Ganoderma lucidum pdrn with anti-wrinkle tightening effect and preparation method thereof

Abstract

This invention discloses a method for preparing Ganoderma lucidum PDRN with anti-wrinkle and firming effects, comprising: S1. Adding defatted Ganoderma lucidum spore powder to a high-salt buffer system, stirring for 20-40 min to obtain an extract; S2. Centrifuging the extract, collecting the supernatant and filtering to obtain a clear liquid; S3. Adding 10%-40% alcohol solvent to the clear liquid, collecting the filtrate through a stepwise microfiltration process; then, adding 40%-200% alcohol solvent to the filtrate, stirring, centrifuging, and collecting the precipitate; resuspending the precipitate multiple times with 70%-80% alcohol solvent, centrifuging, and drying to obtain the Ganoderma lucidum PDRN raw material. This invention eliminates the need for a proteolytic hydrolysis step, simplifying the process and allowing for milder operating conditions. The high-salt buffer system is simple in composition and low in cost, and can efficiently dissolve nucleic acid components. Compared with the traditional one-step alcohol precipitation strategy, the two-stage stepwise alcohol precipitation strategy can effectively separate impurities and target products, improving product purity and batch-to-batch consistency.

Description

Technical Field

[0001] This invention relates to the field of bio-extraction and cosmetic raw material technology, specifically to a process for preparing fungal-derived polydeoxyribonucleic acid (PDRN) using Ganoderma lucidum spore powder as raw material, and particularly to a green preparation method of Ganoderma lucidum PDRN with anti-wrinkle and firming skin care effects. Background Technology

[0002] Polydeoxyribonucleotides (PDRN) are DNA polymers formed by deoxyribonucleotides linked by phosphodiester bonds. They have biological activities such as promoting cell regeneration, tissue repair and collagen synthesis, and have become popular functional ingredients in the fields of medical aesthetics and cosmetics.

[0003] Currently, commercially available PDRN is mainly derived from the testes of salmonid fish, which presents challenges such as limited raw material sources, high preparation costs, risks of animal-derived allergies, and restrictions on its application in religious and cultural contexts. Existing technologies disclose PDRN extraction methods from plants, algae, and fungi (CN122140594A). These processes rely on enzymatic hydrolysis and buffer systems containing surfactants such as CTAB, which are prone to reagent residue irritation. The overall process is complex and costly, and this method is not specifically developed for Ganoderma lucidum spores, failing to address the dual mass transfer resistance issues caused by their dense cell walls and residual lipids. The microbial fermentation preparation route (CN121203909A) requires multiple steps including strain cultivation, fermentation, and cell lysis, resulting in a longer production cycle and higher preparation costs compared to direct extraction processes.

[0004] Ganoderma lucidum is a traditional medicinal and edible fungus with high biocompatibility. Ganoderma lucidum spore oil is a core functional ingredient in health products and cosmetics. Defatted Ganoderma lucidum spore powder, produced after supercritical CO2 oil extraction, is a major byproduct of the industry and is currently mostly processed as feed and fertilizer for low-value disposal. Its endogenous nucleic acid resources have not been effectively developed, making it a potential raw material for the preparation of fungal-derived PDRN.

[0005] Existing technologies still face numerous technical bottlenecks in the preparation of cosmetic-grade PDRN: Ganoderma lucidum spores have dense cell walls composed of chitin and β-glucan, which, combined with residual lipids, form a double mass transfer barrier. Conventional treatment methods have limited cell wall disruption effects, resulting in low nucleic acid dissolution efficiency. Impurities such as polysaccharides, polyphenols, and triterpenes abundant in the spores easily form non-specific binding and co-precipitation with nucleic acids. Traditional water extraction and alcohol precipitation processes are difficult to accurately remove impurities, and the purity and color of the finished product cannot meet cosmetic raw material standards. Conventional preparation processes often rely on reagents such as SDS, CTAB, and proteinase K, which pose a risk of residual irritation. The processes are cumbersome and costly, and some acid-base treatment systems can easily cause off-odors in the finished product. Post-processing is difficult and not conducive to industrial mass production.

[0006] Currently, most commercially available PDRN is animal-derived, which presents challenges such as limited raw material supply, safety of use, and applicable scenarios. Furthermore, there is currently no green and dedicated preparation process suitable for defatted Ganoderma lucidum spore powder. Therefore, there is an urgent need in this field to develop an efficient and green fungal-derived PDRN preparation technology to achieve high-value utilization of Ganoderma lucidum spore byproducts, providing a safe and reliable new route for fungal-derived PDRN preparation in the cosmetics and medical aesthetics fields. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing Ganoderma lucidum PDRN with anti-wrinkle and firming effects. This method uses defatted Ganoderma lucidum spore powder after supercritical CO2 extraction of spore oil as raw material, which has no animal-derived risks and fills the gap in the high-value utilization of defatted Ganoderma lucidum spore powder. At the same time, it overcomes the problem of low extraction efficiency caused by the dual mass transfer resistance of Ganoderma lucidum spores and solves the problems of many impurities, low purity and irritating odor residue in traditional nucleic acid extraction processes.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution: The first aspect of this invention provides a method for preparing Ganoderma lucidum PDRN with anti-wrinkle and firming effects, comprising the following steps: S1. Add defatted Ganoderma lucidum spore powder to a high-salt buffer system, stir for 20-40 min to obtain the extract; S2. After centrifuging the extract, collect the supernatant and filter it to obtain a clear liquid; S3. Add 10%-40% of the mass of the clarified liquid to the clarified liquid, stir, filter through a stepwise microporous membrane, and collect the filtrate; then, add 40%-200% of the mass of the clarified liquid to the filtrate, stir, centrifuge, and collect the precipitate; resuspend repeatedly with 70%-80% alcohol solvent, centrifuge, and dry to obtain Ganoderma lucidum PDRN raw material.

