A method and application for the co-production of plant vesicles and plant polydeoxyribonucleotides

Abstract

This invention relates to the field of biotechnology, specifically to a method and application for the co-production of plant vesicles and plant polydeoxyribonucleotides. This invention achieves the goal of synergistically extracting two high-value-added products from the same raw material, significantly improving the comprehensive utilization rate of the raw material. The plant vesicles obtained by this invention have an average particle size of 50-200 nm and a concentration as high as 8.7 × 10⁻⁶. 11 The plant polydeoxyribonucleotides have high purity and smaller molecular weight, with a molecular weight of <100 bp.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method and application for the co-production of plant vesicles and plant polydeoxyribonucleotides. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Cellular vesicles are nanoscale sac-like structures secreted by cell membranes or endometrial systems. They possess low immunogenicity, natural targeting, and good biocompatibility, making them promising candidates for drug delivery and gene therapy. Compared to animal-derived vesicles (such as exosomes), plant cell vesicles are widely available, inexpensive, and free from ethical controversies, making them a research hotspot in recent years.

[0004] Currently, the extraction of plant vesicles often requires methods such as ultracentrifugation, size exclusion chromatography, or polymer precipitation, which have problems such as complex operation, high equipment requirements, limited yield, and difficulty in completely avoiding organic solvent residues.

[0005] In addition, polydeoxyribonucleotide (PDRN) is a mixture of active polynucleotides that plays a significant role in promoting cell proliferation, collagen synthesis and damage repair.

[0006] Although polydeoxyribonucleic acid (PDA) has gained considerable popularity in recent years, it still faces some limitations and weaknesses in terms of extraction sources, applications, and potential mechanisms. In terms of extraction, its sources are very narrow, mainly consisting of reproductive cells from salmon and trout. These organisms only lay eggs during the breeding season, keeping the price of PDA high. Furthermore, current extraction processes are complex and difficult, and human-derived PDA raises ethical concerns. Even though some studies have proposed extraction methods for PDA from plants, the purity of the extracted DNA is low and cannot meet market demands.

[0007] Therefore, there is an urgent need for a method that can efficiently and greenly co-produce plant vesicles and plant polydeoxyribonucleotides. Summary of the Invention

[0008] To overcome the above problems, the present invention provides a method and application for the co-production of plant vesicles and plant polydeoxyribonucleotides.

[0009] To achieve the above technical objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for co-producing plant vesicles and plant polydeoxyribonucleotides, comprising the following steps: (1) After freezing the plant tissue, grind it, then add phosphate (PBS) buffer solution or physiological saline, homogenize it to obtain a pretreatment solution; add biological enzyme to the pretreatment solution to perform enzymatic hydrolysis to obtain an enzymatic hydrolysate; (2) The enzyme hydrolysate was centrifuged for the first time, and the first supernatant was collected; then the first supernatant was centrifuged for the second time, and the second supernatant and precipitate were collected. (3) The second supernatant was concentrated by ultrafiltration to obtain plant vesicles and filtrate; (4) Mix the precipitate from step (2) and the filtrate from step (3) to obtain a composite solution; perform a third centrifugation on the composite solution to collect the third supernatant; (5) Add PVP-40 to the third supernatant and salt out the third supernatant. Collect the fourth supernatant by centrifugation for the fourth time. Add ethanol to the fourth supernatant, let it stand, and then centrifuge for the fifth time to collect the precipitate and obtain plant polydeoxyribonucleotides.

[0010] In one or more embodiments, in step (1), the freezing method is liquid nitrogen freezing.

[0011] In one or more embodiments, in step (1), the plant tissue includes one or more of the following: rose flower, leaf or stem; green tea leaf; aloe vera; centella asiatica; dendrobium officinale; ginkgo leaf; olive leaf; purslane; wheat germ; coffee fruit; camellia flower; camellia seed; Paris polyphylla; sophora flavescens; American ginseng; black tea; polygonatum sibiricum; ganoderma lucidum; white tomato; ginseng; artemisia annua; forsythia suspensa; or peony.

[0012] In one or more embodiments, in step (1), the mass ratio of plant tissue to phosphate (PBS) buffer solution or physiological saline is 1:(1~3), preferably 1:3.

[0013] In one or more embodiments, in step (1), the homogenization speed is 3000~5000 rpm and the homogenization time is 10~15 min.

[0014] In one or more embodiments, in step (1), the bioenzyme includes one or more of cellulose pectin complex enzyme, pectinase or cellulase, hemicellulase, preferably cellulose pectin complex enzyme.

[0015] In one or more embodiments, in step (1), the enzymatic hydrolysis time is 1 to 10 h.

[0016] In one or more embodiments, in step (2), the rotation speed of the first centrifugation is 300~500 g, and the time of the first centrifugation is 20~30 min.

[0017] In one or more embodiments, in step (2), the rotation speed of the second centrifugation is 10000~15000 g, and the time of the second centrifugation is 20~30 min.

[0018] In one or more embodiments, in step (3), the ultrafiltration membrane used in the ultrafiltration concentration process has a pore size of 2~4 kDa, preferably 3 kDa.

[0019] In one or more embodiments, in step (4), ultrasonic assistance is used during mixing, with an ultrasonic power of 200~400 W and an ultrasonic time of 3~5 min.

