Preparation method and application of rice callus-derived pdrn

Abstract

The application discloses a preparation method and application of PDRN (plant-derived RNA) from rice callus. The preparation method comprises the following steps: inducing and suspending culturing of the rice callus; homogenizing the rice callus suspension cells obtained in step 1; sequentially using cellulase, pectinase and protease K for enzymolysis; nucleic acid purification and precise ultrasonic shearing are performed in sequence, so that PDRN with a molecular weight of 25-500 bp is obtained. Through optimization of the rice callus culture condition and combination of the joint lysis process of "mechanical grinding-composite enzymolysis-protease treatment", efficient and high-purity preparation of the plant source PDRN is realized, the extraction rate can be higher than 0.5 mg / g of fresh weight of the callus, and the purity A260 / A280 is greater than or equal to 1.8. The obtained PDRN has significant effects in terms of anti-wrinkle, soothing, moisturizing, anti-inflammatory and anti-aging.

Description

Technical Field

[0001] This invention belongs to the field of bioactive substance preparation technology, specifically relating to a method for preparing PDRN derived from rice callus and its application. Background Technology

[0002] Polydeoxyribonucleotides (PDRNs) are mixtures of deoxyribonucleotides of varying lengths. Due to their bioactivities, such as promoting tissue repair, anti-inflammation, and promoting angiogenesis, they show broad application prospects in biomedicine and cosmetic repair fields. Currently, the mainstream PDRN sources on the market are animal-derived, such as salmon testes. However, animal-derived PDRNs have several drawbacks: First, the availability of raw materials is limited by season and region, making sustainable supply difficult; second, animal-derived materials pose a risk of introducing animal-derived pathogens and immunogenic substances, potentially threatening the biosafety of the product; furthermore, the extraction process easily introduces impurities such as animal-derived proteins and fats, increasing purification difficulty and cost.

[0003] To overcome the aforementioned shortcomings, research on plant-derived PDRN has gradually become a hot topic. Rice, a widely cultivated gramineous crop globally, possesses callus tissue with rapid proliferation and the potential for large-scale in vitro culture, making it an ideal source of plant-derived PDRN. However, using rice callus tissue for PDRN preparation still faces technical challenges: on the one hand, existing rice callus culture techniques suffer from unstable callus emergence rates and difficulty in controlling the induction conditions for high-quality callus, limiting the stable supply of raw materials; on the other hand, the dense cell wall structure of rice callus makes it difficult for conventional physical or chemical lysis methods to fully and gently release intracellular nucleic acids, resulting in low PDRN extraction efficiency. Currently, there is no mature PDRN extraction process specifically designed for the characteristics of rice callus tissue, nor is there research on the application scenarios for rice-derived PDRN. Summary of the Invention

[0004] The purpose of this invention is to address the raw material deficiencies and process limitations of existing PDRN preparation technologies by providing a method for preparing PDRN derived from rice callus and its applications. This method aims to solve problems such as low callus induction efficiency in rice, numerous impurities in animal-derived PDRN extraction, and low extraction rates in existing technologies, achieving efficient, high-purity, and controllable molecular weight preparation of plant-derived PDRN, and exploring potential application scenarios for rice-derived PDRN.

[0005] The objective of this invention is achieved through the following technical solution: In a first aspect, the present invention provides a method for preparing PDRN derived from rice callus, comprising the following steps: Step 1, Induction and suspension culture of rice callus: Using mature rice embryos as explants, after disinfection, they are inoculated into induction medium and cultured in the dark to obtain callus; after subculturing the callus, suspension culture is performed to obtain rice callus suspension cells. Step 2, Pretreatment: The rice callus suspension cells obtained in Step 1 are homogenized to obtain callus tissue homogenate. Step 3, combined lysis: cellulase and pectinase are added to the callus homogenate obtained in step 2 for the first enzymatic hydrolysis, then the pH is adjusted, and proteinase K is added for the second enzymatic hydrolysis. After solid-liquid separation, the lysate is obtained. Step 4, Nucleic acid purification: The lysis buffer obtained in step 3 is subjected to nucleic acid precipitation, washing and dissolution to obtain nucleic acid extract; Step 5, Precise ultrasonic shearing: The nucleic acid extract obtained in step 4 is subjected to ultrasonic shearing to obtain a solution containing PDRN.

[0006] Preferably, in step 1, the induction medium is N6 medium with 1-3 mg / L of 2,4-dichlorophenoxyacetic acid, 25-35 g / L of sucrose, and 5-8 g / L of agar added, and the pH is 5.8-6.0; and / or, the control conditions for the dark culture include: temperature 25±1℃, culture time 14-20 days; and / or, the suspension culture medium is MS basal medium with 1-3 mg / L of 2,4-dichlorophenoxyacetic acid and 25-35 g / L of sucrose added, and the pH is 5.8-6.0; and / or, the control conditions for the suspension culture include: using a shaker, temperature 25-28℃, and rotation speed 110-130 rpm.

[0007] Preferably, in step 2, the pretreatment step is as follows: rice callus suspension cells are mixed at a ratio of 1g fresh weight to 3-5mL buffer solution and ground into a homogenate under ice bath conditions; more preferably, the buffer solution is a 15-25mM Tris-HCl buffer solution with a pH of 5.5-6.5.

[0008] Preferably, in step 3, the amount of enzyme added for the first enzymatic hydrolysis is: 8-12 U / mL of cellulase and 4-6 U / mL of pectinase, and the enzymatic hydrolysis conditions are: 50-55℃ water bath shaking lysis for 60-90 min; the conditions for the second enzymatic hydrolysis are: adjusting the pH to 7.8-8.2, adding 0.5-1.5 U / mL of proteinase K, and lysing at 58-62℃ for 50-90 min.

[0009] Preferably, in step 4, the nucleic acid purification step is as follows: add 0.6 to 1 volume of isopropanol to the lysis buffer, let stand at -20±5℃ for 20 to 30 min, centrifuge, and collect the precipitate; wash the precipitate with 75% cold ethanol, dry it, and dissolve it in TE buffer. More preferably, the centrifugation control parameters include: centrifugation at 4±1℃ and 8000~12000r / min for 10~20min; More preferably, the pH of the TE buffer solution is 7.8 to 8.2.

