Plant nanovesicle derived from peony, preparation method and application

Abstract

The present application belongs to the technical field of plant exosome, and particularly relates to a plant nanovesicle from peony, a preparation method and application. The present application takes peony as the source of the plant nanovesicle, first crushes peony petals into slurry, then performs enzymatic hydrolysis treatment by using a composite enzyme preparation, and finally obtains the plant nanovesicle from peony by sequentially performing horizontal electrophoresis dialysis, ultrahigh-speed centrifugation and purification. The present application has the beneficial effect of improving the traditional preparation method of the plant nanovesicle, optimizing the preparation process, improving the scale degree of the plant nanovesicle from peony, and having a high content of active ingredients protein and miRNA in the plant nanovesicle from peony prepared by the present application, and a good effect on reducing the damage of human fibroblasts caused by ultraviolet radiation.

Description

Technical Field

[0001] This invention belongs to the field of plant exosome technology, specifically relating to a plant nanovesicle derived from peony, its preparation method, and its application. Background Technology

[0002] Skin aging is an inevitable part of the physiological process; however, it is often influenced by multiple factors, including environment, lifestyle habits, and hormonal changes. Among these factors, exposure to ultraviolet (UV) radiation from the sun is considered one of the main contributing factors. UV radiation can accelerate the appearance of signs of aging such as wrinkles, pigmentation, and decreased elasticity. Currently, anti-aging products mainly include various antioxidants, vitamins C and E, and hyaluronic acid. However, these existing products suffer from problems such as poor stability, poor absorption, and limited effectiveness with long-term use.

[0003] Plant exosomes are extracellular vesicles secreted by plant cells that enable intercellular communication. They range in diameter from 50 to 500 nm. Studies have shown that plant exosomes contain a large number of bioactive substances such as lipids, proteins, enzymes, and nucleic acids. Among them, microRNAs (miRNAs) play an extremely important role in biological processes such as gene expression regulation, intercellular communication, signal transduction, and extracellular matrix regulation, and have therefore become a current research hotspot.

[0004] At present, numerous studies have demonstrated that certain plant exosomes have good effects such as anti-aging, anti-tumor, and prevention of atherosclerosis. For example, patent CN114854667A discloses a plant vesicle derived from kiwifruit and its application in the preparation of anti-tumor drugs; patent CN115584337A discloses the application of plant exosomes such as Polygonum multiflorum, Astragalus membranaceus, ginseng, and Panax notoginseng in anti-skin aging.

[0005] However, traditional internationally recognized methods for extracting high-purity nanovesicles involve ultracentrifugation and sucrose purification. For example, patents CN116855438A and CN117045759A first perform ultracentrifugation on plant materials to separate the nanovesicle-containing fractions, and then use sucrose purification to further improve purity. However, the ultracentrifugation and sucrose purification involved in this method not only require specialized equipment, but are also time-consuming and complex, making it difficult to prepare high-purity plant vesicles on a large scale and severely limiting the feasibility of large-scale production.

[0006] In addition, to further increase the content of active ingredients in plant exosomes, some studies have begun to use enzyme preparations or microbial agents for pretreatment based on the traditional preparation methods of plant nanovesicles. For example, CN115584337A and CN114009754A were pretreated by enzymatic hydrolysis or a combination of enzymatic hydrolysis and fermentation before ultracentrifugation. Although the protein content in the obtained exosomes was increased, the genetic material such as miRNA that plays a role was not significantly increased by the above methods. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a plant nanovesicle derived from peony, its preparation method, and its application.

[0008] The present invention provides a plant nanovesicle derived from peony, which is obtained by: crushing peony petals into a slurry, enzymatically hydrolyzing it with a compound enzyme preparation, and then sequentially performing horizontal electrophoresis dialysis, ultra-high speed centrifugation, and purification.

[0009] This invention improves upon the traditional preparation method of plant exosomes. On one hand, the enzymatic hydrolysis preparation for peony petals was adjusted, employing six different enzymes. Different enzymes have varying degrees of hydrolysis effects on different types of biomacromolecules in the cell walls of different peony petals. The combined effect of these enzymes makes the cell wall hydrolysis of peony petals more thorough, facilitating the more complete dissolution of active substances from the petals. On the other hand, since nucleic acids carry different charges under different pH conditions, resulting in varying forces in an electric field, the enrichment of plant nanovesicles using electrophoretic dialysis maximizes the preservation of the integrity and content of nucleic acids, i.e., miRNA. Finally, high-purity plant nanovesicles derived from peony with high miRNA content were obtained through ultra-high-speed centrifugation.