[0020] In one or more embodiments, in step (4), the rotation speed of the third centrifugation is 10000~15000 g, and the time of the third centrifugation is 20~30 min.

[0021] In one or more embodiments, in step (5), the final concentration of PVP-40 added is 1~2wt%.

[0022] In one or more embodiments, in step (5), the final concentration of sodium chloride during salting out is 3~5 mol / L.

[0023] In one or more embodiments, in step (5), the rotation speed of the fourth centrifugation is 10000~15000 g, and the time of the fourth centrifugation is 20~30 min.

[0024] In one or more embodiments, in step (5), the volume ratio of the fourth supernatant to ethanol is (2~3):1.

[0025] In one or more embodiments, in step (5), the settling time is 20 to 40 minutes.

[0026] In one or more embodiments, in step (5), the rotation speed of the fifth centrifugation is 8000~10000 g, and the time of the fifth centrifugation is 20~40 min.

[0027] In one or more embodiments, the method for co-producing plant vesicles and plant polydeoxyribonucleic acid further includes filtering the plant vesicles obtained in step (3) and the plant polydeoxyribonucleic acid obtained in step (5) sequentially through filter paper, a 1 μm filter membrane, and a 0.22 μm filter membrane to obtain pure plant vesicles and pure plant polydeoxyribonucleic acid.

[0028] A second aspect of the invention provides plant vesicles and plant polydeoxyribonucleotides obtained by the method described in the first aspect.

[0029] In one or more embodiments, the average particle size of the plant vesicles is 50-200 nm.

[0030] In one or more embodiments, the molecular weight of the plant polydeoxyribonucleotide is <100 bp.

[0031] A third aspect of the present invention provides the application of the plant vesicles and plant polydeoxyribonucleotides described in the third aspect in the preparation of cosmetics for repairing and relieving skin irritation.

[0032] A fourth aspect of the present invention provides the application of the plant vesicles and plant polydeoxyribonucleotides described in the third aspect in the preparation of whitening cosmetics.

[0033] A fifth aspect of the invention provides the use of the plant vesicles and plant polydeoxyribonucleotides described in the third aspect in the preparation of anti-aging cosmetics.

[0034] A sixth aspect of the present invention provides a cosmetic comprising the plant vesicles and plant polydeoxyribonucleotides described in the third aspect.

[0035] The cosmetics described in this invention are liquid, lotion, cream, powder, block, or oil-based cosmetics. Liquid cosmetics include, but are not limited to, facial cleansers, bath gels, shampoos, toners, perfumes, cleansing waters, serums, and essences. Lotion cosmetics include, but are not limited to, moisturizers, milk-based products, hair conditioners, and serums. Cream cosmetics include, but are not limited to, face creams, foundation creams, shampoos, concealers, serums, and makeup primers. Powder cosmetics include, but are not limited to, face powder, talcum powder, loose powder, cleansing powder, and setting powder. Block cosmetics include, but are not limited to, pressed powder, lipsticks, and hair waxes. Oil-based cosmetics include, but are not limited to, body oils, hair oils, and facial oils.

[0036] The beneficial effects of this invention are as follows: (1) This invention provides a method for the co-production of plant vesicles and plant polydeoxyribonucleotides, achieving the goal of synergistically extracting two high-value-added products from the same raw material, and significantly improving the comprehensive utilization rate of the raw material. The plant vesicles obtained by this invention have an average particle size of 50~200 nm and a concentration as high as 8.7×10⁻⁶. 11 The plant polydeoxyribonucleotides have high purity and smaller molecular weight, with a molecular weight of <100 bp.

[0037] (2) The plant vesicles prepared by this invention exhibit high stability and good biocompatibility. The plant polydeoxyribonucleotides prepared by this invention not only have good biocompatibility but also possess anti-aging, repairing, and whitening effects. Specifically, plant polydeoxyribonucleotides can promote the proliferation of immortalized keratinocytes, significantly inhibit tyrosinase activity, accelerate the repair of sensitive skin barriers, soothe sensitive skin, reduce redness, reduce skin melanin deposition, and improve skin pigmentation. Therefore, they can be used to prepare cosmetics and have good application prospects. Attached Figure Description

[0038] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0039] Figure 1 Transmission electron microscopy image of plant vesicles in rose petals; Figure 2 The particle size and concentration of plant vesicles in rose petals; Figure 3 The ultraviolet absorption spectrum of polydeoxyribonucleotides in rose petals; Figure 4 Agarose gel electrophoresis image of polydeoxyribonucleotides in rose petals; Figure 5 The image shows the results of a cell proliferation experiment; where a is the stock solution of polydeoxyribonucleic acid from rose petals, b is the stock solution of polydeoxyribonucleic acid from green tea leaves, and c is the sodium DNA solution. Figure 6 The figure shows the results of the tyrosinase inhibition experiment; Figure 7 A graph showing the cumulative permeation amount Qn over time in a skin permeability test. Figure 8 The image shows the results of a double-blind trial involving volunteers. In the image, a represents the transdermal water loss rate, b represents the skin erythema index, and c represents the skin melanin index. Detailed Implementation

[0040] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0041] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0042] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0043] Example 1 A method for co-producing plant vesicles and plant polydeoxyribonucleotides from rose petals: (1) Take fresh rose petals, freeze them with liquid nitrogen, grind them into powder, soak them in 3 times their weight of physiological saline, and homogenize them in a homogenizer (3000 rpm, 15 min) to obtain a pretreatment solution; Cellulose pectin complex enzyme was added to the pretreatment solution at a dosage of 1 wt%, the pH was adjusted to 5, the temperature was 55 ℃, and the reaction was carried out for 5 h to obtain the enzymatic hydrolysate.