[0010] Preferably, in step 5, the nucleic acid extract obtained in step 4 is placed in an ice bath and ultrasonically sheared using an ultrasonic cell disruptor; more preferably, the process parameters for ultrasonic shearing are: power 200-400W, working time 2-4s, interval 4-6s, and total shearing time 30-60min.

[0011] More preferably, the method also includes step 6, PDRN purification: 1,2-hexanediol and sodium phytate are added to the PDRN solution obtained in step 5, the pH is adjusted and then filtered to obtain a purified PDRN solution derived from rice callus tissue. More preferably, the final weight-volume concentration of 1,2-hexanediol in the system is 1-3%, and the final weight-volume concentration of sodium phytate in the system is 0.1-0.5%. More preferably, the pH is adjusted to 7.0-7.5; Even more preferably, filtration is performed using a 0.22μm sterile filter membrane.

[0012] Optionally, the 1,2-hexanediol may be partially or completely replaced with at least one of propylene glycol and trehalose.

[0013] In a second aspect, the present invention provides rice callus-derived PDRN prepared by the preparation method described in the first aspect, wherein the PDRN has a molecular weight of 25-500 bp.

[0014] Thirdly, the present invention provides the use of the PDRN described in the second aspect in the preparation of at least one skin product as follows: 1) Anti-wrinkle skin products; 2) Soothing skin products; 3) Moisturizing skin products; 4) Anti-inflammatory skin products; 5) Anti-aging skin products.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: Firstly, the raw materials are highly efficient and stable: By optimizing the induction medium (N6+2.0mg / L 2,4-D) and culture conditions, this invention enables the callus emergence rate of mature rice embryos to reach over 90%, and the callus tissue to proliferate rapidly. Combined with suspension culture technology, it can achieve large-scale and sustainable expansion of raw materials in vitro, completely solving the problem of supply limitations of plant-derived raw materials.

[0016] Secondly, the extraction rate and purity are high: This invention targets the dense cell wall structure of rice callus and designs a combined lysis process of "mechanical grinding - enzymatic hydrolysis (cellulase, pectinase) - protease treatment," which can gently and fully release intracellular nucleic acids. Combined with purification methods of isopropanol precipitation and ethanol washing, impurities such as polysaccharides, polyphenols, and proteins are effectively removed. The obtained PDRN extraction rate can reach over 0.5 mg / g of fresh callus weight, with a purity of A260 / A280 ≥ 1.8 and A260 / A230 ≥ 2.0, meeting the requirements for biopharmaceutical grade purity.

[0017] Thirdly, the molecular weight is precisely controllable: This invention establishes a "high-purity nucleic acid-precise ultrasonic shearing" process. Based on high-purity DNA, ultrasonic treatment is performed. By precisely controlling the total shearing time, PDRN with a molecular weight range of 25-500bp can be stably and repeatedly prepared, meeting the differentiated requirements of PDRN molecular weight for different application scenarios such as cosmetic external use and medical wound healing.

[0018] Fourth, it is green, environmentally friendly, and low-cost: The reagents used in the preparation process of this invention are mostly conventional biological reagents, and organic solvents such as ethanol and isopropanol can be recycled, without generating any toxic or harmful waste. At the same time, rice, as a widely cultivated food crop worldwide, is readily available and inexpensive, significantly reducing the preparation cost of PDRN compared to animal-derived raw materials such as salmon testes. The process steps are simple and easy to scale up for industrial production.

[0019] Fifth, it has high biocompatibility: This invention uses plant-derived raw materials, which completely eliminates the risk of animal-derived pathogens and immunogenicity, and has better biocompatibility with humans and broader application prospects.

[0020] In summary, this invention optimizes rice callus culture conditions, establishes a combined lysis process of "mechanical grinding-compound enzymatic hydrolysis-protease treatment," and combines it with graded purification and precise ultrasonic shearing technology to achieve efficient, high-purity, and controllable molecular weight preparation of plant-derived PDRN. The obtained PDRN exhibits significant effects in anti-wrinkle, soothing, moisturizing, anti-inflammatory, and anti-aging aspects, providing a novel solution for the green and sustainable production of PDRN and its application in the field of skin health. Attached Figure Description

[0021] Figure 1 The graph shows the inhibition rate of PDRN derived from rice callus prepared in Example 2 of this invention against elastase activity. The horizontal axis represents the concentration of the tested sample (ppm), and the vertical axis represents the elastase inhibition rate (%). The results show that the inhibition rate increases in a concentration-dependent manner within the concentration range of 6.25~100 ppm.

[0022] Figure 2The graph shows the inhibition rate of PDRN derived from rice callus prepared in Example 2 of this invention on hyaluronidase activity. The horizontal axis represents the concentration of the tested sample (ppm), and the vertical axis represents the hyaluronidase inhibition rate (%). The results show that the inhibition rate increases in a concentration-dependent manner within the concentration range of 5 to 100 ppm.

[0023] Figure 3 The graph shows the change in the moisture retention rate of PDRN derived from rice callus prepared in Example 2 of this invention over time. The horizontal axis represents time (h), and the vertical axis represents the moisture retention rate (%). With 5% glycerol solution as a control, the results show that the moisture retention rate of the tested sample was higher than that of the control group at each time point.

[0024] Figure 4 The effect of PDRN, a rice callus-derived protein prepared in Example 2 of this invention, on the viability of RAW264.7 cells.

[0025] Figure 5 The figure shows the effect of PDRN derived from rice callus prepared in Example 2 of this invention on the expression of inflammation-related genes induced by LPS in RAW264.7 cells. The horizontal axis represents different treatment groups, and the vertical axis represents the relative expression level of each inflammation-related gene. The results show that the 25 ppm and 100 ppm test samples significantly downregulated the expression of COX-2, IL-1α, NOS2, IL-1β, IL-6, and TNF-α genes, and upregulated the expression of IL-10 gene.

[0026] Figure 6 The effect of PDRN, a rice callus-derived protein prepared in Example 2 of this invention, on the activity of HSF cells.

[0027] Figure 7 Immunofluorescence staining images (magnification 200×) showing the effect of rice callus-derived PDRN prepared in Example 2 of the present invention on type I collagen synthesis in HSF cells, showing DAPI-stained cell nuclei, Collagen I green fluorescence, and Merge images.

