneously extract plant active ingredients such as PDRN, polysaccharides, polyphenols, and flavonoids.

[0059] (3) The extraction method of the present invention can inhibit browning and oxidation during the extraction process, and obtain a cosmetic raw material liquid with clear color and high stability;

[0060] (4) The combination of PDRN and active ingredient extracts from different sources can achieve synergistic effects of anti-inflammatory and soothing through their target complementarity, which has high application value in the cosmetics field. Attached Figure Description

[0061] Figure 1 These are the electrophoresis results of PDRN from different sources. Detailed Implementation

[0062] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0063] The sources of the active ingredients in the products involved in the following examples and comparative examples are as follows (only the active ingredients are shown; other necessary excipients contained in commercially available raw materials are not described):

[0064] PVP is a product called povidone K30 purchased from the company's product name, Shanxi Jinyang Pharmaceutical Excipients Co., Ltd.

[0065] The cellulase was purchased from Jiahe Xuri Company and its product is marketed as food-grade cellulase.

[0066] The pectinase was purchased from Jiahe Xuri Company and its product is marketed as food-grade pectinase.

[0067] The neutral protease was purchased from Jiahe Xuri Company and its product was marketed as food-grade neutral protease.

[0068] The β-glucanase was purchased from Jiahe Xuri Company under the product name "food grade β-glucanase";

[0069] The chitinase was purchased from Kangtai Biological Company under the product name "food grade chitinase".

[0070] Preparation Example 1

[0071] This preparation example provides an extraction buffer, which comprises, by concentration, 10 g / L trehalose, 1 g / L L-cysteine, 0.8 g / L ascorbic acid, 3 g / L sodium citrate, 18 g / L CTAB, 25 g / L PVP, and 12 g / L NaCl, and by molar concentration, 0.1 mol / L Tris-HCl buffer and 0.02 mol / L EDTA-disodium, with water as the solvent.

[0072] The preparation method is as follows: Tris-HCl buffer, EDTA-disodium, NaCl, sodium citrate and water are mixed, and then CTAB, PVP and trehalose are mixed at 55℃ until dissolved. Then, L-cysteine ​​and ascorbic acid are mixed at room temperature, the pH is adjusted to 8, and the mixture is filtered to remove bacteria.

[0073] Preparation Example 2

[0074] This preparation example provides an extraction buffer, which comprises, by concentration, 5 g / L trehalose, 2 g / L L-cysteine, 0.5 g / L ascorbic acid, 5 g / L sodium citrate, 30 g / L CTAB, 10 g / L PVP, and 18 g / L NaCl, and by molar concentration, 0.5 mol / L Tris-HCl buffer and 0.01 mol / L EDTA-disodium, with water as the solvent.

[0075] The preparation method is the same as in Preparation Example 1.

[0076] Preparation Example 3

[0077] This preparation example provides an extraction buffer, which comprises, by concentration, 20 g / L trehalose, 0.5 g / L L-cysteine, 2 g / L ascorbic acid, 0.5 g / L sodium citrate, 8 g / L CTAB, 40 g / L PVP, and 5 g / L NaCl, and by molar concentration, 0.05 mol / L Tris-HCl buffer and 0.05 mol / L EDTA-disodium, with water as the solvent.

[0078] The preparation method is the same as in Preparation Example 1.

[0079] Comparative Preparation Example 1

[0080] This comparative preparation example provides an extraction buffer that differs from Preparation Example 1 only in that it does not contain CTAB, and the reduced mass of CTAB is proportionally allocated to the mass of PVP, trehalose, L-cysteine, and ascorbic acid, while other components and contents remain unchanged.

[0081] The preparation method is the same as in Preparation Example 1.

[0082] Comparative Preparation Example 2

[0083] This comparative preparation example provides an extraction buffer that differs from Preparation Example 1 only in that it does not contain PVP, and the reduced mass of PVP is proportionally allocated to the mass of CTAB, trehalose, L-cysteine, and ascorbic acid, while the other components and contents remain unchanged.