[0044] (2) The enzyme hydrolysate was centrifuged for the first time (500 g, 30 min) and the first supernatant was collected; then the first supernatant was centrifuged for the second time (10000 g, 30 min) and the second supernatant and precipitate were collected.

[0045] (3) The second supernatant was concentrated by ultrafiltration. The ultrafiltration membrane used in the ultrafiltration concentration process had a pore size of 3 kDa to obtain crude rose petal plant vesicle suspension and filtrate.

[0046] (4) Mix the precipitate from step (2) and the filtrate from step (3), sonicate at 4 ℃ (400 w) for 5 min, repeat 5 times to obtain a composite solution, and centrifuge the composite solution for the third time (15000 g, 30 min) to collect the third supernatant.

[0047] (5) Add PVP-40 to the third supernatant at an addition rate of 1 wt%, and add sodium chloride for salting out. The final concentration of sodium chloride is 5 mol / L. Stir at 55 ℃ for 2 h, and collect the fourth supernatant by centrifugation (10000 g, 30 min).

[0048] The fourth supernatant was added to anhydrous ethanol at -20 °C and stirred. After standing for 30 min, flocculent polydeoxyribonucleotides were precipitated. The volume ratio of the fourth supernatant to anhydrous ethanol was 2.5:1.

[0049] The mixture was centrifuged for the fifth time (10000 g, 40 min), the precipitate was collected, washed three times with 75% ethanol aqueous solution, and the precipitate was dissolved in pure water to obtain crude rose petal polydeoxyribonucleotide stock solution.

[0050] (6) The crude rose petal plant vesicle suspension and the crude rose petal polydeoxyribonucleic acid stock solution were filtered sequentially through filter paper, 1 μm filter membrane and 0.22 μm filter membrane to obtain the pure rose petal plant vesicle stock solution and the pure rose petal polydeoxyribonucleic acid stock solution.

[0051] Example 2 A method for co-producing plant vesicles and plant polydeoxyribonucleic acid from rose leaves: (1) Take fresh rose leaves, freeze them with liquid nitrogen, grind them into powder, soak them in 3 times their weight of physiological saline, and homogenize them in a homogenizer (5000 rpm, 10 min) to obtain a pretreatment solution; Cellulose pectin complex enzyme was added to the pretreatment solution at a dosage of 1 wt%, the pH was adjusted to 5, the temperature was 55 ℃, and the reaction was carried out for 3 h to obtain the enzymatic hydrolysate.

[0052] (2) The enzyme hydrolysate was centrifuged for the first time (300 g, 30 min) and the first supernatant was collected; then the first supernatant was centrifuged for the second time (12000 g, 20 min) and the second supernatant and precipitate were collected.

[0053] (3) The second supernatant was concentrated by ultrafiltration. The ultrafiltration membrane used in the ultrafiltration concentration process had a pore size of 3 kDa to obtain crude rose leaf plant vesicle suspension and filtrate.

[0054] (4) Mix the precipitate from step (2) and the filtrate from step (3), sonicate at 4 ℃ (300 w) for 5 min, repeat 5 times to obtain a composite solution, and centrifuge the composite solution for the third time (15000 g, 20 min) to collect the third supernatant.

[0055] (5) Add PVP-40 to the third supernatant at a dosage of 2 wt%, and add sodium chloride for salting out. The final concentration of sodium chloride is 4 mol / L. Stir at 55 ℃ for 3 h, and centrifuge for the fourth time (10000 g, 30 min) to collect the fourth supernatant.

[0056] The fourth supernatant was added to anhydrous ethanol at -20 °C and stirred. After standing for 30 min, flocculent polydeoxyribonucleotides were precipitated. The volume ratio of the fourth supernatant to anhydrous ethanol was 2.5:1.

[0057] The mixture was centrifuged for the fifth time (10000 g, 40 min), the precipitate was collected, washed three times with 75% ethanol aqueous solution, and the precipitate was dissolved in pure water to obtain crude rose leaf polydeoxyribonucleic acid stock solution.

[0058] (6) The crude plant vesicle suspension of rose leaves and the crude polydeoxyribonucleic acid stock solution of rose leaves were filtered sequentially through filter paper, 1 μm filter membrane and 0.22 μm filter membrane to obtain the pure plant vesicle stock solution of rose leaves and the pure polydeoxyribonucleic acid stock solution of rose leaves.

[0059] Example 3 A method for co-producing plant vesicles and plant polydeoxyribonucleotides from green tea leaves: (1) Take fresh green tea leaves, freeze them with liquid nitrogen, grind them into powder, soak them in 3 times their weight of physiological saline, and homogenize them in a homogenizer (3000 rpm, 15 min) to obtain a pretreatment solution; Cellulose pectin complex enzyme was added to the pretreatment solution at a dosage of 1 wt%, the pH was adjusted to 5, the temperature was 55 ℃, and the reaction was carried out for 8 h to obtain the enzymatic hydrolysate.