[0009] Further, in step S1, the defatted Ganoderma lucidum spore powder refers to Ganoderma lucidum (… Ganoderma lucidum The defatted Ganoderma lucidum spore powder obtained by cryogenic ultra-micro low-temperature vibration to break the cell walls, followed by supercritical CO2 fluid extraction to remove the spore oil, has a residual oil content of ≤3% and a water content of ≤8%. If unextracted raw Ganoderma lucidum spore powder is used, the raw oil will encapsulate nucleic acids, resulting in a significant decrease in extraction purity.

[0010] Further, in step S1, the method for preparing the defatted Ganoderma lucidum spore powder is as follows: a. Cryogenic ultra-micro low-temperature vibration cell wall breaking: The dried Ganoderma lucidum spore powder is pre-cooled to -40℃ to -20℃ and maintained for 20 min to 40 min to induce low-temperature embrittlement of the spore wall; then it is put into a high-frequency vibration crusher equipped with a cooling jacket and vibrated at a frequency of 35 Hz to 50 Hz for 10 min to 25 min at a temperature not higher than -10℃ to obtain broken Ganoderma lucidum spore powder; In step a above, if the cryogenic temperature is below -40℃, energy consumption increases dramatically and the improvement in cell wall breakage rate is limited; if it is above -20℃, the embrittlement effect is insufficient and the cell wall breakage rate decreases significantly.

[0011] Furthermore, the preferred parameters for cryogenic ultra-micro low-temperature oscillation cell disruption are: the material temperature is reduced to -30℃, the oscillation frequency is 42 Hz, and the oscillation and disruption time is 25 min.

[0012] b. Highly selective defatting extraction with supercritical CO2 fluid: The broken-cell Ganoderma lucidum spore powder is added to a supercritical CO2 extraction vessel, supercritical CO2 fluid is introduced, the extraction working pressure is adjusted to 28 MPa~35 MPa, the extraction temperature is controlled at 38℃~45℃, the CO2 mass flow rate is controlled at 20~30 kg / (h·kg raw material), and continuous cyclic extraction is performed for 1.5 h~3 h; after the extraction is completed, the defatted Ganoderma lucidum spore powder is collected.

[0013] Furthermore, in step b above, the preferred parameters for supercritical CO2 fluid degreasing extraction are: extraction working pressure of 32 MPa, extraction temperature of 40℃, and continuous cyclic extraction for 2 h.

[0014] In this invention, cryogenic cell disruption and supercritical defatting are not simply sequential operations, but rather exhibit a synergistic effect. Cryogenic embrittlement creates microcracks in the spore wall, increasing the specific surface area for contact between the CO2 fluid and lipids; while during lipid extraction, the osmotic pressure of supercritical CO2 further expands these microcracks, making it easier for the subsequent aqueous buffer to contact the nucleic acids. Without this synergistic pretreatment, even with the same buffer extraction and alcohol precipitation conditions, the purity of PDRN would significantly decrease.

[0015] Further, in step S1, the high-salt buffer system is a sodium chloride-sodium citrate-citric acid buffer system or an acetate-sodium acetate buffer system; its pH is preferably 4.5-5.2, more preferably 4.8.

[0016] In the high-salt buffer system, the concentration of sodium chloride is 0.1-2 mol / L, for example, it can be 0.1 mol / L, 0.5 mol / L, 1.0 mol / L, 1.5 mol / L, 2.0 mol / L, etc., preferably 0.5-1.5 mol / L, more preferably 0.8-1.2 mol / L, and even more preferably 1.0 mol / L.

[0017] Further, in step S1, the high-salt buffer system is a sodium chloride-sodium citrate-citric acid buffer system, which is prepared by adding sodium chloride to water to a concentration of 0.1-2 mol / L, adding sodium citrate to a concentration of 0.01-1 mol / L, adjusting the pH to 4.5-5.2 with citric acid, and stirring at 200-600 rpm until clear to obtain the high-salt buffer system.

[0018] DNA is stable within a pH range of 4–11, undergoing depurination degradation only under strongly acidic conditions below pH 4. Neutral buffer systems cannot achieve both simultaneous impurity removal and efficient extraction. High salt concentrations can effectively disrupt the binding of nucleic acids and proteins, releasing nucleic acids into the solution. Acidic pH conditions promote the denaturation and precipitation of impurity proteins and acidic polysaccharides within Ganoderma lucidum, facilitating the removal of these impurities.