[0084] The preparation method is the same as in Preparation Example 1.

[0085] Comparative preparation example 3

[0086] This comparative preparation example provides an extraction buffer that differs from Preparation Example 1 only in that it does not contain trehalose, and the reduced mass of trehalose is proportionally allocated to the mass of CTAB, PVP, L-cysteine, and ascorbic acid, while other components and contents remain unchanged.

[0087] The preparation method is the same as in Preparation Example 1.

[0088] Comparative preparation example 4

[0089] This comparative preparation example provides an extraction buffer that differs from Preparation Example 1 only in that it does not contain L-cysteine, and the reduced mass of L-cysteine ​​is proportionally allocated to the mass of CTAB, PVP, trehalose, and ascorbic acid, while the other components and contents remain unchanged.

[0090] The preparation method is the same as in Preparation Example 1.

[0091] Comparative preparation example 5

[0092] This comparative preparation example provides an extraction buffer that differs from Preparation Example 1 only in that it does not contain ascorbic acid, and the reduced mass of ascorbic acid is proportionally allocated to the mass of CTAB, PVP, trehalose, and L-cysteine, while the other components and contents remain unchanged.

[0093] The preparation method is the same as in Preparation Example 1.

[0094] Example 1

[0095] This embodiment provides a method for simultaneously extracting PDRN and active ingredients, the method comprising:

[0096] (1) The rose petals were crushed and passed through an 80-mesh sieve. The crushed rose petals were mixed with an enzyme-containing aqueous solution at a material-liquid ratio of 1 g:10 mL. The enzymes included cellulase, pectinase and neutral protease in a mass ratio of 1.2:0.8:0.3. The enzyme content in the enzyme-containing aqueous solution was 2.3% by mass. The solution was enzymatically hydrolyzed at 40°C for 3.5 h to obtain the enzymatic hydrolysate.

[0097] (2) The enzymatic hydrolysate was subjected to ultrasonic treatment. The ultrasonic power was 250 W, the temperature was 55℃, the working time was 2 s, the interval was 3 s, and 120 cycles were performed to obtain the ultrasonic product.

[0098] (3) The ultrasonic product was mixed with the extraction buffer solution described in Preparation Example 1 at a volume ratio of 1:1 and extracted at 55°C for 20 min to obtain the extract.

[0099] (4) The extract was treated at 65℃ for 15 min, filtered, and the filtrate was centrifuged at 10000 rpm for 8 min at 4℃. The supernatant was filtered through a 0.45 μm filter membrane to obtain the crude extract.

[0100] (5) The crude extract was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa. The material of the ultrafiltration membrane was polyethersulfone. The crude extract was concentrated to 1 / 3 of its original volume using the ultrafiltration membrane, and then water was added to the original volume. The above operation was repeated twice to obtain rose extract.

[0101] Example 2

[0102] This embodiment provides a method for simultaneously extracting PDRN and active ingredients, the method comprising:

[0103] (1) The rose petals were crushed and passed through an 80-mesh sieve. The crushed rose petals were mixed with an enzyme-containing aqueous solution at a material-liquid ratio of 1 g:15 mL. The enzymes included cellulase, pectinase and neutral protease in a mass ratio of 1:0.5:0.5. The enzyme content in the enzyme-containing aqueous solution was 2% by mass. The solution was enzymatically hydrolyzed at 37°C for 5 h to obtain the enzymatic hydrolysate.

[0104] (2) The enzymatic hydrolysate was subjected to ultrasonic treatment. The ultrasonic power was 200 W, the temperature was 50℃, the working time was 5 s, the interval was 3 s, and 120 cycles were performed to obtain the ultrasonic product.

[0105] (3) The ultrasonic product was mixed with the extraction buffer solution described in Preparation Example 2 at a volume ratio of 1:1.5, and extracted at 50°C for 30 min to obtain the extract.

[0106] (4) The extract was treated at 60℃ for 20 min, filtered, and the filtrate was centrifuged at 8000 rpm for 10 min at 4℃. The supernatant was filtered through a 0.45 μm filter membrane to obtain the crude extract.