[0028] Figure 8 The graph shows the statistical results of the relative expression of type I collagen in HSF cells by PDRN derived from rice callus prepared in Example 2 of this invention. The horizontal axis represents different treatment groups, and the vertical axis represents the relative expression of type I collagen (normalized with the control group as the benchmark). The results show that 25 ppm and 100 ppm of the tested samples significantly promoted the expression of type I collagen. Detailed Implementation

[0029] In this embodiment of the invention, the rice variety is Zhonghua 11, which is sourced from Baige Gene Technology (Jiangsu) Co., Ltd.

[0030] Furthermore, it should be noted that unless specific conditions are specified in the examples, they should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0031] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These descriptions are for illustrative purposes only and not for limiting the scope of protection of the present invention. Furthermore, it should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning understood by those skilled in the art.

[0032] Example 1: In this embodiment, rice callus PDRN with a molecular weight of 25-500 bp was prepared. The specific process is as follows: Step 1, Explant Disinfection and Callus Induction: 100 plump, fresh, mature rice embryos (variety: Zhonghua 11) were selected as explants, and the glumes were removed. The explants were first disinfected with 75% ethanol for 30 seconds, then rinsed once with sterile water; next, they were disinfected with 0.1% mercuric chloride solution for 9 minutes, rinsed four times with sterile water, and the surface moisture was blotted dry with sterile filter paper. The disinfected explants were inoculated onto induction medium (N6 basal medium supplemented with 2 mg / L 2,4-D, 30 g / L sucrose, 7 g / L agar, pH 5.9, autoclaved at 121℃ for 20 minutes) and cultured in the dark at 25℃ for 18 days to obtain loose, pale yellow, high-quality rice callus tissue, with a callus rate of 92%.

[0033] Step 2, Subculture and Suspension Culture: The primary callus tissue was transferred to fresh induction medium as described above, and subcultured every 15 days for two consecutive times to obtain callus tissue with uniform proliferation. The subcultured callus tissue was then transferred to MS suspension medium (MS as the basal medium, supplemented with 2 mg / L 2,4-D and 30 g / L sucrose, pH 5.9, autoclaved at 121℃ for 20 min) and cultured on a shaker at 26±1℃ and 120 rpm to obtain a large number of rice callus suspension cells.

[0034] Step 3, Pretreatment: Weigh 100g of fresh callus suspension cells, rinse twice with sterile water, and blot dry. Add pre-cooled buffer solution (20mM Tris-HCl, pH 6.0) at a ratio of 3mL per 1g of fresh weight, and grind thoroughly into a homogenate in a mortar under ice bath conditions.

[0035] Step 4, Combined Lysis: Transfer the homogenate to an Erlenmeyer flask, add cellulase (final concentration 10 U / mL) and pectinase (final concentration 5 U / mL), mix well, and incubate at 52°C with shaking for 75 min, shaking every 15 min during lysis. After lysis, cool to room temperature in an ice bath and adjust the pH to 8.0 with NaOH solution. Then add proteinase K (final concentration 1 U / mL) and incubate at 60°C with shaking for 60 min. After the reaction, separate the solid and liquid phases using a filter cloth or centrifugation, and collect the lysate.

[0036] Step 5, Nucleic Acid Purification: Add 0.8 volumes of pre-chilled isopropanol to the lysis buffer, mix by inversion, and incubate at -20°C for 30 min. Then centrifuge at 10,000 rpm for 15 min at 4°C, discard the supernatant, and collect the nucleic acid precipitate. Wash the precipitate twice with 75% cold ethanol, and air dry upside down at room temperature until no ethanol residue remains. Dissolve the precipitate in an appropriate amount of TE buffer (pH 8.0) to obtain the nucleic acid extract. UV spectrophotometry showed A260 / A280 = 1.86, indicating high purity.

[0037] Step 6, Precise Ultrasonic Shearing: Place the nucleic acid extract in an ice bath and shear using an ultrasonic cell disruptor. The parameters were set as follows: power 300W, 3s operation time, 5s interval, total shearing time 45min. After shearing, a PDRN solution was obtained. Agarose gel electrophoresis analysis showed that the molecular weight of the product was mainly concentrated in the range of 25-500bp.

[0038] Step 7, PDRN purification: Add 1,2-hexanediol (final concentration 2%, w / v) and sodium phytate (final concentration 0.2%, w / v) to the ultrasonically sheared PDRN solution, stir well, and adjust the pH to 7.2. Finally, filter under sterile conditions through a 0.22 μm filter membrane to obtain the purified PDRN solution derived from rice callus.

[0039] The experimental results of this embodiment are as follows: (1) The formula for calculating the callus emergence rate of rice callus tissue obtained in step 1 is: The callus formation rate is calculated as (number of explants that produced callus tissue / total number of explants inoculated) × 100%. A total of 100 explants were inoculated, of which 92 produced callus tissue, resulting in a callus formation rate of 92%.

[0040] (2) Detection results of PDRN purified solution: (2-1) PDRN extraction rate: 0.5 mg / g fresh weight of callus tissue; The extraction rate is calculated by measuring the concentration and volume of PDRN obtained after extraction-purification-ultrasonic shearing, calculating the total amount of PDRN, and then dividing it by the fresh weight of the original extracted rice callus tissue.

[0041] (2-2) Purity measured by ultraviolet spectrophotometer: A260 / A280 = 1.85, A260 / A230 = 2.1; (2-3) The PDRN solution obtained in step 6 was detected by agarose gel electrophoresis, and the molecular weight of PDRN was found to be 25-500 bp.

[0042] Example 2: This embodiment is a replication and scale-up experiment of Example 1. Another batch of fresh, mature rice embryos (variety: Zhonghua 11) was used, with 1000 explants. The procedure was repeated exactly as in Example 1. The experimental results are as follows: (1) A total of 1,000 explants were inoculated, of which 935 produced callus tissue, with a callus formation rate of 93.5%.

[0043] (2) In the PDRN purified solution, the PDRN extraction rate was 0.6 mg / g fresh weight of callus; the purity detected by UV spectrophotometer was A260 / A280 = 1.89, A260 / A230 = 2.3; the molecular weight of PDRN detected by agarose gel electrophoresis was 25-500 bp.