[0060] (2) The enzyme hydrolysate was centrifuged for the first time (500 g, 20 min) and the first supernatant was collected; then the first supernatant was centrifuged for the second time (10000 g, 30 min) and the second supernatant and precipitate were collected.

[0061] (3) The second supernatant was concentrated by ultrafiltration. The ultrafiltration membrane used in the ultrafiltration concentration process had a pore size of 3 kDa to obtain crude green tea leaf plant vesicle suspension and filtrate.

[0062] (4) Mix the precipitate from step (2) and the filtrate from step (3), sonicate at 4 ℃ (400 w) for 5 min, repeat 5 times to obtain a composite solution, and centrifuge the composite solution for the third time (15000 g, 20 min) to collect the third supernatant.

[0063] (5) Add PVP-40 to the third supernatant at an addition rate of 1 wt%, and add sodium chloride for salting out. The final concentration of sodium chloride is 5 mol / L. Stir at 55 ℃ for 2 h, and collect the fourth supernatant by centrifugation (10000 g, 30 min).

[0064] The fourth supernatant was added to anhydrous ethanol at -20 °C and stirred. After standing for 30 min, flocculent polydeoxyribonucleotides were precipitated. The volume ratio of the fourth supernatant to anhydrous ethanol was 2.5:1.

[0065] The mixture was centrifuged for the fifth time (10000 g, 40 min), the precipitate was collected, washed three times with 75% ethanol aqueous solution, and the precipitate was dissolved in pure water to obtain crude green tea leaf polydeoxyribonucleotide stock solution.

[0066] (6) The crude green tea leaf plant vesicle suspension and the crude green tea leaf polydeoxyribonucleic acid stock solution were filtered sequentially through filter paper, 1 μm filter membrane and 0.22 μm filter membrane to obtain the pure green tea leaf plant vesicle stock solution and the pure green tea leaf polydeoxyribonucleic acid stock solution.

[0067] Figure 1 This is a transmission electron microscope (TEM) image of the rose petal vesicles prepared in Example 1. Figure 1 As can be seen, the plant vesicles of rose petals are classic disc-shaped vesicles.

[0068] Particle size and concentration were analyzed using a Nanosight NS300 nanoparticle tracking analyzer. The rose petal vesicle resuspension was diluted 200-fold with PBS buffer, dispersed by filtration through a 0.22 μm filter membrane, and the particle size and concentration of the rose petal vesicles were determined. The results were collected and recorded. The results are as follows: Figure 2 As shown, from Figure 2 It can be seen that the average particle size of the plant vesicles in rose petals is 50~200 nm, and the concentration is as high as 8.7×10⁻⁶. 11 per mL.

[0069] Figure 3 The ultraviolet absorption spectrum of polydeoxyribonucleotides in rose petals, from Figure 3 As can be seen, the polydeoxyribonucleotides in rose petals have a characteristic absorption peak at 260 nm.

[0070] Figure 4 Agarose gel electrophoresis image of polydeoxyribonucleotides in rose petals, from Figure 4 It can be seen that the molecular weight of rose petal polydeoxyribonucleotides is <100 bp, which is much smaller than the molecular weight of sodium DNA (salmon-derived polydeoxyribonucleotides).

[0071] Example 4 Stability of rose petal vesicles: Plant vesicles in rose petals (8.7 × 10⁻⁶) 11 Add 1,2-pentanediol (2 wt%) and 1,2-hexanediol (1.2 wt%) to the solution to obtain a mixture.

[0072] Storage condition settings: Temperature: Set to 4℃ or 25℃ (room temperature).

[0073] Time: Samples were taken and tested on day 0, day 7, day 14, and day 30.

[0074] Other conditions: All samples were stored away from light and no stabilizers were added.

[0075] Detection indicators and methods: Particle size and concentration: The average particle size and particle concentration of vesicles in the samples were measured using a Nanosight NS300 nanoparticle tracking analyzer. Each sample was measured three times and the average value was taken.

[0076] Data analysis: Concentration retention rate (%) = (Concentration on day N / Concentration on day 0) × 100%; Particle size change is expressed as average particle size (nm).

[0077] The results are shown in Table 1. As can be seen from Table 1, after 30 days of storage at 4 ℃, the particle concentration retention rate in the rose petal plant vesicle suspension was about 95%, and there was no statistically significant difference in the average particle size (p>0.05); after 30 days of storage at 25 ℃, the particle concentration retention rate in the rose petal plant vesicle suspension was about 90%, and there was no statistically significant difference in the average particle size (p>0.05).

[0078] Table 1. Average particle size of plant vesicles in rose petals

[0079] Under the condition of adding preservatives, rose petal plant vesicles showed high stability whether stored at 4 ℃ or at room temperature in the dark. The particle concentration and particle size did not change significantly, and the suspension did not show any signs of decay or deterioration. It can be seen that this preservative system can be used for long-term (within 30 days) cold storage and room temperature storage of rose vesicles.

[0080] Example 5 Biocompatibility testing: Cell line: Human immortalized keratinocytes (HaCaT) cultured in DMEM medium containing 10% fetal bovine serum.