[0107] (5) Use an ultrafiltration membrane with a molecular weight cutoff of 3 kDa to ultrafilter the crude extract. The material of the ultrafiltration membrane is polyethersulfone. Use the ultrafiltration membrane to concentrate the crude extract to 1 / 3 of the original volume, then add water to the original volume. Repeat the above operation twice to obtain the final product.

[0108] Example 3

[0109] This embodiment provides a method for simultaneously extracting PDRN and active ingredients, the method comprising:

[0110] (1) The rose petals were crushed and passed through an 80-mesh sieve. The crushed rose petals were mixed with an enzyme-containing aqueous solution at a material-liquid ratio of 1 g:20 mL. The enzymes included cellulase, pectinase and neutral protease in a mass ratio of 1.5:1:0.2. The enzyme content in the enzyme-containing aqueous solution was 3.5% by mass. The solution was enzymatically hydrolyzed at 50°C for 3 h to obtain the enzymatic hydrolysate.

[0111] (2) The enzymatic hydrolysate was subjected to ultrasonic treatment. The ultrasonic power was 300 W, the temperature was 60℃, the working time was 3 s, the interval was 2 s, and 120 cycles were performed to obtain the ultrasonic product.

[0112] (3) The ultrasonic product was mixed with the extraction buffer solution described in Preparation Example 3 at a volume ratio of 2:1, and extracted at 60°C for 10 min to obtain the extract.

[0113] (4) The extract was treated at 70℃ for 10 min, filtered, and the filtrate was centrifuged at 12000 rpm for 4 min at 4℃. The supernatant was filtered through a 0.45 μm filter membrane to obtain the crude extract.

[0114] (5) Use an ultrafiltration membrane with a molecular weight cutoff of 3 kDa to ultrafilter the crude extract. The material of the ultrafiltration membrane is polyethersulfone. Use the ultrafiltration membrane to concentrate the crude extract to 1 / 3 of the original volume, then add water to the original volume. Repeat the above operation twice to obtain the final product.

[0115] Example 4

[0116] This embodiment provides a method for simultaneously extracting PDRN and active ingredients. The only difference between this method and Example 1 is that the rose petals are replaced with Astragalus root, while the other operations remain unchanged.

[0117] Example 5

[0118] This embodiment provides a method for simultaneously extracting PDRN and active ingredients. The only difference between this method and Example 1 is that rose petals are replaced with tea leaves, while other operations remain unchanged.

[0119] Example 6

[0120] This embodiment provides a method for simultaneously extracting PDRN and active ingredients. The only difference between this method and Example 1 is that step (1) is to "crush black truffles through an 80-mesh sieve, mix the crushed black truffles with an enzyme-containing aqueous solution at a material-liquid ratio of 1 g:10 mL, wherein the enzymes include β-glucanase, chitinase and neutral protease in a mass ratio of 1.2:0.8:0.3, the enzyme mass percentage in the enzyme-containing aqueous solution is 2.3%, and hydrolyze at 40°C for 3.5 h to obtain an enzymatic hydrolysate". Other operations remain unchanged.

[0121] Example 7

[0122] This embodiment provides a method for simultaneously extracting PDRN and active ingredients. The only difference between this method and Example 1 is that the rose petals are replaced with kelp, while the other operations remain unchanged.

[0123] Comparative Example 1

[0124] This comparative example provides a method for simultaneously extracting PDRN and active ingredients. The only difference between this method and Example 1 is that step (3) is to "mix the ultrasonic product with the extraction buffer described in Comparative Preparation Example 1 at a volume ratio of 1:1, extract at 55°C for 20 min, and obtain the extract". All other operations remain unchanged.

[0125] Comparative Example 2

[0126] This comparative example provides a method for simultaneously extracting PDRN and active ingredients. The only difference between this method and Example 1 is that step (3) is to "mix the ultrasonic product with the extraction buffer described in Comparative Preparation Example 2 at a volume ratio of 1:1, extract at 55°C for 20 min to obtain the extract". Other operations remain unchanged.