[0044] Comparative Example 1: This comparative example illustrates a freeze-thaw-grinding method used in the experimental exploration of this invention to extract PDRN from rice callus tissue, as detailed below: Step 1, Explant Disinfection and Callus Induction: 100 plump, fresh, mature rice embryos (variety: Zhonghua 11) were selected as explants, and the glumes were removed. The explants were first disinfected with 75% ethanol for 30 seconds, then rinsed once with sterile water; next, they were disinfected with 0.1% mercuric chloride solution for 9 minutes, rinsed four times with sterile water, and the surface moisture was blotted dry with sterile filter paper. The disinfected explants were inoculated onto induction medium (N6 basal medium supplemented with 2 mg / L 2,4-D, 30 g / L sucrose, 7 g / L agar, pH 5.9, autoclaved at 121℃ for 20 minutes) and cultured in the dark at 25℃ for 18 days to obtain loose, pale yellow, high-quality rice callus tissue, with a callus rate of 85%.

[0045] Step 2, Subculture and Suspension Culture: The primary callus tissue was transferred to fresh induction medium as described above, and subcultured every 15 days for two consecutive times to obtain callus tissue with uniform proliferation. The subcultured callus tissue was then transferred to MS suspension medium (MS as the basal medium, supplemented with 2 mg / L 2,4-D and 30 g / L sucrose, pH 5.9, autoclaved at 121℃ for 20 min) and cultured on a shaker at 26±1℃ and 120 rpm to obtain a large number of rice callus suspension cells.

[0046] Step 3, Pretreatment: Weigh 100g of fresh callus suspension cells, rinse twice with sterile water, blot dry, and grind into a fine powder in liquid nitrogen. Continuously replenish with liquid nitrogen during grinding to prevent the powder from melting. After grinding, quickly add 150mL of DNA extraction solution (containing the following final concentrations: 2% SDS, 50mM EDTA, 100mM Tris-HCl, pH 8.0) to the powder, mix well, and let stand at room temperature for 10 minutes to allow the powder to fully dissolve.

[0047] Step 4: Freeze the above mixture at -80℃ for 30 minutes, then thaw it in a 37℃ water bath for 20 minutes to complete one freeze-thaw cycle. Repeat the freeze-thaw operation 3 times, gently inverting the conical flask 5-6 times after each thaw cycle to promote complete cell lysis. After freeze-thaw, add an equal volume of phenol-chloroform-isoamyl alcohol mixture (volume ratio 25:24:1) to the mixture, gently invert and mix for 10 minutes, centrifuge at 4℃ and 8000 rpm for 15 minutes, carefully aspirate the upper aqueous phase (avoiding contact with the intermediate protein layer), transfer it to a new conical flask, and repeat the phenol-chloroform-isoamyl alcohol extraction operation twice. Collect the final supernatant for subsequent purification steps.

[0048] Step 5, Nucleic Acid Purification: Add 0.8 volumes of pre-chilled isopropanol to the lysis buffer, mix by inversion, and incubate at -20°C for 30 min. Then centrifuge at 10,000 rpm for 15 min at 4°C, discard the supernatant, and collect the nucleic acid precipitate. Wash the precipitate twice with 75% cold ethanol, and air dry upside down at room temperature until no ethanol residue remains. Dissolve the precipitate in an appropriate amount of TE buffer (pH 8.0) to obtain the nucleic acid extract. UV spectrophotometry showed A260 / A280 = 1.86, indicating high purity.

[0049] Step 6, Precise Ultrasonic Shearing: Place the nucleic acid extract in an ice bath and shear using an ultrasonic cell disruptor. The parameters were set as follows: power 300W, 3s operation time, 5s interval, total shearing time 45min. After shearing, a PDRN solution was obtained. Agarose gel electrophoresis analysis showed that the molecular weight of the product was mainly concentrated in the range of 25-500bp.

[0050] Step 7, PDRN purification: Add 1,2-hexanediol (final concentration 2%, w / v) and sodium phytate (final concentration 0.2%, w / v) to the ultrasonically sheared PDRN solution, stir well, and adjust the pH to 7.2. Finally, filter under sterile conditions through a 0.22 μm filter membrane to obtain the purified PDRN solution derived from rice callus.

[0051] The experimental results of this comparative example are as follows: (1) The callus formation rate of rice tissue obtained in step 1 was 85%.

[0052] (2) In the PDRN purification solution, the PDRN extraction rate was 0.3 mg / g fresh weight of callus tissue; the purity detected by UV spectrophotometer was A260 / A280 = 1.88, A260 / A230 = 1.5; the molecular weight of PDRN detected by agarose gel electrophoresis was 25-500 bp.

[0053] The extraction rate of this comparative example can also reach 85%, but the PDRN extraction rate is lower, 40% lower than the result of Example 1, and the UV spectrophotometer detection results show that there are more impurities such as polysaccharides and polyphenols remaining.

[0054] Comparative Example 2: This comparative example illustrates a single ultrasonic disruption method used in the experimental exploration of this invention to prepare PDRN, as detailed below: Step 1, Explant Disinfection and Callus Induction: 100 plump, fresh, mature rice embryos (variety: Zhonghua 11) were selected as explants, and the glumes were removed. The explants were first disinfected with 75% ethanol for 30 seconds, then rinsed once with sterile water; next, they were disinfected with 0.1% mercuric chloride solution for 9 minutes, rinsed four times with sterile water, and the surface moisture was blotted dry with sterile filter paper. The disinfected explants were inoculated onto induction medium (N6 basal medium supplemented with 2 mg / L 2,4-D, 30 g / L sucrose, 7 g / L agar, pH 5.9, autoclaved at 121℃ for 20 minutes) and cultured in the dark at 25℃ for 18 days to obtain loose, pale yellow, high-quality rice callus tissue, with a callus rate of 80%.