[0081] Sample processing: Rose petal polydeoxyribonucleotide stock solution (5000 ppm) was added at concentrations of 1 wt%, 2.5 wt%, 5 wt%, and 10 wt%.

[0082] The amounts of polydeoxyribonucleic acid stock solution (5000 ppm, prepared in Experimental Example 3) added were 1 wt%, 2.5 wt%, 5 wt%, and 10 wt%, respectively.

[0083] Rose petal plant vesicle suspension (8.7×10⁻⁶) 11 (pcs / mL), with addition amounts of 1wt%, 2.5wt%, 5wt%, and 10wt%, respectively.

[0084] A blank control group (containing only culture medium) and a positive control group (containing 0.1% Triton X-100) were set up.

[0085] Cell seeding and treatment: HaCaT cells were seeded in 96-well plates at a density of 1 × 10⁶ cells per well. 4 Cells were cultured for 24 h until adherence. The original culture medium was discarded, and 200 μL of culture medium containing different concentrations of the sample was added to each well, with 6 replicates per group. After another 24 h of culture, 20 μL of thiazolyl blue (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) (MTT) solution (5 mg / mL) was added to each well, and incubation continued for 4 h. The supernatant was discarded, and 150 μL of DMSO was added to each well, shaken for 10 min to dissolve the crystals. The absorbance (OD value) of each well was measured at 490 nm using a microplate reader.

[0086] Cell viability (%) = (OD value of experimental group / OD value of blank control group) × 100%. The experiment was repeated 3 times, and the data are expressed as mean ± standard deviation. Statistical analysis was performed using the t-test.

[0087] The results are shown in Table 2. In the test concentration range (1.0%–10.0%) of rose petal polydeoxyribonucleic acid stock solution (5000 ppm), the HaCaT cell survival rate was above 90%, with no significant difference compared to the blank control group (p > 0.05). Similarly, in the test concentration range (1.0%–10.0%) of green tea leaf polydeoxyribonucleic acid stock solution (5000 ppm), the HaCaT cell survival rate was above 90%, with no significant difference compared to the blank control group (p > 0.05). In all concentration groups (1.0%–10.0%), the cell survival rate of rose petal plant vesicle suspension remained above 88%, with no statistically significant difference compared to the control group (p > 0.05). The cell survival rate in the positive control group was below 20%, indicating that the experimental system was effective.

[0088] Table 2 Cell viability

[0089] In summary, within the experimentally defined concentration range, the polydeoxyribonucleic acid stock solution of rose petals, polydeoxyribonucleic acid stock solution of green tea leaves, and plant vesicle suspension of rose petals did not show significant cytotoxicity to HaCaT cells, indicating that the two products have good biocompatibility and providing support for their safe use in topical skin preparations.

[0090] Example 6 Cell proliferation experiment: Experimental Groups: Blank control group: high glucose medium DMEM + 10% FBS.

[0091] Positive control group: high glucose culture medium DMEM + 15% fetal bovine serum.

[0092] Experimental group: The amounts of rose petal polydeoxyribonucleic acid stock solution (5000 ppm), green tea leaf polydeoxyribonucleic acid stock solution (5000 ppm), and sodium DNA solution (5000 ppm) were added at 10 wt%, 2.5 wt%, 1.00 wt%, 0.50 wt%, and 0.10 wt%, respectively.

[0093] Cell seeding: HaCaT cells in the logarithmic growth phase were digested and centrifuged, and then prepared into 2×10⁻⁶ plates with complete culture medium. 5 Cell suspensions of [number] cells / mL were seeded into 96-well plates according to the experimental design and incubated at 37 ℃ in a 5% CO2 incubator for 24±2 h. The culture medium in the plates was discarded, and the samples were added according to the previously described experimental groups. The plates were then incubated at 37 ℃ in a 5% CO2 incubator for 24±2 h and 48±2 h. After incubation, the culture medium was discarded, and the absorbance of each well was read using a microplate reader according to the cytotoxicity assay procedure in Example 5.

[0094] Data analysis: Quantitative data are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was performed, and p-values ​​were used to determine statistical significance; p < 0.05 was considered statistically significant. The experiment started at 24 h with a proliferation rate of 0, which was used to statistically analyze cell proliferation at subsequent time points (e.g., 48 h, 72 h).

[0095] Cell proliferation rate (%) = (OD value of test group - average OD value of 24 h blank control group) / average OD value of 24 h blank control group × 100%.

[0096] The results are as follows Figure 5 As shown, the cell proliferation rates of rose petal polydeoxyribonucleic acid stock solution at concentrations of 10wt%, 2.5wt%, 1.0wt%, 0.5wt%, and 0.10wt% were 36.25%, 23.24%, 18.58%, 13.98%, and 8.17% at 24 h, respectively; and 142.87%, 121.51%, 97.54%, 78.83%, and 57.34% at 48 h, respectively, which were significantly higher than those of the blank control group.

[0097] The polydeoxyribonucleic acid stock solution from green tea leaves, at concentrations of 10wt%, 2.5wt%, 1.0wt%, 0.5wt%, and 0.10wt%, showed cell proliferation rates of 37.13%, 22.65%, 1.38%, 18.33%, 14.01%, and 7.93% at 24 h, respectively; and 138.77%, 119.34%, 99.82%, 76.83%, and 55.46% at 48 h, respectively; significantly higher than the blank control group.