[0019] This invention selects a pH range of 4.5-5.2 to ensure that DNA is within a stable range while allowing for a safety margin, and simultaneously achieves the best removal effect of contaminating proteins. When the pH is below 4.0, DNA will undergo depurination and degradation, resulting in a decrease in PDRN yield; when the pH is above 5.2, the denaturation of contaminating proteins is insufficient, leading to a decrease in removal efficiency.

[0020] Further, in step S1, in the sodium chloride-sodium citrate-citric acid buffer system, the concentration range of sodium chloride is 0.8-1.2 mol / L, the concentration range of sodium citrate is 0.01-0.5 mol / L, and the pH range of the buffer system is 4.76-4.84, preferably 4.8.

[0021] Furthermore, in step S1, the mass ratio of the defatted Ganoderma lucidum spore powder to the high-salt buffer system ranges from 1:8 to 1:12, preferably 1:10. Within this range, sufficient contact between the raw material and the buffer system and extraction efficiency can be ensured, while avoiding a system that is too dilute, which would reduce the efficiency of subsequent alcohol precipitation.

[0022] Furthermore, in step S1, the stirring time is preferably 30 min. In a preferred embodiment, extraction can also be performed using methods such as microwave-assisted extraction, probe-type ultrasonic extraction, or homogenization extraction.

[0023] Further, in step S2, the extract is centrifuged at 4000-5000 rpm for 15-25 min, the supernatant is collected, and then filtered sequentially through a Buchner funnel and a stepped microporous filter to obtain a clear liquid; wherein the pore sizes of the stepped microporous filter membrane are 5 μm, 0.8 μm, and 0.45 μm, respectively. Preferably, the centrifugation speed is 4500 rpm and the centrifugation time is 20 min.

[0024] Traditional one-step high-concentration ethanol precipitation will simultaneously precipitate polysaccharide and protein impurities. In step S3 of this invention, low-concentration ethanol is first used for pre-removal of impurities to remove large-molecule polysaccharides and impurities, and then the ethanol concentration is increased to precipitate PDRN fragments, achieving fractional separation. This is the core innovative step to improve product purity.

[0025] Further, in step S3, an alcohol solvent equivalent to 10%-40% of the mass of the clarified liquid is added to the clarified liquid. After stirring at 200-400 rpm for 1-10 min, the precipitated impurities and submicron colloids are removed by stepwise microporous membrane filtration. The stepwise filtration involves sequentially passing the solution through 0.45 μm, 0.22 μm, and 0.1 μm microporous membranes, and the filtrate is collected. Next, an alcohol solvent equivalent to 40%-200% of the mass of the clarified liquid is added to the filtrate. After stirring at 200-400 rpm for 1-10 min, the solution is centrifuged at 4000-5000 rpm for 15-25 min, and the precipitate is collected. After resuspending the precipitate multiple times with 70%-80% alcohol solvent and centrifuging, the precipitate is dried to obtain Ganoderma lucidum PDRN raw material.

[0026] Furthermore, in step S3, the alcohol solvent includes, but is not limited to, alcohols such as ethanol and isopropanol, and is preferably anhydrous ethanol.

[0027] In this invention, a low-concentration alcohol solvent is used for pre-removal of impurities, which can remove polysaccharides and impurities that may interfere with subsequent nucleic acid precipitation. If high-concentration alcohol solvent precipitation is performed directly, the purity of the product will decrease significantly.

[0028] Furthermore, in step S3, the amount of alcohol solvent added for the first time is 10%-20% of the mass of the clarified liquid, preferably 20%. This range can effectively remove impurities such as polysaccharides and proteins, while avoiding the loss of target nucleic acid components.

[0029] Furthermore, in step S3, the amount of alcohol solvent added a second time is 40%-100% of the mass of the clarified solution, preferably 60%. This range ensures sufficient precipitation of nucleic acid components and achieves a high PDRN recovery rate.

[0030] Furthermore, in step S3, the drying time is 2-6 hours. Within this range, sufficient evaporation of residual ethanol in the precipitate is ensured, while excessive drying leading to PDRN degradation is avoided.

[0031] The second aspect of the present invention provides a Ganoderma lucidum PDRN raw material with anti-wrinkle and firming effects prepared by the preparation method described above.

[0032] The product obtained by this invention is cosmetic-grade Ganoderma lucidum PDRN raw material, with a total protein residue of less than 1%, a total polysaccharide residue of less than 0.5%, no animal-derived components, and no irritating odor.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention eliminates the need for a proteolytic hydrolysis step, simplifying the process flow, providing mild operating conditions (room temperature operation), and requiring less sophisticated equipment, making it suitable for industrial-scale production. The high-salt buffer system is simple in composition and low in cost, and can efficiently dissolve nucleic acid components. Compared to traditional one-step alcohol precipitation, the two-stage step-growth alcohol precipitation strategy effectively separates impurities and the target product, improving product purity and batch-to-batch consistency.