[0055] Step 2, Subculture and Suspension Culture: The primary callus tissue was transferred to fresh induction medium as described above, and subcultured every 15 days for two consecutive times to obtain callus tissue with uniform proliferation. The subcultured callus tissue was then transferred to MS suspension medium (MS as the basal medium, supplemented with 2 mg / L 2,4-D and 30 g / L sucrose, pH 5.9, autoclaved at 121℃ for 20 min) and cultured on a shaker at 26±1℃ and 120 rpm to obtain a large number of rice callus suspension cells.

[0056] Step 3, Pretreatment: Weigh 100g of fresh callus suspension cells, rinse twice with sterile water, and blot dry. Add pre-cooled buffer solution (20mM Tris-HCl, pH 6.0) at a ratio of 3mL per 1g of fresh weight, and grind thoroughly into a homogenate in a mortar under ice bath conditions.

[0057] Step 4, Ultrasonic Disruption: Fix the conical flask containing the pretreated cell suspension under the probe of the ultrasonic cell disruptor, ensuring the probe is immersed in the suspension (avoid contact with the bottom of the flask). Set the ultrasonic parameters as follows: power 500W, 5s operation, 5s interval, total shearing time 30min. Maintain an ice bath throughout the ultrasonic process to prevent nucleic acid degradation due to temperature rise. After ultrasonic disruption, centrifuge the suspension at 4℃ and 10000r / min for 20min. Carefully aspirate the supernatant, remove the bottom precipitate (undisrupted cells and cell debris), and collect the supernatant for subsequent purification steps.

[0058] Step 5, Nucleic Acid Purification: Add 0.8 volumes of pre-chilled isopropanol to the lysis buffer, mix by inversion, and incubate at -20°C for 30 min. Then centrifuge at 10,000 rpm for 15 min at 4°C, discard the supernatant, and collect the nucleic acid precipitate. Wash the precipitate twice with 75% cold ethanol, and air dry upside down at room temperature until no ethanol residue remains. Dissolve the precipitate in an appropriate amount of TE buffer (pH 8.0) to obtain the nucleic acid extract. UV spectrophotometry showed A260 / A280 = 1.86, indicating high purity.

[0059] Step 6, Precise Ultrasonic Shearing: Place the nucleic acid extract in an ice bath and shear using an ultrasonic cell disruptor. The parameters were set as follows: power 300W, 3s operation time, 5s interval, total shearing time 45min. After shearing, a PDRN solution was obtained. Agarose gel electrophoresis analysis showed that the molecular weight of the product was mainly concentrated in the range of 25-500bp.

[0060] Step 7, PDRN purification: Add 1,2-hexanediol (final concentration 2%, w / v) and sodium phytate (final concentration 0.2%, w / v) to the ultrasonically sheared PDRN solution, stir well, and adjust the pH to 7.2. Finally, filter under sterile conditions through a 0.22 μm filter membrane to obtain the purified PDRN solution derived from rice callus.

[0061] The experimental results of this comparative example are as follows: (1) The callus formation rate of rice tissue obtained in step 1 was 80%.

[0062] (2) In the PDRN purified solution, the PDRN extraction rate was 0.1 mg / g fresh weight of callus tissue; the purity detected by UV spectrophotometer was A260 / A280 = 1.87, A260 / A230 = 2.3; the molecular weight of PDRN detected by agarose gel electrophoresis was 25-500 bp.

[0063] The recovery rate of this comparative example can reach 80%, but the PDRN extraction rate is extremely low, 80% lower than the result of Example 1, which is difficult to meet the application requirements.

[0064] The above examples and comparative examples demonstrate that the present invention has achieved significant results in terms of PDRN extraction efficiency, purity, and stability by optimizing rice callus culture, employing a combined process of "enzymatic hydrolysis-mechanical disruption" and "gradual purification-precision ultrasonic shearing".

[0065] Application Example 1 The PDRN obtained in Example 1 was functionally verified as follows: (1) Anti-wrinkle effect Elastase has the ability to degrade various proteins such as collagen and elastin. The degradation of elastin in skin tissue by elastase is closely related to the skin aging process. Therefore, counteracting the degradation of elastin by elastase and restoring skin elasticity is one of the important ways to delay skin aging.

[0066] Whether a test sample has a firming and anti-wrinkle effect can be determined by the elastase inhibition rate. The higher the elastase inhibition rate, the stronger the firming and anti-wrinkle effect of the substance, and vice versa.

[0067] The PDRN purified solution obtained in Example 1 was diluted with buffer (100 mM Tris-HCl, pH 8.0) to form gradient solutions of different concentrations: 3.125 ppm, 6.25 ppm, 12.5 ppm, 25 ppm, 50 ppm, and 100 ppm. The experimental group consisted of: a positive control (10 mg / mL epigallocatechin gallate (EGCG) solution) and a blank control group (100 mM Tris-HCl, pH 8.0).

[0068] Blank buffer, sample diluents of different concentrations, positive control solution, and substrate AAA-PNA (from Aladdin) were added to 96-well plates. The sample group consisted of 90 µL of blank buffer and 30 µL of AAA-PNA; the positive control group consisted of 90 µL of EGCG solution and 30 µL of AAA-PNA; the blank group consisted of only 120 µL of buffer. After vortexing and mixing, the plates were incubated at 25 °C for 20 min. Then, 20 µL of 0.1 mg / mL elastase (from Shanghai Yuanye) solution was added, and the plates were immediately vortexed and the OD was measured. 400nm After incubation for 10 minutes, OD was measured again. 400nm The effect of the sample on elastase activity was determined.

[0069] Elastase inhibition rate (%) = 1 - [(absorbance value of sample group after 10 min of incubation) - (absorbance value of sample group before 0 min of incubation)] / [(absorbance value of blank group after 10 min of incubation) - (absorbance value of blank group before 0 min of incubation)], where the sample group refers to the experimental group and the positive control group.

[0070] The results are as follows Figure 1 As shown, in the elastase inhibition test, the rice callus-derived PDRN prepared in Example 2 exhibited a significant concentration-dependent inhibitory effect on elastase activity within the concentration range of 6.25–100 ppm. When the sample concentration was 100 ppm, the inhibition rate reached 49.6%, indicating that this PDRN can effectively inhibit the degradation of elastin by elastase, thereby exerting a firming and anti-wrinkle effect.