[0098] At concentrations of 10wt%, 2.5wt%, 1.0wt%, 0.5wt%, and 0.10wt%, the cell proliferation rates at 24 h were 42.28%, 35.59%, 28.83%, 24.67%, and 21.42%, respectively; and at 48 h, they were 173.58%, 155.37%, 119.87%, 98.92%, and 73.43%, respectively, which were significantly higher than those of the blank control group.

[0099] In summary, within the concentration range set in the experiment, both rose petal polydeoxyribonucleic acid stock solution and green tea leaf polydeoxyribonucleic acid stock solution significantly promoted the cell proliferation of human immortalized keratinocytes in HaCaT cells, with effects comparable to those of sodium DNA (salmon-derived PDRN).

[0100] Example 7 Tyrosinase activity inhibition experiment: Experimental Groups: Blank control group: Sodium dihydrogen phosphate-citric acid buffer (pH=6.8); Positive control group: Kojic acid was diluted to 0.2 mg / mL with sodium dihydrogen phosphate-citrate buffer; Experimental group: Rose petal polydeoxyribonucleic acid stock solution (5000 ppm), green tea leaf polydeoxyribonucleic acid stock solution (5000 ppm), and sodium DNA solution (5000 ppm) were added at amounts of 5.0 wt%, 0.50 wt%, and 0.10 wt%, respectively.

[0101] Add 50 μL of sample solution of the same concentration to the sample wells (T) and sample background wells (T0), and add 50 μL of positive control solution of the same concentration to the positive control wells (P) and positive control background wells (P0). Add 50 μL of disodium hydrogen phosphate-citrate buffer to the enzyme reaction wells (C) and solvent background wells (C0).

[0102] Add 25 μL of tyrosinase solution to the sample well (T), enzyme reaction well (C), and positive control well (P). Replace the sample background well (T0), solvent background well (C0), and positive control background well (P0) with 25 μL of disodium hydrogen phosphate-citrate buffer. Mix the sample and tyrosinase thoroughly and incubate at 37 °C for 10 min. Then add 100 μL of levodopa solution to each well.

[0103] Assay: Transfer the ELISA plate into the microplate reader and incubate at 37 °C for 10 minutes. Measure the absorbance at 475 nm.

[0104] Data analysis: Quantitative data are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was performed on the data, and the p-value was used to determine statistical differences. P < 0.05 indicates statistical significance.

[0105] Cell proliferation rate (%) = (OD value of test group - average OD value of blank control group) / average OD value of blank control group × 100%.

[0106] The results are as follows Figure 6 As shown, in the tyrosinase inhibition experiment, 5.0%, 0.5%, and 0.1% of rose petal polydeoxyribonucleotide stock solution (5000 ppm) significantly inhibited tyrosinase activity, with inhibition rates of 79.64%, 51.18%, and 14.34%, respectively; 5%, 0.5%, and 0.1% of green tea leaf polydeoxyribonucleotide stock solution (5000 ppm) significantly inhibited tyrosinase activity, with inhibition rates of 77.13%, 48.81%, and 10.59%, respectively; and 5% concentration of sodium DNA (5000 ppm) significantly inhibited tyrosinase activity, with an inhibition rate of 35.57%.

[0107] Example 8 Franz diffusion cell method for evaluating skin permeability: Experimental Groups: Control group: PBS buffer solution; Rose petal polydeoxyribonucleic acid (PDA) composition: Rose petal PDA stock solution (5000 ppm); Green tea leaf polydeoxyribonucleic acid composition: Green tea leaf polydeoxyribonucleic acid stock solution (5000ppm); Sodium DNA group: Sodium DNA preparation solution (5000 ppm); Prepared mouse skin tissue, epidermis facing upwards, was fixed on the Franz diffusion cell of a transdermal transdermal analyzer, and 2 mL of sample solution was applied. The receiving cell was filled with receiving solution (physiological saline), and 1 mL samples were taken at 15, 30, 45, 60, 90, 120, and 180 min, with an equal volume of isothermal physiological saline added. The sample solution was filtered through a 0.45 μm microporous membrane and subjected to HPLC to determine the drug content, calculate the cumulative release rate, and fit the equation.

[0108] Data analysis: Calculate the cumulative permeation per unit area (Qn), fit the transdermal kinetic curve, and calculate the steady-state transdermal rate (Jss) and hysteresis time (Tlag).

[0109] The cumulative permeability Qn of the experimental group over 180 min varies with time as shown in the curve. Figure 7 As shown, all three active substance groups exhibited time-dependent permeation behavior. Among them, the cumulative permeation of the rose petal polydeoxyribonucleotide group was significantly higher than that of the green tea leaf polydeoxyribonucleotide group and the sodium DNA group at all time points, showing the best mucosal permeation performance.

[0110] Permeation kinetic parameters: The steady-state transdermal rate (Jss) and lag time (Tlag) of each group were obtained by calculating the linear regression of the steady-state phase (45-120 min). The results are shown in Table 3. The rose petal polydeoxyribonucleotide group had the highest Jss and the shortest Tlag, indicating that it could penetrate the mucosal barrier more quickly and reach steady-state permeation.