[0010] In the above-mentioned plant nanovesicles derived from peony provided by the present invention, preferably, the composite enzyme preparation is selected from at least three of cellulase, pectinase, hemicellulase, β-1,3-glucanase, ligninase, and cell wall protease.

[0011] Preferably, the compound enzyme preparation is a mixed enzyme preparation of cellulase, pectinase, hemicellulase, β-1,3-glucanase, ligninase, and cell wall protease.

[0012] More preferably, the mass fractions of the various enzymes used in the compound enzyme preparation are: 60% cellulase, 10% pectinase, 10% hemicellulase, 10% β-1,3-glucanase, 5% ligninase, and 5% cell wall protease.

[0013] Preferably, the added mass of the compound enzyme preparation accounts for 0.1 to 0.4% of the mass of the peony petals.

[0014] Preferably, the specific operating steps of horizontal electrophoresis dialysis are as follows: the enzymatic hydrolysate is placed into a dialysis bag with a capacity of 100-300 kDa, the dialysis bag is placed in an electrophoresis tank, and electrophoretic separation is performed using a current of 200-300 mA. The electrophoretic direction is changed and the electrophoresis buffer is replaced every hour.

[0015] More preferably, the electrophoresis buffer is prepared using deionized water and contains 7-9 g / L glycine and 1-3 g / L tris(hydroxymethyl)aminomethane hydrochloride (Tris hydrochloride).

[0016] More preferably, the electrophoresis buffer is prepared using deionized water and contains 7.2 g / L glycine and 1.5 g / L Tris hydrochloride.

[0017] Preferably, the centrifugation temperature of the ultra-high speed centrifuge is 0-10℃, the centrifugation force is 100,000-180,000g, and the centrifugation time is 1-3h.

[0018] Furthermore, the present invention also provides a method for preparing the above-mentioned plant vesicles derived from peony, specifically including the following steps:

[0019] (1) Wash and dry the peony petals and then crush them to obtain a slurry;

[0020] (2) Add a compound enzyme preparation to the slurry obtained in (1) for enzymatic hydrolysis to obtain the hydrolysate;

[0021] (3) Centrifuge and filter the enzymatic hydrolysate obtained in (2) to obtain the enzymatic hydrolysate supernatant;

[0022] (4) Perform horizontal electrophoresis dialysis on the enzymatic hydrolysis supernatant obtained in (3) to obtain dialysate;

[0023] (5) The dialysis solution obtained in (4) was centrifuged at ultra-high speed, the supernatant was discarded, and the precipitate was resuspended in phosphate buffer solution to obtain plant nanovesicles derived from peony.

[0024] In the above preparation method, preferably, in (1), the peony petals are washed and dried at a low temperature of 40-60°C, then crushed to 80-120 mesh, and then a 0.01M phosphate buffer solution with pH 7.2 is added. The mixture is then homogenized using a tissue homogenizer. The mass-volume ratio of peony petals to phosphate buffer solution is 1g: 5-10mL.

[0025] Preferably, the peony petals mentioned in (1) are peony petals produced in Heze, and the pulverization is ultra-fine pulverization.

[0026] As a preferred embodiment, in (1), the tissue homogenizer is used to homogenize the tissue for 3 to 8 minutes.

[0027] Preferably, in (2), after adding the compound enzyme preparation, the pH of the slurry is adjusted to 5.5-6.0, and enzymatic hydrolysis is performed for 0.5-3 hours.

[0028] Preferably, in (3), the enzymatic hydrolysate obtained in (2) is centrifuged at 0-10°C with a centrifugal force of 3000-5000g for 15-30 minutes, and then filtered through a filter membrane with a pore size of 1μm to obtain the enzymatic hydrolysate supernatant.

[0029] Preferably, in (5), the phosphate buffer solution used for resuspension has a pH of 7.2 and a concentration of 0.01 mol / L.

[0030] In addition, the application of peony-derived plant nanovesicles obtained by the above method in the preparation of anti-skin aging products is also a key technical content that this invention aims to protect.