[0071] (2) Soothing effect Hyaluronidase is a hydrolytic enzyme that degrades hyaluronic acid. It is a major component of the extracellular matrix of connective tissue cells and is associated with most IgE-mediated type I and T cell-mediated type IV hypersensitivity reactions. Inhibiting its activity can ensure normal hyaluronic acid content and function. Therefore, inhibiting hyaluronidase activity is, to some extent, related to the soothing, anti-allergic, and anti-inflammatory effects of products.

[0072] The in vitro inhibition test of hyaluronidase can determine whether a test sample has a soothing effect by the hyaluronidase inhibition rate. The higher the hyaluronidase inhibition rate, the stronger the soothing effect of the substance, and vice versa.

[0073] The PDRN purified solution sample obtained in Example 2 was diluted with purified water to form gradient solutions of different concentrations: 5 ppm, 10 ppm, 25 ppm, 50 ppm, 75 ppm, and 100 ppm. These were used as the experimental group; the positive control was 5 mg / ml dipotassium glycyrrhizate solution; and the blank control was phosphate buffer (pH approximately 5.35).

[0074] In a 96-well plate, samples from the experimental group, positive control group, and blank control group were mixed with 10 U / mL hyaluronidase solution, vortexed, and incubated at 37°C for 10 min. Then, 0.3 mg / mL hyaluronic acid salt solution was added, and the plate was incubated at 37°C for 45 min. Finally, 1 g / L BSA solution was added, and the plate was incubated for another 10 min. OD was then measured. 600nm The value was used to determine the inhibitory effect of the tested sample on hyaluronidase.

[0075] The inhibition rate is calculated as follows: Hyaluronidase inhibition rate (%) = [1 - (CD) ÷ (AB)] × 100% In the formula: A - absorbance of the reaction solution of the blank control group (with enzyme); B - absorbance of the reaction solution of the blank control group (without enzyme); C - absorbance of the reaction solution of the sample group (with enzyme); D - absorbance of the reaction solution of the sample group (without enzyme). Here, "sample group" refers to both the experimental group and the positive control group.

[0076] The hyaluronic acid solution was prepared as follows: 1.5 mg of hyaluronic acid was accurately weighed and dissolved in 5 mL of phosphate buffer, resulting in a concentration of 0.3 mg / mL. The amounts of each component added and the mixing methods for each group are further shown in Table 1. Table 1. Amounts of each component added and mixing methods in each group Note: "——" indicates that it has not been added.

[0077] The results are as follows Figure 2 As shown, in the hyaluronidase inhibition test, the PDRN derived from rice callus prepared in Example 2 exhibited a concentration-dependent inhibitory effect on hyaluronidase activity within the concentration range of 5-100 ppm. When the sample concentration was 100 ppm, the inhibition rate reached 39.1%, indicating that the PDRN can effectively inhibit hyaluronidase activity, reduce hyaluronic acid degradation, and thus exert a soothing and anti-allergic effect.

[0078] (3) Moisturizing effect Using a 5% glycerin solution, a common moisturizer, as a control, the moisturizing properties of the tested samples were determined by an in vitro weighing method to evaluate their moisturizing efficacy. A higher moisturizing rate indicates a better moisturizing effect.

[0079] After being placed under constant temperature and humidity conditions (temperature 22±2℃, humidity 40±5%) for a certain period of time, the mass of the PDRN purified solution sample obtained in Example 2 and the control sample before and after placement were weighed, and the moisturizing effect of the tested sample was calculated. The formula for calculating the moisturizing rate is: Moisture retention rate (%) = Mt / M0 × 100%, where Mt is the mass of the sample after being placed under constant temperature and humidity (22±2℃, humidity 40±5%) for a certain time t, and M0 is the initial mass of the sample before placement.

[0080] The results are as follows Figure 3 As shown, in the moisturizing test reaction system, under constant temperature and humidity (22±2℃, humidity 40±5%) conditions, the moisturizing rates of the tested samples at 1h, 2h, 4h, 6h, and 8h were 97.95%, 90.92%, 85.77%, 81.45%, and 78.68%, respectively. The moisturizing rate curves were higher than those of the 5% glycerol (control sample), indicating that the PDRN in rice callus tissue had a better moisturizing effect compared to the control sample.

[0081] (4) Anti-inflammatory effects Prostaglandin-inner-peroxide synthase 2 (Cydoxygenase-2, COX-2): also known as cyclooxygenase 2, is an enzyme that is rapidly expressed by monocytes, macrophages, fibroblasts, etc. after the body is stimulated by inflammatory factors. It is called an inducible enzyme and is one of the key enzymes that cause inflammatory responses. It can promote inflammatory responses and lead to tissue damage.

[0082] NOS2 is a subtype of nitric oxide synthase that produces nitric oxide (NO). In macrophages, NO mediates tumor-killing and bactericidal effects, participates in inflammatory responses, and can enhance the synthesis of pro-inflammatory mediators such as IL-6 and IL-8.

[0083] Interleukin-1α (IL-1α): A cytokine belonging to the chemokine family, also known as hematopoietic 1. IL-1α is mainly produced by activated macrophages, neutrophils, epithelial cells, and endothelial cells, and is abundant in the epidermis of normal individuals. Pro-inflammatory cytokines mainly stimulate the expression of genes related to inflammation and autoimmune diseases.

[0084] Interleukin-1β (IL-1β): There are two subtypes of interleukin-1 (IL-1α and II-1β), of which I-1β is the most common secreted form. It is produced by monocytes / macrophages and lymphocytes. When macrophages are stimulated (such as by LPS), they can produce IL-1, which causes granulocyte degranulation, mediates the process of tissue damage, and attracts inflammatory cells to enter the lesion site, thereby causing damage to the body.

[0085] Interleukin-6 (IL-6) is a pleiotropic inflammatory cytokine produced by T cells, monocytes, and giant cells, and is a key factor in the inflammatory response. IL-6 can alleviate inflammatory damage induced by bacterial endotoxins because it inhibits the production of IL-1 and TNF-α by macrophages. The effect of IL-6 on the body is related to its level. Normal levels of IL-6 can provide some protection and help the body fight inflammation. However, excessive IL-6 can harm the body, produce inflammatory effects, and lead to various diseases.