[0111] Table 3 Permeation kinetic parameters

[0112] Example 7 (1) Preparation of rose petal polydeoxyribonucleotide soothing and repairing essence: Table 4 shows the formula for the Rose Petal Polydeoxyribonucleic Acid Soothing and Repairing Essence.

[0113] Table 4. Formula of Rose Petal Polydeoxyribonucleotide Soothing and Repairing Essence

[0114] Preparation method: Aqueous phase preparation: Deionized water was heated to 75 °C, and glycerol, panthenol, sodium hyaluronate and dipotassium glycyrrhizate were added and stirred until completely dissolved.

[0115] Gel matrix preparation: Xanthan gum was slowly dispersed in the above aqueous phase and stirred continuously until it swelled completely to form a uniform and transparent matrix.

[0116] Adding active ingredients: After the system cools down to below 35°C, add the rose petal polydeoxyribonucleotide stock solution and stir slowly until homogeneous.

[0117] System conditioning: Add 1,2-hexanediol and p-hydroxyacetophenone, and stir until the system is homogeneous.

[0118] Filling: Adjust the pH to 5.5~6.0, add deionized water to the total volume, filter, and fill into light-proof essence bottles.

[0119] (2) Preparation of Rose Petal Vesicle Barrier Repairing and Softening Cream: Table 5 shows the formula for the Rose Petal Vesicle Barrier Repairing Softening Cream.

[0120] Table 5. Rose Petal Vesicle Barrier Repairing Softening Cream Formula

[0121] Preparation method: Oil phase preparation: Ceramide NP, squalane, shea butter, jojoba oil and cetearyl alcohol are mixed, heated to 78~80 ℃ and stirred until completely melted and homogeneous.

[0122] Aqueous phase preparation: Add glycerol, allantoin, and xanthan gum to deionized water, heat to 78-80 ℃, and stir until completely dissolved.

[0123] Emulsification: The aqueous phase is slowly added to the oil phase while being emulsified with a homogenizer for 5 minutes to form a stable cream matrix.

[0124] Addition of active ingredients: After the system has cooled to below 40 ℃, slowly add the rose vesicle suspension and stir until homogeneous.

[0125] Preservation and finishing: Add 1,2-pentanediol and continue stirring until the system is fine and uniform. After cooling, fill into sterile packaging.

[0126] (3) Preparation of green tea leaf polydeoxyribonucleotide soothing and repairing essence: Table 6 shows the formula for the Green Tea Leaf Polydeoxyribonucleic Acid Soothing and Repairing Essence.

[0127] Table 6. Green Tea Leaf Polydeoxyribonucleic Acid Soothing and Repairing Essence

[0128] Preparation method: Aqueous phase preparation: Deionized water was heated to 75 °C, and glycerol, panthenol, sodium hyaluronate and dipotassium glycyrrhizate were added and stirred until completely dissolved.

[0129] Gel matrix preparation: Xanthan gum was slowly dispersed in the above aqueous phase and stirred continuously until it swelled completely to form a uniform and transparent matrix.

[0130] Adding active ingredients: After the system cools down to below 35°C, add the green tea leaf polydeoxyribonucleotide stock solution and stir slowly until homogeneous.

[0131] System conditioning: Add 1,2-hexanediol and p-hydroxyacetophenone, and stir until the system is homogeneous.

[0132] Filling: Adjust the pH to 5.5-6.0, add deionized water to the total volume, filter, and fill into light-proof essence bottles.

[0133] Comparative Example 1 The preparation method is the same as that of the rose petal polydeoxyribonucleic acid soothing and repairing essence in Example 7, but the formula is different. In this formula, the rose petal polydeoxyribonucleic acid stock solution is replaced with physiological saline.

[0134] Example 8 Double-blind trial for volunteers: Experimental Groups: Group A (blank control group): Blank essence from Comparative Example 1.

[0135] Group B (Rose PDRN Essence Group): Rose petal polydeoxyribonucleic acid soothing and repairing essence.

[0136] Sixty female volunteers aged 25-35 years with mild to moderate sensitive skin, meeting the inclusion criteria, were recruited. Informed consent was obtained, and participants underwent a lactic acid stinging test and baseline assessment. Those who passed were randomly assigned to two groups of 30 each. The intervention period was 28 days. Blinding was implemented: neither the participants nor the assessing researchers (those performing instrument measurements and image analysis) knew their group assignments.

[0137] Directions for use: After cleansing your face in the morning and evening, take 0.5 mL of the designated product and apply it evenly to the entire face. Use continuously for 28 days.

[0138] Assessment indicators and time points: Transdermal water loss rate, erythema index (EI), and melanin index (MI) of subjects were assessed using a measuring instrument on days 0, 14, and 28, respectively.

[0139] Results of the double-blind trial of volunteers as follows Figure 8As shown, the transdermal water loss rate in the test group of the rose petal polydeoxyribonucleic acid soothing and repairing essence decreased significantly on day 14 (compared to baseline and group A at the same time), and the effect was more pronounced on day 28; this indicates that rose petal polydeoxyribonucleic acid can effectively accelerate the repair of sensitive skin barrier. The erythema index in the test group of the rose petal polydeoxyribonucleic acid soothing and repairing essence showed a significant decrease on both day 14 and day 28; this supports the efficacy of the rose petal polydeoxyribonucleic acid soothing and repairing essence in soothing sensitive skin and reducing redness. The melanin index in the test group of the rose petal polydeoxyribonucleic acid soothing and repairing essence showed a significant decrease on both day 14 and day 28; this supports the efficacy of the rose petal polydeoxyribonucleic acid soothing and repairing essence in reducing skin melanin deposition, improving skin pigmentation, and combating skin photoaging.