[0127] Comparative Example 3

[0128] This comparative example provides a method for simultaneously extracting PDRN and active ingredients. The only difference between this method and Example 1 is that step (3) is to "mix the ultrasonic product with the extraction buffer described in Comparative Preparation Example 3 at a volume ratio of 1:1, extract at 55°C for 20 min to obtain the extract". Other operations remain unchanged.

[0129] Comparative Example 4

[0130] This comparative example provides a method for simultaneously extracting PDRN and active ingredients. The only difference between this method and Example 1 is that step (3) is to "mix the ultrasonic product with the extraction buffer described in Comparative Preparation Example 4 at a volume ratio of 1:1, extract at 55°C for 20 min to obtain the extract". Other operations remain unchanged.

[0131] Comparative Example 5

[0132] This comparative example provides a method for simultaneously extracting PDRN and active ingredients. The only difference between this method and Example 1 is that step (3) is to "mix the ultrasonic product with the extraction buffer described in Comparative Preparation Example 5 at a volume ratio of 1:1, extract at 55°C for 20 min to obtain the extract". Other operations remain unchanged.

[0133] Comparative Example 6

[0134] This comparative example provides a method for simultaneously extracting PDRN and active ingredients. The only difference between this method and Example 1 is that step (1) is to "crush rose petals through an 80-mesh sieve, mix the crushed rose petals with water at a material-to-liquid ratio of 1 g:10 mL, treat at 40°C for 3.5 h to obtain a treated liquid", and step (2) is to "ultrasonically treat the treated liquid with an ultrasonic power of 300 W and a temperature of 55°C, working for 2 s, intermittent for 3 s, for 120 cycles to obtain an ultrasonic product". Other operations remain unchanged.

[0135] Comparative Example 7

[0136] This comparative example provides a method for simultaneously extracting PDRN and active ingredients. The only difference between this method and Example 1 is that step (1) is "to crush rose petals through an 80-mesh sieve, mix the crushed rose petals with an enzyme-containing aqueous solution at a material-to-liquid ratio of 1 g:10 mL, wherein the enzymes include cellulase, pectinase and neutral protease in a mass ratio of 1.2:0.8:0.3, and the enzyme mass percentage in the enzyme-containing aqueous solution is 2.3%, and to hydrolyze at 40°C for 4 h to obtain an enzymatic hydrolysate", without step (2), and all other operations remain unchanged.

[0137] Application Example 1

[0138] This application example provides a composition comprising, by weight, 2 parts of rose extract as described in Example 1, 2 parts of astragalus root extract as described in Example 4, 2 parts of tea extract as described in Example 5, and 0.5 parts of black truffle extract as described in Example 6.

[0139] The preparation method is as follows: the raw materials are physically mixed to obtain the product.

[0140] Application Example 2

[0141] This application example provides a composition comprising, by weight, 0.5 parts of rose extract as described in Example 1, 1 part of astragalus root extract as described in Example 4, 0.5 parts of tea extract as described in Example 5, and 1 part of black truffle extract as described in Example 6.

[0142] The preparation method is described in Application Example 1.

[0143] Application Example 3

[0144] This application example provides a composition comprising, by weight, 1 part of rose extract as described in Example 1, 0.5 parts of astragalus root extract as described in Example 4, 1 part of tea extract as described in Example 5, and 0.2 parts of black truffle extract as described in Example 6.

[0145] The preparation method is described in Application Example 1.

[0146] Comparative Application Example 1

[0147] This comparative application example provides a composition that differs from Application Example 1 only in that it does not contain the rose extract described in Example 1, and the reduced mass of the rose extract is proportionally allocated to the mass of the astragalus root extract described in Example 4, the tea extract described in Example 5, and the black truffle extract described in Example 6.

[0148] The preparation method is described in Application Example 1.