[0086] Interleukin-10 (IL-10): a subfamily of class II cytokines, also known as human cytokine synthesis inhibitor, is an anti-inflammatory cytokine that can enhance B cell survival, proliferation, and antibody production. IL-10 can block NF-κB activity and participate in the regulation of the JAK-STAT signaling pathway.

[0087] Tumor necrosis factor-α (TNF-α): One of the most potent inflammatory mediators in the body, exhibiting extremely strong inflammatory and damaging effects. It is primarily secreted by monocytes / macrophages and lymphocytes under the stimulation of inflammatory factors such as endotoxins. TNF-α can activate the MAPKS and NF-κB pathways through specific cell membrane receptors, causing cellular alterations and promoting the inflammatory cascade, inducing the production of new inflammatory mediators or cytokines.

[0088] (4-1) Cell viability assay: The PDRN purified solution sample obtained in Example 2 was diluted with complete culture medium to form gradient solutions of different concentrations: 1.56 ppm, 3.13 ppm, 6.25 ppm, 12.5 ppm, 25 ppm, 50 ppm, and 100 ppm.

[0089] RAW264.7 cells were seeded into 96-well plates at a seeding density of 1 × 10⁶ cells / well. 4 Cells / well; after 24 hours, discard the old culture medium and add complete culture medium containing different concentrations of the test samples. After 24 hours, OD is measured by MTT assay. 570nm and OD 630nm The effect of the tested samples on the viability of RAW264.7 cells was determined by t-test analysis.

[0090] Experimental results are as follows Figure 4 As shown, when the concentration of PDRN in rice callus tissue is <100ppm, it has no inhibitory effect on the activity of RAW264.7 cells. Subsequent tests can be conducted at concentrations <100ppm.

[0091] (4-2) Cellular inflammation-related gene expression assay: RAW264.7 cells were resuspended in fresh complete culture medium and seeded into 24-well plates at a seeding density of 4 × 10⁶ cells / well. 4 Count cells per well, then discard the culture medium in the 24-well plate and proceed with the experiment. The specific procedure is as follows: Group setup, induction and drug administration: 1) Experimental group: PDRN purified solution sample and LPS storage solution were added to cells, and then diluted with complete culture medium so that the final concentrations of PDRN in the system were 5ppm, 25ppm and 100ppm, and the final concentration of LPS in the system was 50ng / mL. 2) Positive control: Dexamethasone and LPS storage solution were added to the cells, and then diluted with complete culture medium to a final concentration of 100 µg / mL for dexamethasone and 50 ng / mL for LPS. 3) Model group: Only LPS storage solution was added to the cells, and the solution was diluted with complete culture medium to a final LPS concentration of 50 ng / mL.

[0092] 4) Control group: only cells and complete culture medium; then place the 24-well plate in a CO2 incubator and continue to incubate for 24 hours.

[0093] The LPS storage solution is prepared as follows: accurately weigh 1 mg of LPS powder (Maclean) and dissolve it in 1 mL of sterile PBS solution to prepare a 1 mg / mL LPS stock solution for later use.

[0094] After 24 hours of culture, total mRNA was extracted from cells (Meiji Bio, R4111-02), quantified, and reverse transcribed (using Evo M-MLV reverse transcription reagent premix, Aike Rui, AG11706) to obtain cDNA for Real-Time PCR. A SYBR Green Pro Taq HS premixed qPCR kit (Aike Rui, AG11701) was used, and the primers used are shown in Table 2. The relative expression levels of inflammation-related genes were analyzed using the 2-AACI method. The average Ct value of three replicates for each cDNA sample and each gene was used as the amplification result. The amplification level of the GAPDH gene was used as the internal reference gene. The gene cycle threshold ΔCt = Ctgene - CtGAPDDH was calculated, and the relative gene expression level ΔΔCt = ΔCttest - ΔCtblank. -^△△CT The ratio of expression levels in the test group to those in the control group was analyzed and calculated.

[0095] Table 2 Primer Sequences Based on the RAW264.7 cell inflammation model, the experimental results are as follows: Figure 5 As shown, the results indicated that the expression of all inflammatory genes in the model group was significantly higher than that in the control group, indicating that the model was valid. Compared with the model group, 25 ppm and 100 ppm of rice callus PDRN downregulated COX2, IL-1α, NOS2, IL-1β, IL-6 and TNF-α genes in RAW264.7 cells, respectively (P<0.05; P<0.01), and 100 ppm of rice callus PDRN promoted IL-10, indicating that rice callus PDRN has a clear inhibitory effect on inflammatory mediators / inflammatory factors.

[0096] (5) Anti-aging effects Type I collagen is a major structural protein in the dermis, synthesized in fibroblasts. Normal skin is predominantly composed of type I collagen. Collagen is the main insoluble fibrous protein in the ECM and connective tissue. Type I collagen is not only a product of cell secretion but also a key signaling source and environment regulating cell survival, proliferation, differentiation, migration, and function.

[0097] (5-1) Cell viability test: After seeding healthy HSF cells into 96-well plates and culturing for 24 hours, the cell seeding density was 1×10⁶ cells / well. 4 Cells / well were collected, and the culture medium in the 24-well plate was discarded. PDRN purified solution was added to the sample and diluted with complete culture medium to achieve final PDRN concentrations of 25 ppm, 75 ppm, 100 ppm, 150 ppm, 200 ppm, and 50 ppm. The control group consisted only of cells and complete culture medium. After 24 hours, OD570 and OD630 values ​​were measured by MTT assay to analyze the effect of the tested samples on HSF cell tolerance.

[0098] Experimental results are as follows Figure 6 As shown, when the PDRN concentration in rice callus tissue is ≤100ppm, it has no inhibitory effect on HSF cell activity. Subsequent detection can be performed at a concentration ≤100ppm. (5-2) Immunofluorescence assay for type I collagen in cells: Drug administration and incubation: HSF cells in good growth condition were seeded into 12-well plates. After the cells reached 85% density, the old culture medium was discarded, and PDRN purified solution was added. The solution was diluted with complete culture medium to make the final concentrations of PDRN in the system 25 ppm and 100 ppm, respectively. The cells were then cultured for another 48 h.