[0034] 2. In a zebrafish model, the upregulation rate of the col1a1a gene in the 10 μg / mL concentration group prepared by this invention reached 190.43%, which was 2.90 times that of the blank group and 15.64 percentage points higher than that of salmon-derived PDRN at the same concentration (174.79%). At the cellular level (HSF UVB photoaging model), the type I collagen synthesis promotion rate in the 10 μg / mL concentration group reached 86.73%, and the collagen secretion was 1.87 times that of the model group, which was 5.25 percentage points higher than that of salmon-derived PDRN at the same concentration. This indicates that the collagen-promoting and anti-wrinkle activity of Ganoderma lucidum PDRN is superior to that of salmon-derived PDRN.

[0035] 3. This invention uses Ganoderma lucidum spore oil processing by-products as raw materials to realize the resource utilization of processing by-products; it eliminates the multi-step process of proteolysis and deproteinization required by traditional animal-derived PDRN, shortening the overall production time; the ethanol used in the process can be recovered through distillation, with a recovery rate of ≥85%, further reducing production costs.

[0036] 4. The entire process of this invention uses only ethanol as an organic solvent, with no emissions of toxic or harmful reagents such as CTAB and SDS, making it green and environmentally friendly; the product contains no animal-derived ingredients and leaves no irritating odor residue, meeting the standards for green cosmetic raw materials. Detailed Implementation

[0037] The present invention will be further described below with reference to specific embodiments, so that those skilled in the art can better understand and implement the present invention, but the embodiments are not intended to limit the present invention.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0039] Unless otherwise specified, the experimental methods used in the following examples and comparative examples are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0040] Cell wall breakage rate: Microscopic counting was employed. A suitable amount of the disrupted spore powder was taken, mixed with water to prepare a uniform spore suspension, and smeared. Under a 400x optical microscope, avoiding the edges of the smear and areas of spore aggregation, 10 fields of view were randomly selected for observation. Spores with ruptured outer shells and exposed intracellular material were defined as disrupted spores. The number of intact spores and the total number of disrupted spores in each field of view were counted and calculated using the following formula:

[0041] Where: N 破壁 N represents the total number of broken spores obtained from all fields of view; 完整 The total number of complete spores obtained from the entire field of view.

[0042] (2) Residual oil content: The Soxhlet extraction gravimetric method was used. A precise amount of defatted spore powder sample m, obtained through supercritical CO2 extraction, was placed into a pre-weighed filter paper tube and sealed. The constant weight of the empty filter paper tube was recorded as m. 滤纸 The sample was placed in a Soxhlet extractor and extracted with n-hexane as solvent under reflux in a water bath for at least 8 hours, or until the last few drops of extract evaporated onto the filter paper without leaving any oil residue and the sample mass no longer changed. After extraction, the filter paper tube was removed, and after most of the solvent was evaporated at room temperature, it was dried in a 105 ℃ forced-air drying oven to constant weight. After cooling to room temperature, the total mass m2 of the filter paper tube and the completely defatted matrix was weighed.

[0043] Formula for calculating total residual oil content:

[0044] Where: m is the mass of the spore powder sample to be tested before extraction; m2 is the total mass of the filter paper tube and defatted matrix after complete extraction and drying; m 滤纸 The constant weight of the air filter paper tube.

[0045] (3) Total protein residue: A series of standard solutions were prepared using bovine serum albumin (BSA) using the BCA colorimetric method, and the absorbance was measured at a wavelength of 562 nm to establish a standard curve. The absorbance of the sample solution was measured using the same method, and the protein concentration of the test solution was obtained by substituting it into the standard curve. The total residual protein in the sample was calculated by combining the sample dilution factor, and the result was expressed as BSA equivalent.

[0046] (4) Total sugar residue: A series of standard solutions were prepared using the phenol-sulfuric acid colorimetric method, and the absorbance was measured at a wavelength of 490 nm to establish a standard curve. The absorbance of the sample solution was measured using the same method, and the results were substituted into the standard curve to obtain the total sugar concentration of the test solution. The total residual sugar in the sample was calculated by combining the sample dilution factor, and the results were expressed as glucose equivalents.

[0047] (5) Characterization of molecular weight range: Agarose gel electrophoresis was used, with DNA markers selected as molecular weight references. Agarose gels (containing Nucleic Acid Stain) with a volume fraction of 1.5-3.0% were prepared. 10 μL of the sample to be tested was mixed with 2 μL of 6× Loading Buffer and then loaded onto the gel. Simultaneously, DNA markers were added to one well. Electrophoresis was performed in a 1×TAE buffer system at a constant voltage of 90V for 30-60 min. After electrophoresis, the gel was observed using a gel imaging system, and the molecular weight distribution range of the sample was analyzed by comparing it with the marker bands. Example 1

[0048] This embodiment provides a method for preparing Ganoderma lucidum PDRN, including the following steps: Step S1 (Pretreatment): Take dried Ganoderma lucidum ( Ganoderma lucidum The spore powder was pre-cooled to -30°C in an ultra-low temperature chamber and maintained for 30 min. It was then rapidly transferred to a vibrating mill equipped with a -10°C cooling jacket, and the mill was pulverized for 15 min at a vibration frequency of 42 Hz. The cell wall breakage rate was measured to be 98.6% under a microscope. The broken spore powder was then loaded into a supercritical extraction vessel, and CO2 was introduced. The working pressure was controlled at 32 MPa, the working temperature at 40°C, and the CO2 flow rate at 25 kg / h. Continuous extraction was performed for 2.0 h to remove the spore oil. The defatted Ganoderma lucidum spore powder in the vessel was collected, and its residual oil rate was determined to be 1.45%.