[0149] Comparative Application Example 2

[0150] This comparative application example provides a composition that differs from Application Example 1 only in that it does not contain the Astragalus root extract of Example 4, and the reduced mass of the Astragalus root extract is proportionally allocated to the mass of the rose extract of Example 1, the tea extract of Example 5, and the black truffle extract of Example 6.

[0151] The preparation method is described in Application Example 1.

[0152] Comparative Application Example 3

[0153] This comparative application example provides a composition that differs from Application Example 1 only in that it does not contain the tea extract described in Example 5, and the reduced mass of the tea extract is proportionally allocated to the mass of the rose extract described in Example 1, the astragalus root extract described in Example 4, and the black truffle extract described in Example 6.

[0154] The preparation method is described in Application Example 1.

[0155] Comparative Application Example 4

[0156] This comparative application example provides a composition that differs from Application Example 1 only in that it does not contain the black truffle extract described in Example 6, and the reduced mass of the black truffle extract is proportionally allocated to the mass of the rose extract described in Example 1, the astragalus root extract described in Example 4, and the tea extract described in Example 5.

[0157] The preparation method is described in Application Example 1.

[0158] Test Example 1

[0159] Extract component analysis results

[0160] Test method:

[0161] PDRN content detection method:

[0162] A highly specific double-stranded DNA fluorescent dye (PicoGreen) is used. This dye exhibits extremely weak fluorescence in its free state, binding specifically only to the minor groove of double-stranded DNA (dsDNA). After binding, the fluorescence intensity is significantly enhanced (enhancement factor >1000-fold), and the fluorescence intensity shows a strong linear positive correlation with the double-stranded DNA content. A fluorescence intensity-nucleic acid content standard curve is established using standards of known concentrations. By measuring the fluorescence signal of the sample and substituting it into the curve, the nucleic acid (PDRN) content can be accurately calculated. This method is unaffected by impurities such as proteins, polysaccharides, pigments, and salt ions, and is suitable for the quantitative detection of low concentrations and small fragments of nucleic acids in complex biological matrices.

[0163] Prepare working solutions according to the number of samples to be tested, using a detection buffer:fluorescent dye ratio of 199:1 (volume ratio). Vortex to mix and allow to stand in the dark for later use. Prepare three reaction systems (2 standards + 1 sample) for each sample to be tested. Prepare working solutions fresh each time. Take three 0.2 mL PCR tubes without nuclease and label them as Standard 1, Standard 2, and the sample to be tested, respectively. Add the samples according to the following systems: Standard 1: 190 μL working solution + 10 μL Standard #1; Standard 2: 190 μL working solution + 10 μL Standard #2; Sample to be tested: 198 μL working solution + 2 μL nucleic acid sample. Vortex to mix after adding the samples, and briefly centrifuge to ensure the liquid settles at the bottom of the tube. Incubate the PCR tubes at room temperature (20-25℃) in the dark for 2 minutes to avoid fluorescence dye attenuation caused by light. Strictly control the incubation time; excessive incubation can affect detection stability.

[0164] Turn on the Qubit 3.0 fluorescence quantitative analyzer and select the "dsDNA HS" detection mode; add standard 1 and standard 2 sequentially. The instrument will automatically read the fluorescence signal and establish a linear standard curve (R²). 2 ≥0.99 is considered valid); place the sample to be tested in the instrument, and the instrument will directly read the sample nucleic acid concentration (unit: ng / μL); if the sample concentration exceeds the linear range of the kit (0-100 ng / μL), dilute with nuclease-free pure water and retest. The final concentration = detection value × dilution factor. Final nucleic acid extraction amount (mg) = nucleic acid concentration (ng / μL) × 10g total volume obtained from sample extraction (mL) × 10 -3 The PDRN extraction amounts in Table 1 are the nucleic acid amounts extracted from 10g of plants, fungi, or algae.