[0031] The beneficial effects of this invention are as follows:

[0032] (1) A method for preparing plant nanovesicles derived from peony is provided, which improves the traditional preparation method of plant nanovesicles. The method combines enzymatic hydrolysis, horizontal electrophoresis dialysis and ultra-high speed centrifugation. The whole preparation process is more efficient, has a higher degree of scalability and low cost, and provides a more feasible way for the preparation of plant-derived nanovesicles.

[0033] (2) A high-protein and high-purity peony plant nanovesicle with high protein and miRNA content was obtained, with the highest protein content being 48.03 mg / mL and the highest miRNA content being 177.65 ng / mL.

[0034] (3) The cell viability of the peony-derived plant nanovesicles prepared in this invention was relatively high after MTT assay, reaching up to about 69.7%. Further in-depth research on the composition and mechanism of action of peony plant nanovesicles has shown that the peony plant nanovesicles prepared in this invention contain active substances such as miRNA, which are encapsulated in a double-layer lipid vesicle, making it more conducive to the absorption of active substances by the body and their effects. The experimental results of this invention show that the prepared peony plant nanovesicles can reduce skin inflammation and type I collagen loss in mice caused by ultraviolet radiation, and can also effectively reduce damage to human fibroblasts caused by ultraviolet radiation. Attached Figure Description

[0035] Figure 1 This is a transmission electron microscope image of plant nanovesicles derived from peony prepared in Example 1 of this invention;

[0036] Figure 2 The average particle size analysis results are for the plant nanovesicles derived from peony prepared in Example 1 of this invention;

[0037] Figure 3 The average potential analysis results are for the plant nanovesicles derived from peony prepared in Example 1 of this invention;

[0038] Figure 4 The effect of plant nanovesicles derived from peony prepared in each experimental group of this invention on the survival rate of human fibroblasts irradiated with ultraviolet light;

[0039] Figure 5 The effects of peony-derived plant nanovesicles prepared in each experimental group of this invention on inflammatory factors in the skin homogenate of a photo-aged mouse model.

[0040] Figure 6 This paper describes the effect of peony-derived plant nanovesicles prepared in each experimental group of this invention on collagen levels in skin homogenates of photoaging model mice. Detailed Implementation

[0041] To enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments.

[0042] Example 1

[0043] (1) Peony petals produced in Heze, Shandong Province were washed and dried at low temperature at 40℃, then ultra-finely pulverized to 80 mesh. Then, a 0.01M phosphate buffer solution with pH 7.2 was added to the solution. The mass-volume ratio of peony petals to phosphate buffer solution was 1g:10mL. Then, the solution was homogenized for 5min using a tissue homogenizer.

[0044] (2) Add a compound enzyme preparation to the uniform slurry of peony petals obtained in (1) for enzymatic hydrolysis. The mass of the compound enzyme preparation added accounts for 0.1% of the mass of peony petals in (1). The compound enzyme preparation is a mixture of cellulase, pectinase, protease, β-1,3-glucanase, ligninase and cell wall protease. The mass fraction of each enzyme is: 60% cellulase, 10% pectinase, 10% hemicellulase, 10% β-1,3-glucanase, 5% ligninase and 5% cell wall protease. Then adjust the pH of the slurry to 5.5 and enzymatically hydrolyze at 45℃ for 0.5h to obtain the enzymatic hydrolysate.

[0045] (3) Centrifuge the enzymatic hydrolysate obtained in (2) at 4℃ and 5000g for 15 min. After centrifugation, filter the solution using a 1μm pore size filter membrane to obtain the enzymatic hydrolysate supernatant.

[0046] (4) The supernatant of the enzyme digestion filtered in (3) is put into a 100kDa dialysis bag. The dialysis bag is placed in an electrophoresis tank for electrophoresis. The electrophoresis current is 200mA. The electrophoresis direction is changed and the electrophoresis buffer is replaced every hour. The electrophoresis buffer is prepared with deionized water and contains 7.2g / L glycine and 1.5g / L Tris hydrochloride.

[0047] (5) The dialysis liquid obtained in (4) was centrifuged at 4°C and 100,000g for 2 hours. The supernatant was discarded, and the precipitate was resuspended in a phosphate buffer solution with a pH of 7.2 and a concentration of 0.01 mol / L to obtain plant nanovesicles derived from peony.