[0049] Step S2: Add sodium chloride to purified water to a final concentration of 1.0 mol / L, then add sodium citrate, adjust the pH to 4.8 with citric acid, and stir at 300 rpm for about 5 minutes until clear to obtain a high-salt buffer system.

[0050] Step S3: Weigh defatted Ganoderma lucidum spore powder and add it to a high-salt buffer system with a material-to-liquid ratio of 1:10. Stir in a multi-functional extraction tank for 30 minutes at room temperature.

[0051] Step S4: Centrifuge the extract at 4500 rpm for 20 min and collect the supernatant; pass it sequentially through a Buchner funnel, a 5 μm microporous membrane, a 0.8 μm microporous membrane, and a 0.45 μm microporous membrane to obtain a clear solution.

[0052] Step S5-1 (Impurity Removal): Weigh the total mass of the clarified liquid, add 20% of the total mass of the clarified liquid in anhydrous ethanol, stir at 200-400 rpm for 5 min, remove the precipitated impurities and retain submicron colloids by filtration through a series of microporous membranes, and then filter through 0.45 μm, 0.22 μm and 0.1 μm microporous membranes in sequence, and collect the filtrate.

[0053] Step S5-2 (Precipitation of nucleic acid): Add 60% anhydrous ethanol (total mass of the clarified liquid) to the filtrate obtained in step S5-1, stir at 200-400 rpm for 5 min, centrifuge at 4500 rpm for 20 min, and collect the precipitate; purify Ganoderma lucidum PDRN, resuspend the precipitate in 70% ethanol, centrifuge, and discard the supernatant; resuspend again in 70% ethanol, centrifuge, and dry thoroughly to obtain Ganoderma lucidum PDRN raw material.

[0054] The Ganoderma lucidum PDRN raw material prepared in Example 1 was tested and found to have A260 / A280=1.92, A260 / A230=2.08, protein residue of 0.094%, polysaccharide residue of 0.286%, pH value of 6.48, and molecular weight distribution range of 50-300 kDa. Examples 2-4

[0055] Following the same process as in Example 1, the sodium chloride concentration in step S2 was set to 0.1 mol / L, 0.5 mol / L, and 2.0 mol / L respectively, while other parameters remained unchanged, to prepare Ganoderma lucidum PDRN, resulting in Examples 2-4.

[0056] Table 1

[0057] Referring to Table 1, increasing the sodium chloride concentration from 0.1 mol / L to 1.0 mol / L improves the ionic strength of the system, promoting the complete dissolution of PDRN from Ganoderma lucidum and resulting in a continuous increase in PDRN yield. The A260 / A280 and A260 / A230 ratios also increase simultaneously, while the proportion of impurities decreases. When the concentration reaches 2.0 mol / L, the excessively high salt concentration causes some ribonucleotides to precipitate, leading to a decrease in PDRN yield and consequently, a reduction in quality indicators. Considering both yield and quality indicators, a sodium chloride concentration of 1.0 mol / L is optimal. Examples 5-6

[0058] Following the process of Example 1, the pH in step S2 was set to 4.5 and 5.2 respectively, while other parameters remained unchanged, to prepare Ganoderma lucidum PDRN, resulting in Examples 5 and 6.

[0059] Table 2

[0060] Referring to Table 2, the pH of the buffer system affects the PDRN dissolution efficiency and the quality control of A260 / A280 and A260 / A230. When the pH is 4.8, the PDRN yield in the extract is high, and the A260 / A230 ratio is optimal, with the lowest levels of polysaccharide and polyphenol impurities. When the pH is lowered to 4.5, the PDRN yield decreases, the A260 / A230 ratio decreases, and the proportion of impurities increases. When the pH is raised to 5.2, the A260 / A230 ratio remains at a relatively good level, but the PDRN yield decreases significantly. Examples 7-8

[0061] Following the process of Example 1, the proportion of impurity-removing ethanol in step S5-1 was set to 10% and 40% respectively, while other parameters remained unchanged, to prepare Ganoderma lucidum PDRN, resulting in Examples 7 and 8.

[0062] Table 3

[0063] Referring to Table 3, the quality of Ganoderma lucidum PDRN prepared was best when the proportion of impurity-removing ethanol was 20%. When the proportion of impurity-removing ethanol was low (10%), impurities were not removed sufficiently, resulting in a decrease in OD260 / 230; when the proportion of impurity-removing ethanol was high (40%), some nucleic acids were co-precipitated, resulting in a decrease in PDRN yield. Examples 9-14

[0064] Following the process of Example 1, the proportion of precipitated ethanol in step S5-2 was set to 20%, 40%, 80%, 100%, 150%, and 200%, respectively, while other parameters remained unchanged, to prepare Ganoderma lucidum PDRN, resulting in Examples 9-14.