[0140] In summary, the transdermal water loss rate of the test group containing rose petal polydeoxyribonucleotides was significantly reduced, skin sensitivity was significantly improved, and facial pigmentation was reduced. This indicates that the plant-derived polydeoxyribonucleotides extracted in this invention have repairing, whitening, and anti-aging effects, providing an innovative raw material solution for the development of high-end cosmetics, medical devices, and functional skin care products, and has significant application value and market prospects.

[0141] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for the co-production of plant vesicles and plant polydeoxyribonucleotides, characterized in that, Includes the following steps: (1) After freezing the plant tissue, grind it, then add phosphate (PBS) buffer solution or physiological saline, homogenize it to obtain a pretreatment solution; add biological enzyme to the pretreatment solution to perform enzymatic hydrolysis to obtain an enzymatic hydrolysate; (2) The enzyme hydrolysate was centrifuged for the first time, and the first supernatant was collected; then the first supernatant was centrifuged for the second time, and the second supernatant and precipitate were collected. (3) The second supernatant was concentrated by ultrafiltration to obtain plant vesicles and filtrate; (4) Mix the precipitate from step (2) and the filtrate from step (3) to obtain a composite solution; perform a third centrifugation on the composite solution to collect the third supernatant; (5) Salt out the third supernatant, and collect the fourth supernatant by centrifugation for the fourth time; add ethanol to the fourth supernatant, let it stand, and centrifuge for the fifth time to collect the precipitate and obtain plant polydeoxyribonucleotides.

2. The method as described in claim 1, characterized in that, In step (1), the freezing method is liquid nitrogen freezing; Alternatively, in step (1), the plant tissue includes one or more of the following: rose flower, leaf or stem, green tea leaf, aloe vera, centella asiatica, dendrobium officinale, ginkgo leaf, olive leaf, purslane, wheat germ, coffee fruit, camellia flower, camellia seed, Paris polyphylla, sophora flavescens, American ginseng, black tea, polygonatum sibiricum, ganoderma lucidum, white tomato, ginseng, artemisia annua, forsythia, or peony. Alternatively, in step (1), the mass ratio of plant tissue to phosphate (PBS) buffer solution or physiological saline is 1:(1~3), preferably 1:3; Alternatively, in step (1), the homogenization speed is 3000~5000 rpm and the homogenization time is 10~15 min; Alternatively, in step (1), the bio-enzyme includes one or more of cellulose pectin complex enzyme, pectinase or cellulase, hemicellulase, preferably cellulose pectin complex enzyme; Alternatively, in step (1), the enzymatic hydrolysis time is 1~10 h.

3. The method as described in claim 1, characterized in that, In step (2), the rotation speed of the first centrifugation is 300~500 g, and the time of the first centrifugation is 20~30 min; Alternatively, in step (2), the rotation speed of the second centrifugation is 10000~15000 g, and the time of the second centrifugation is 20~30 min; Alternatively, in step (3), the ultrafiltration membrane used in the ultrafiltration concentration process has a pore size of 2~4 KDa, preferably 3 KDa.

4. The method as described in claim 1, characterized in that, In step (4), ultrasonic assistance is used during mixing, with an ultrasonic power of 200~400 W and an ultrasonic time of 3~5 min; Alternatively, in step (4), the rotation speed of the third centrifugation is 10000~15000 g, and the time of the third centrifugation is 20~30 min; Alternatively, in step (5), the final concentration of sodium chloride during salting out is 3~5 mol / L; Alternatively, in step (5), the rotation speed of the fourth centrifugation is 10000~15000 g, and the time of the fourth centrifugation is 20~30 min; Alternatively, in step (5), the volume ratio of the fourth supernatant to ethanol is (2~3):1; Alternatively, in step (5), the settling time is 20~40 min; Alternatively, in step (5), the rotation speed of the fifth centrifugation is 8000~10000 g, and the time of the fifth centrifugation is 20~40 min.

5. Plant vesicles and plant polydeoxyribonucleotides, characterized in that, Obtained by the method described in any one of claims 1 to 4.

6. The plant vesicles and plant polydeoxyribonucleotides as described in claim 5, characterized in that, The average particle size of the plant vesicles is 50~200 nm; Alternatively, the molecular weight of the plant polydeoxyribonucleotide is <100 bp.

7. The application of the plant vesicles and plant polydeoxyribonucleotides described in claim 5 in the preparation of cosmetics for repairing and relieving skin irritation.

8. The application of the plant vesicles and plant polydeoxyribonucleotides as described in claim 5 in the preparation of whitening cosmetics.

9. The application of the plant vesicles and plant polydeoxyribonucleotides as described in claim 5 in the preparation of anti-aging cosmetics.

10. A cosmetic product, characterized in that, Includes the plant vesicles and plant polydeoxyribonucleotides as described in claim 5.

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