[0165] Methods for detecting flavonoid content:

[0166] Accurately weigh an appropriate amount of sample, add ethanol for ultrasonic extraction, filter, and dilute to volume to obtain the test solution; separately prepare a series of reference solutions by adding ethanol to rutin reference standard. Accurately measure the test solution and reference solutions respectively, add 5% sodium nitrite solution, 10% aluminum nitrate solution and 4% sodium hydroxide solution sequentially, dilute to volume with water, shake well and let stand, and measure the absorbance at a wavelength of 510 nm. Using the corresponding reagents as blanks, calculate the total flavonoid content (calculated as rutin) in the sample according to the standard curve method. The flavonoid extraction amounts in Table 1 are all the mass of flavonoids extracted from 10g of plants, fungi or algae.

[0167] X = C × V × D

[0168] Wherein, C: concentration obtained from the standard curve (mg / mL); V: total volume of the test sample extracted from 10g of raw material (mL); D: dilution factor;

[0169] Polyphenol content determination:

[0170] Accurately weigh an appropriate amount of sample, add ethanol for ultrasonic extraction, filter, and dilute to volume to prepare the test solution; separately prepare a series of reference solutions by adding water to gallic acid reference standard. Accurately measure the test solution and reference solutions respectively, add Folin-phenol reagent and sodium carbonate solution, dilute to volume with water, and allow to stand in the dark for color development. Measure the absorbance at 760 nm wavelength. Using the corresponding reagent as a blank, calculate the total polyphenol content (calculated as gallic acid) in the sample according to the standard curve method. The polyphenol extraction amounts in Table 1 are the mass of 10g of polyphenols extracted from plants, fungi, or algae.

[0171] X = C × V × D

[0172] Wherein, C: concentration obtained from the standard curve (mg / mL); V: total volume of the test sample extracted from 10g of raw material (mL); D: dilution factor;

[0173] Polysaccharide content determination:

[0174] Accurately weigh an appropriate amount of sample, add water, and extract ultrasonically. After deproteinization, centrifuge the extract and dilute the supernatant to a final volume to obtain the test solution. Separately, prepare a series of reference solutions by adding water to glucose reference standard. Accurately measure the test solution and reference solutions separately, add 5% phenol solution, quickly add concentrated sulfuric acid, shake well, allow to stand for color development, cool to room temperature, and measure the absorbance at 490 nm. Using the corresponding reagent as a blank, calculate the total polysaccharide content (calculated as glucose) in the sample according to the standard curve method. The polysaccharide extraction amounts in Table 1 are the mass of polysaccharides extracted from 10g of plants or fungi.

[0175] X = C × V × D

[0176] Wherein, C: concentration obtained from the standard curve (mg / mL); V: total volume of the test sample extracted from 10g of raw material (mL); D: dilution factor;

[0177] Table 1

[0178]

[0179] Different plants contain different types and amounts of active ingredients, including PDRN, flavonoids, polyphenols, polysaccharides, saponins, and amino acids. This invention tested the content of PDRN, flavonoids, polyphenols, and polysaccharides in the extract, and the results are shown in Table 1. The method described can simultaneously achieve good extraction effects on PDRN, flavonoids, polyphenols, and polysaccharides. The composition of the extract has the most significant impact on the extraction effect of PDRN and polyphenols. The absence of any one of CTAB, PVP, trehalose, L-cysteine, and ascorbic acid will significantly reduce the PDRN content in the extract. Enzymatic hydrolysis and ultrasound have a certain synergistic effect on PDRN extraction. The electrophoresis results of Examples 1, 2, 4, 5, 6, 7 and Comparative Examples 6-7 are shown below. Figure 1 As shown.