[0065] Table 4

[0066] Referring to Table 4, when the ethanol addition ratio is below 40%, the PDRN yield is very low; when the ethanol addition ratio is above 60%, the A260 / A230 ratio continues to decrease significantly, indicating that polysaccharide, polyphenolic impurities, salts, or organic solvent residues co-precipitate along with nucleic acids. Using higher ethanol concentrations (e.g., 100%, 150%, 200%) can improve the PDRN yield, but it brings two drawbacks: firstly, it increases ethanol consumption, significantly increasing raw material costs; secondly, it increases co-precipitated impurities, requiring additional purification steps to remove impurities, lengthening the process and increasing production load. Considering the product A260 / A280 and A260 / A230 quality indicators, PDRN yield, raw material costs, and subsequent purification pressure, a 60% ethanol addition ratio is optimal for the alcohol precipitation stage. Examples 15-18

[0067] Following the process of Example 1, the mass ratio of defatted Ganoderma lucidum spore powder to the high-salt buffer system in step S2 was set to 1:5, 1:8, 1:12, and 1:15, respectively, while other parameters remained unchanged, to prepare Ganoderma lucidum PDRN, resulting in Examples 15-18.

[0068] Table 5

[0069] Referring to Table 5, when the material-to-liquid ratio is 1:5, extraction is insufficient. When the material-to-liquid ratio is increased to 1:15, although the PDRN yield increases slightly, the amount of buffer system used increases by 50%, and the subsequent ethanol consumption during precipitation also increases, thus increasing the production scale and material costs. Considering the PDRN yield, product quality standards, and the economics of industrial production, the material-to-liquid ratio is determined to be 1:8~1:12, with 1:10 being the preferred ratio. Comparative Example 1

[0070] Comparative Example 1 skipped the S1-2 supercritical defatting step and directly used Ganoderma lucidum spore powder after cryogenic cell wall disruption for extraction. The remaining steps were the same as in Example 1.

[0071] Detection results: PDRN yield was 0.021%, A260 / A280=2.09, A260 / A230=0.84.

[0072] Conclusion: The lack of supercritical defatting treatment resulted in a significant decrease in extraction efficiency due to the encapsulation of nucleic acids in the spore oil, demonstrating the necessity of the supercritical defatting step. Comparative Example 2

[0073] Comparative Example 2 was extracted with Tris-HCl buffer (containing 1.0 mol / L NaCl) at pH 7.0, and precipitated directly with 80% ethanol in one step. The remaining steps were the same as in Example 1.

[0074] Detection results: PDRN yield was 0.085%, A260 / A280=1.86, A260 / A230=1.74.

[0075] Conclusion: The sample quality of the traditional neutral buffer + one-step alcohol precipitation method was significantly lower than that of the sample in Example 1, proving that the acidic high-salt + two-stage step-growth alcohol precipitation process can improve the yield and purity of PDRN. Comparative Example 3

[0076] Comparative Example 3 used a Tris-HCl buffer solution at pH 7.0 (containing 1.0 mol / L NaCl), and the remaining steps were the same as in Example 1 (the two-stage alcohol precipitation step was retained unchanged).

[0077] Detection results: PDRN yield was 0.083%, A260 / A280=1.85, A260 / A230=1.80.

[0078] Conclusion: When using a traditional neutral buffer, both the PDRN yield and the A260 / 230 index decreased, indicating that the acidic high-salt buffer system of the present invention has a significant impact on the PDRN yield and purity. Comparative Example 4

[0079] Comparative Example 4 used the same acidic high-salt buffer system with pH 4.8 as Example 1, but skipped the 20% ethanol pre-purification step and directly added 80% anhydrous ethanol by weight of the system to precipitate nucleic acids in one step. The remaining steps were the same as in Example 1.

[0080] Detection results: PDRN yield was 0.131%, A260 / A280=1.89, A260 / A230=1.84.

[0081] Conclusion: When PDRN is precipitated directly in one step without pre-removal, the yield is slightly higher than that of two-stage ethanol precipitation, but the A260 / 230 index decreases, indicating that polysaccharide and polyphenol impurities co-precipitate with nucleic acids, resulting in a decrease in product purity. Comparative Example 5

[0082] Comparative Example 5 used room temperature mechanical crushing (without cryogenic pretreatment), and the remaining steps were the same as in Example 1.

[0083] Test results: Cell wall breakage rate 25.3%, PDRN yield 0.016%, A260 / A280=1.80, A260 / A230=1.62.

[0084] Conclusion: The cryogenic cell wall disruption step can significantly improve the cell wall disruption rate of Ganoderma lucidum spore powder, thereby improving the PDRN yield and quality indicators. Comparative Example 6

[0085] Comparative Example 6 replaced the sodium chloride-sodium citrate-citric acid buffer system in step S1 with an acetate-sodium acetate buffer system (pH 4.8, containing 1.0 mol / L NaCl), and the remaining steps were the same as in Example 1.

[0086] Test results: PDRN yield was 0.112%, A260 / A280=1.88, A260 / A230=1.94, but the product showed obvious residual acetic acid odor after sensory evaluation.