[0099] Cellular immunofluorescence: Discard the old culture medium, wash cells three times with PBS, and fix cells in each well with 4% paraformaldehyde; discard the 4% paraformaldehyde, wash three times with PBS, and block with blocking buffer; discard the blocking buffer, wash three times with PBST, and incubate with a diluted solution containing Collagen I primary antibody; discard the primary antibody dilution, wash three times with PBST, and continue incubation with a diluted solution containing fluorescent secondary antibody; discard the fluorescent secondary antibody dilution, wash three times with PBST, and incubate with an appropriate amount of PBST. Observe and photograph under a fluorescence microscope.

[0100] Based on the HSF cell model, the results are as follows: Figure 7 , Figure 8 As shown, compared with the control group, the green fluorescence signal of type I collagen in cells was significantly enhanced and more widely distributed after treatment with 25 ppm and 100 ppm samples for 48 h. Quantitative analysis results showed that the relative expression level of type I collagen in the 25 ppm and 100 ppm sample treatment groups was significantly increased to 1.52 times and 2.08 times that of the control group, respectively (P < 0.01 or P < 0.001).

[0101] The above results indicate that PDRN derived from rice callus can significantly promote the synthesis of type I collagen in fibroblasts, demonstrating a clear anti-aging effect.

[0102] In summary, this invention utilizes a five-step process of "induction and suspension culture—pretreatment—combined lysis—nucleic acid purification—precise ultrasonic shearing" to obtain a PDRN derived from rice callus tissue through the synergistic effect of each step. Experiments have confirmed that this PDRN derived from rice callus tissue has certain effects in anti-wrinkle, soothing, moisturizing, anti-inflammatory, and anti-aging.

[0103] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for preparing PDRN derived from rice callus, characterized in that, Includes the following steps: Step 1, Induction and suspension culture of rice callus: Using mature rice embryos as explants, after sterilization, they were inoculated into induction medium and cultured in the dark to obtain callus tissue; After subculturing the callus tissue, suspension culture was performed to obtain rice callus suspension cells. Step 2, Pretreatment: The rice callus suspension cells obtained in Step 1 are homogenized to obtain callus tissue homogenate. Step 3, combined lysis: cellulase and pectinase are added to the callus homogenate obtained in step 2 for the first enzymatic hydrolysis, then the pH is adjusted, and proteinase K is added for the second enzymatic hydrolysis. After solid-liquid separation, the lysate is obtained. Step 4, Nucleic acid purification: The lysis buffer obtained in step 3 is subjected to nucleic acid precipitation, washing and dissolution to obtain nucleic acid extract; Step 5, Precise ultrasonic shearing: The nucleic acid extract obtained in step 4 is subjected to ultrasonic shearing to obtain a solution containing PDRN.

2. The preparation method according to claim 1, characterized in that, In step 1, the induction medium is N6 medium with 1-3 mg / L of 2,4-dichlorophenoxyacetic acid, 25-35 g / L of sucrose, and 5-8 g / L of agar added, and the pH is 5.8-6.0; and / or, the control conditions for the dark culture include: temperature 25±1℃, culture time 14-20 days; and / or, the suspension culture medium is MS basal medium with 1-3 mg / L of 2,4-dichlorophenoxyacetic acid and 25-35 g / L of sucrose added, and the pH is 5.8-6.0; and / or, the control conditions for the suspension culture include: using a shaker, temperature 25-28℃, and rotation speed 110-130 rpm.

3. The preparation method according to claim 1, characterized in that, In step 2, the pretreatment step is as follows: rice callus suspension cells are mixed with 3-5 mL of buffer solution at a ratio of 1 g fresh weight, and then ground into a homogenate under ice bath conditions; preferably, the buffer solution is a Tris-HCl buffer solution with a concentration of 15-25 mM and a pH of 5.5-6.

5.

4. The preparation method according to claim 1, characterized in that, In step 3, the enzyme addition for the first enzymatic hydrolysis is: 8-12 U / mL of cellulase and 4-6 U / mL of pectinase, and the enzymatic hydrolysis conditions are: 50-55℃ water bath with shaking for 60-90 min; the conditions for the second enzymatic hydrolysis are: adjusting the pH to 7.8-8.2, adding 0.5-1.5 U / mL of proteinase K, and lysing at 58-62℃ for 50-90 min.

5. The preparation method according to claim 1, characterized in that, In step 4, the nucleic acid purification step is as follows: add 0.6 to 1 volume of isopropanol to the lysis buffer, let it stand at -20±5℃ for 20 to 30 minutes, then centrifuge and collect the precipitate; Wash the precipitate with 75% cold ethanol, dry it, and then dissolve it in TE buffer. Preferably, the centrifugation control parameters include: centrifugation at 4±1℃ and 8000~12000r / min for 10~20min; Preferably, the pH of the TE buffer solution is 7.8 to 8.

2.

6. The preparation method according to claim 1, characterized in that, In step 5, the nucleic acid extract obtained in step 4 is placed in an ice bath and ultrasonically sheared using an ultrasonic cell disruptor; preferably, the ultrasonic shearing process parameters are: power 200-400W, working time 2-4s, interval 4-6s, and total shearing time 30-60min.

7. The preparation method according to any one of claims 1 to 6, characterized in that, It also includes step 6, PDRN purification: 1,2-hexanediol and sodium phytate are added to the PDRN solution obtained in step 5, the pH is adjusted and then filtered to obtain the purified PDRN solution derived from rice callus tissue. Preferably, the final weight-volume concentration of 1,2-hexanediol in the system is 1-3%, and the final weight-volume concentration of sodium phytate in the system is 0.1-0.5%. Preferably, the pH is adjusted to 7.0-7.5; Preferably, a 0.22μm sterile filter membrane is used for filtration.

8. The preparation method according to claim 7, characterized in that, The 1,2-hexanediol may be partially or completely replaced with at least one of propylene glycol and trehalose.

9. The rice callus-derived PDRN prepared by the preparation method according to any one of claims 1-8, wherein the PDRN has a molecular weight of 25-500 bp.

10. Use of the PDRN of claim 9 in the preparation of at least one skin product as described in claim 9: 1) Anti-wrinkle skin products; 2) Soothing skin products; 3) Moisturizing skin products; 4) Anti-inflammatory skin products; 5) Anti-aging skin products.

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