[0180] Test Example 2

[0181] Soothing efficacy test

[0182] Test method:

[0183] Hyaluronidase was dissolved in 0.4% BSA solution to prepare a 0.5 U / mL enzyme working solution. Hyaluronic acid (HA) substrate was diluted with purified water to 0.5 mg / mL. 50 μL of the test sample, 50 μL of the hyaluronidase working solution, and 100 μL of the hyaluronic acid (HA) substrate were added to establish a sample group, a blank control group, and a positive control group, with three replicates for each group. After pre-incubation at 37℃ for 30 min, 50 μL of 0.1 mol / L NaOH solution was added to terminate the reaction. 50 μL of 0.2 mol / L acetylacetone was added and the mixture was boiled in a water bath for 15 min. After cooling, 150 μL of DMAB (p-dimethylaminobenzaldehyde) chromogenic reagent was added, and the mixture was shaken well. The mixture was then incubated at 37℃ for 20 min for color development. The absorbance was measured at 528 nm using a microplate reader. The inhibition rate of hyaluronidase was calculated using the following formula.

[0184] Inhibition rate (%) = [1 - (Absorbance of sample group / Absorbance of blank control group)] × 100%

[0185] Sample group: Dilute with pure water to a sample concentration of 3%;

[0186] Positive control (epigallocatechin gallate, purity >98%): diluted with water to a positive control concentration of 1 mg / mL;

[0187] Blank control: pure water.

[0188] This method directly assesses the potential for soothing and repairing efficacy by quantifying the sample's ability to inhibit HA decomposition. The test results are shown in Table 2.

[0189] Table 2

[0190]

[0191] The results of hyaluronidase inhibition are shown in Table 2. The combination of rose extract, astragalus root extract, tea extract and black truffle extract showed the best inhibitory effect on hyaluronidase, which was close to that of the positive control. Furthermore, rose extract, astragalus root extract, tea extract and black truffle extract had a certain synergistic effect on the above effects.

[0192] Test Example 3

[0193] Anti-inflammatory effect test

[0194] Test method:

[0195] This study used immortalized human keratinocytes (HaCaT) as the research subject and constructed an in vitro model related to skin inflammation using LPS stimulation. Under the inflammatory conditions induced by LPS at a concentration of 2 μg / mL, cell supernatants from the sample group, blank control group, LPS-induced group, and positive control group were collected, and the secretion of key inflammatory factors (TNF-α) was quantitatively analyzed using an ELISA kit. Using a 96-well ELISA plate pre-coated with anti-human TNF-α monoclonal antibody as the solid-phase carrier, the following were prepared: biotinylated anti-human TNF-α detection antibody, HRP-labeled streptavidin, PBS-Tween 20 concentrated washing buffer, sample dilution buffer, TMB chromogenic solution, and 2 M H2SO4 stop solution. The specific procedure was as follows: 50 μL of standard or test sample was added to each well, and the plate was incubated at 37°C in the dark for 60 min. The plate was then washed five times with 350 μL / well washing buffer and patted dry. 100 μL of biotinylated detection antibody working solution was added, and the plate was incubated at 37°C in the dark for 30 min. After washing, 100 μL of HRP-streptavidin working solution was added, and the plate was incubated at 37°C in the dark for 30 min. After washing again, 100 μL of TMB chromogenic solution was added, and the plate was incubated at 37°C in the dark for 10 min. 50 μL of stop solution was added and mixed thoroughly. The absorbance was measured at 450 nm using an ELISA reader within 15 min, and the inhibition rate of TNF-α was calculated.

[0196] TNF-α inhibition rate (%) = (Absorbance of LPS group - Absorbance of sample group) / (Absorbance of sample group - Absorbance of blank group) × 100%

[0197] The positive control group received 1 mg / mL dexamethasone.

[0198] By comparing factor expression levels among groups, the anti-inflammatory potential of samples in skin-related cell models was systematically evaluated.

[0199] Table 3

[0200]

[0201] The inhibitory effects of TNF-α are shown in Table 3. The combination of rose extract, astragalus root extract, tea extract and black truffle extract showed the best inhibitory effect on TNF-α, which was close to that of the positive control. Furthermore, rose extract, astragalus root extract, tea extract and black truffle extract had a certain synergistic effect on the above effects.