[0087] Conclusion: Although the acetic acid-sodium acetate system can extract nucleic acids, the product has a residual acetic acid odor and requires additional deodorization treatment.

[0088] As can be seen from Comparative Examples 1 to 6, the optimal process of this invention is superior to each of the comparative examples in terms of PDRN yield, A260 / A280, A260 / A230 and residual impurities (protein, polysaccharide), which proves the necessity and inventiveness of the core steps of this invention, namely, cryogenic cell disruption + supercritical defatting synergistic pretreatment, acidic high-salt buffer extraction, and two-stage step-growth alcohol precipitation. Experimental Example 1: Evaluation of the Collagen-Promoting Efficacy of Ganoderma Lucidum PDRN in Zebrafish

[0089] This experiment used a zebrafish qRT-PCR collagen gene expression model to evaluate the collagen-promoting, firming, and anti-wrinkle activity of Ganoderma lucidum PDRN. Healthy AB strain zebrafish juveniles (4 dpf) were selected as the experimental model. The test samples were diluted with pure water, with 25 juveniles per group. Samples were collected after 24 h of incubation at 28℃ in the dark. Total RNA was extracted in an enzyme-free environment. β-actin As an internal reference gene, the zebrafish type I collagen gene was detected by qRT-PCR. col1a1a The relative expression level.

[0090] The experiment was statistically analyzed using SPSS software. One-way ANOVA was used for comparisons among multiple groups, Dunnett's test was used for within-group controls, and Tukey's test was used for pairwise comparisons between groups. The significance level was set at α=0.05. The validity criteria for the experiment were: zebrafish juvenile survival rate ≥90%, and the expression level of the col1a1a gene in the sample group significantly higher than that in the blank control group. p <0.05).

[0091] The Ganoderma lucidum PDRN prepared in Example 1 was formulated into concentration gradient test solutions of 1 μg / mL, 5 μg / mL, and 10 μg / mL. A blank control group and a positive control group of 10 μg / mL salmon-derived PDRN were set up. Gene expression was quantified using the ΔΔCt method, with a relative gene expression fold of 2. -ΔΔCt The upregulation rate (%) of the col1a1a gene = [2 -ΔΔCt -1]×100%, with the blank group as the expression benchmark, the experimental results are shown in Table 6.

[0092] Table 6

[0093] As shown in Table 6, all concentrations of Ganoderma lucidum PDRN significantly upregulated the expression of the col1a1a gene in zebrafish (p < 0.05), with the efficacy exhibiting a clear concentration-dependent effect. The collagen gene upregulation rate in the 10 μg / mL Ganoderma lucidum PDRN group reached 190.43%, which was 2.90 times that of the control group and 15.64 percentage points higher than that of salmon-derived PDRN at the same concentration. The difference between groups was statistically significant (p < 0.05). Under the conditions of this experiment, Ganoderma lucidum PDRN possessed excellent collagen-promoting and anti-wrinkle activities, and its efficacy was superior to that of traditional salmon-derived PDRN. Experimental Example 2: Evaluation of the cellular level collagen-promoting efficacy of Ganoderma lucidum PDRN

[0094] This study used a human skin fibroblast (HSF) UVB photoaging model to evaluate the firming, anti-wrinkle, and collagen-promoting activities of Ganoderma lucidum PDRN. Samples were dissolved in pure water to prepare concentration gradient test solutions of 1 μg / mL, 5 μg / mL, and 10 μg / mL. HSF cells were cultured with the drug for 4 days after UVB photoaging modeling, with the entire experimental period lasting 3 weeks. Each group had at least 3 replicates. The content of type I collagen (COL-1) in the cell supernatant was detected by ELISA at a wavelength of 450 nm, and the COL-1 protein concentration was calculated using a standard curve.

[0095] Statistical analysis was performed using SPSS software. One-way ANOVA was used for comparisons among multiple groups, and Dunnett's and Tukey's tests were used to assess differences between groups. The significance level was set at α=0.05. The effectiveness of the model was determined by a significant decrease in COL-1 content in the model group compared to the control group. p <0.05), the COL-1 content in the positive control group was significantly higher than that in the model group ( p <0.05) was the judgment criterion; the COL-1 content in the sample group was significantly higher than that in the model group ( p A value <0.05 indicates that the substance has collagen-promoting and anti-wrinkle activity.

[0096] The type I collagen synthesis promotion rate was calculated using the photoaging model group as a baseline. The calculation formula was: Type I collagen synthesis promotion rate (%) = [(COL-1 concentration in the sample group - COL-1 concentration in the model group) / COL-1 concentration in the model group] × 100%. A blank control group, a photoaging model group, and a 10 μg / mL salmon-derived PDRN positive control group were set up in the experiment. The results are shown in Table 7.

[0097] Table 7

[0098] As shown in Table 7, within the concentration range of 1 μg / mL to 10 μg / mL, Ganoderma lucidum PDRN significantly upregulated type I collagen secretion in photoaged fibroblasts (p < 0.05), with the effect being concentration-dependent. At a concentration of 10 μg / mL, Ganoderma lucidum PDRN promoted collagen synthesis by 86.73%, and the collagen secretion amount was 1.87 times that of the model group, which was 5.25 percentage points higher than that of salmon-derived PDRN at the same concentration, with significant differences between groups (p < 0.05). Cellular level studies confirmed that Ganoderma lucidum PDRN can significantly improve collagen loss caused by photoaging, exhibiting excellent collagen-promoting, anti-wrinkle, and firming activities.