[0202] The above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A method for simultaneously extracting PDRN and active ingredients from plants, algae, or fungi, characterized in that, The method includes: (1) Mix the sample with an enzyme-containing aqueous solution for enzymatic hydrolysis to obtain the enzymatic hydrolysate; (2) After sonicating the enzyme hydrolysate, it is mixed with the extraction buffer to extract the solution. (3) After filtering the extract, centrifuge it and then ultrafilter the supernatant to obtain the final product; The sample includes at least one of plants, algae, or fungi; The extraction buffer comprises trehalose, L-cysteine, ascorbic acid, sodium citrate, hexadecyltrimethylammonium bromide, polyvinylpyrrolidone, NaCl, Tris-HCl buffer, and disodium EDTA, with water as the solvent.

2. The method for simultaneously extracting PDRN and active ingredients from plants, algae, or fungi according to claim 1, characterized in that, The extraction buffer comprises, by concentration, trehalose 2-20 g / L, L-cysteine ​​0.1-2 g / L, ascorbic acid 0.3-2 g / L, sodium citrate 0.5-5 g / L, hexadecyltrimethylammonium bromide 5-30 g / L, polyvinylpyrrolidone 10-40 g / L, and NaCl 2-20 g / L, and by molar concentration, Tris-HCl buffer 0.01-0.5 mol / L and EDTA-disodium 0.01-0.05 mol / L, with water as the solvent; The volume ratio of the enzymatic hydrolysate after ultrasonic treatment to the extraction buffer is (0.5-2):(0.5-2).

3. The method for simultaneously extracting PDRN and active ingredients from plants, algae, or fungi according to claim 1, characterized in that, The plants include any one or a combination of at least two of the following: rose, camellia, peony, honeysuckle, peony, chamomile, gentian, gentian, astragalus, rhodiola, ginseng, dendrobium, tea, artemisia, aloe, or mint. The algae include any one or a combination of at least two of Spirulina, kelp, Chlorella, or Ulva; The fungi include black truffles.

4. The method for simultaneously extracting PDRN and active ingredients from plants, algae, or fungi according to claim 3, characterized in that, The sample was a combination of rose petals, astragalus root, tea leaves, and black truffles.

5. The method for simultaneously extracting PDRN and active ingredients from plants, algae, or fungi according to claim 1, characterized in that, The enzyme is a combination of cellulase, pectinase and neutral protease or a combination of β-glucanase, chitinase and neutral protease. The mass ratio of cellulase, pectinase, and neutral protease is (1-1.5):(0.5-1):(0.2-0.5). The mass ratio of β-glucanase, chitinase and neutral protease is (1-1.5):(0.5-1):(0.2-0.5).

6. The method for simultaneously extracting PDRN and active ingredients from plants, algae, or fungi according to claim 1, characterized in that, The ratio of the sample to the enzyme-containing aqueous solution is (1-2) g:(10-20) mL; The enzyme-containing aqueous solution contains 1.5-4% enzyme by mass. The enzymatic hydrolysis is performed at a temperature of 37-50℃ for 3-5 hours. The ultrasonic treatment has a power of 200-300W, a temperature of 50-60℃, a working time of 2-5 seconds, an interval of 2-5 seconds, and a total ultrasonic treatment time of 10-40 minutes. The extraction temperature is 50-60℃ and the time is 10-30 min.

7. The method for simultaneously extracting PDRN and active ingredients from plants, algae, or fungi according to claim 1, characterized in that, Before filtration, the extract is further treated at 60-70℃ for 10-20 minutes. The ultrafiltration membrane used in this ultrafiltration process has a molecular weight cutoff of 3-10 kDa.

8. The extract prepared by the method according to any one of claims 1-7, which simultaneously extracts PDRN from plants, algae or fungi and active ingredients.

9. The extract according to claim 8, characterized in that, The extract may include single extracts or mixed extracts; The mixed extracts include rose extract, astragalus extract, tea extract, and black truffle extract; The mass ratio of the rose extract, astragalus extract, tea extract, and black truffle extract is (0.5-2):(0.5-2):(0.5-2):(0.2-1).

10. The use of the extract according to claim 8 or 9 in the preparation of products with soothing and anti-inflammatory effects.

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