[0099] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A method for preparing Ganoderma lucidum PDRN with anti-wrinkle and firming effects, characterized in that, Includes the following steps: S1. Add defatted Ganoderma lucidum spore powder to a high-salt buffer system, stir for 20-40 min to obtain an extract; the defatted Ganoderma lucidum spore powder is defatted Ganoderma lucidum spore powder obtained after supercritical CO2 extraction of spore oil; S2. After centrifuging the extract, collect the supernatant and filter it to obtain a clear liquid; S3. Add 10%-40% of the mass of the clarified liquid to the clarified liquid, stir, filter through a stepwise microporous membrane, and collect the filtrate; then, add 40%-200% of the mass of the clarified liquid to the filtrate, stir, centrifuge, and collect the precipitate; resuspend repeatedly with 70%-80% alcohol solvent, centrifuge, and dry to obtain Ganoderma lucidum PDRN raw material.

2. The method for preparing Ganoderma lucidum PDRN with anti-wrinkle and firming effects according to claim 1, characterized in that, In step S1, the method for preparing the defatted Ganoderma lucidum spore powder is as follows: a. Pre-cool the dried Ganoderma lucidum spore powder to -40℃ to -20℃ and maintain it for 20 min to 40 min. Then put it into a high-frequency vibration crusher and vibrate and crush it at a frequency of 35 Hz to 50 Hz for 10 min to 25 min at a temperature not higher than -10℃ to obtain broken Ganoderma lucidum spore powder. b. Add the broken-cell wall Ganoderma lucidum spore powder to a supercritical CO2 extraction vessel, introduce supercritical CO2 fluid, adjust the extraction working pressure to 28 MPa~35 MPa, control the extraction temperature at 38℃~45℃, control the CO2 mass flow rate at 20~30 kg / (h·kg raw material), and continuously circulate for extraction for 1.5 h~3 h; after extraction, collect the defatted Ganoderma lucidum spore powder.

3. A method for preparing Ganoderma lucidum PDRN with anti-wrinkle and firming effects according to claim 1, characterized in that, The high-salt buffer system is a sodium chloride-sodium citrate-citric acid buffer system; in the high-salt buffer system, the concentration of sodium chloride is 0.1-2 mol / L, and the pH is 4.5-5.

2.

4. The method for preparing Ganoderma lucidum PDRN with anti-wrinkle and firming effects according to claim 3, characterized in that, The preparation method of the sodium chloride-sodium citrate-citric acid buffer system is as follows: Add sodium chloride to water to a concentration of 0.1-2 mol / L, add sodium citrate to a concentration of 0.01-1 mol / L, adjust the pH to 4.5-5.2 with citric acid, and stir at 200-600 rpm until clear to obtain the high-salt buffer system.

5. A method for preparing Ganoderma lucidum PDRN with anti-wrinkle and firming effects according to claim 4, characterized in that, In the sodium chloride-sodium citrate-citric acid buffer system, the concentration range of sodium chloride is 0.8-1.2 mol / L, the concentration range of sodium citrate is 0.01-0.5 mol / L, and the pH range of the buffer system is 4.76-4.

84.

6. The method for preparing Ganoderma lucidum PDRN with anti-wrinkle and firming effects according to claim 1, characterized in that, The mass ratio of defatted Ganoderma lucidum spore powder to the high-salt buffer system is 1:8-1:

12.

7. A method for preparing Ganoderma lucidum PDRN with anti-wrinkle and firming effects according to claim 1, characterized in that, In step S2, the extract is centrifuged at 4000-5000 rpm for 15-25 min, the supernatant is collected, and filtered sequentially through a Buchner funnel and a stepped microporous membrane to obtain a clear liquid; wherein the pore sizes of the stepped microporous membrane are 5 μm, 0.8 μm, and 0.45 μm, respectively.

8. The method for preparing Ganoderma lucidum PDRN with anti-wrinkle and firming effects according to claim 1, characterized in that, In step S3, the alcohol solvent is ethanol or isopropanol; and / or, The amount of alcohol solvent added for the first time is 10%-20% of the mass of the clarified liquid, and the amount of alcohol solvent added for the second time is 40%-100% of the mass of the clarified liquid; and / or, The drying time is 2-8 hours.

9. A method for preparing Ganoderma lucidum PDRN with anti-wrinkle and firming effects according to claim 1, characterized in that, In step S3, the pore sizes of the stepped microporous filter membrane are 0.45 μm, 0.22 μm, and 0.1 μm, respectively.

10. The Ganoderma lucidum PDRN with anti-wrinkle and firming effects prepared by the preparation method according to any one of claims 1 to 9, characterized in that, The Ganoderma lucidum PDRN with anti-wrinkle and firming effects contains less than 1% total protein residue, less than 0.5% total polysaccharide residue, no animal-derived components, and no irritating odor.

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