[0048] Transmission electron microscopy (TEM) images of peony-derived plant nanovesicles prepared using the method of this embodiment are attached. Figure 1 As shown.

[0049] From the appendix Figure 1 As can be seen in the scale bar (200nm), the plant nanovesicles derived from peony prepared in this invention are individual nanoscale particles, exhibiting the spherical morphology characteristic of exosomes.

[0050] Furthermore, the average particle size and average potential of the obtained peony-derived plant nanovesicles were analyzed, as shown in the attached figure. Figure 2-3 As shown.

[0051] Appendix Figure 2 In the graph, the horizontal axis represents particle size, and the vertical axis represents the percentage of plant nanovesicles. Figure 2 As can be seen from the figure, the average particle size of the peony plant nanovesicles prepared in this embodiment is 179.7 nm.

[0052] Appendix Figure 3 In the graph, the horizontal axis represents different potentials, and the vertical axis represents the percentage of plant nanovesicles. Figure 3 As can be seen, the average potential of the prepared peony plant nanovesicles is -10.7mV.

[0053] The spherical vesicle structure and negative zeta potential under transmission electron microscopy are typical characteristics of plant nanovesicles. The above data is sufficient to prove that the method used in this embodiment can successfully extract peony nanovesicles.

[0054] Example 2

[0055] (1) Peony petals produced in Heze, Shandong Province were washed, dried at low temperature at 40℃, and ultra-finely pulverized to 80 mesh. Then, 0.01M phosphate buffer solution with pH 7.2 was added to the solution. The mass-volume ratio of peony petals to phosphate buffer solution was 1g:10mL. The solution was homogenized for 5min using a tissue homogenizer.

[0056] (2) Add a compound enzyme preparation to the homogenate obtained in (1) for enzymatic hydrolysis. The mass of the compound enzyme preparation added accounts for 0.2% of the mass of the peony petals in (1). The compound enzyme preparation used is a mixture of 70% cellulase, 15% hemicellulase and 15% β-1,3-glucanase. Adjust the pH of the slurry to 5.8 and enzymatically hydrolyze at 50℃ for 1.5h to obtain the enzymatic hydrolysate.

[0057] (3) Centrifuge the enzymatic hydrolysate obtained in (2) at 4℃ and 5000g for 20 min. After centrifugation, filter the solution using a 1μm pore size filter membrane to obtain the enzymatic hydrolysate supernatant.

[0058] (4) The supernatant of the enzyme digestion filtered in (3) is put into a 100kDa dialysis bag. The dialysis bag is placed in an electrophoresis tank for electrophoresis. The electrophoresis current is 200mA. The electrophoresis direction is changed and the electrophoresis buffer is replaced every hour. The electrophoresis buffer is prepared with deionized water and contains 7.2g / L glycine and 1.5g / L Tris hydrochloride.

[0059] (5) The dialysis liquid obtained in (4) was centrifuged at 4°C and 150,000g for 2 hours. The supernatant was discarded, and the precipitate was resuspended in a phosphate buffer solution with a pH of 7.2 and a concentration of 0.01 mol / L to obtain plant nanovesicles derived from peony.

[0060] Example 3

[0061] (1) Peony petals produced in Heze, Shandong Province were washed and dried at low temperature at 40℃; ultra-finely pulverized to 120 mesh, and then 0.01M phosphate buffer solution with pH 7.2 was added. The mass-volume ratio of peony petals to phosphate buffer solution was 1g:10mL. The mixture was homogenized for 5min using a tissue homogenizer.

[0062] (2) Add a compound enzyme preparation to the uniform slurry of peony petals obtained in (1) for enzymatic hydrolysis. The mass of the compound enzyme preparation added accounts for 0.4% of the mass of peony petals in (1). The compound enzyme preparation used is a mixture of 70% cellulase, 15% ligninase and 15% cell wall protease. Adjust the pH of the slurry to 6.0 and enzymatically hydrolyze at 45℃ for 3 hours to obtain the enzymatic hydrolysate.

[0063] (3) Centrifuge the enzymatic hydrolysate obtained in (2) at 4℃ and 5000g for 20 min. After centrifugation, filter the solution using a 1μm pore size filter membrane to obtain the enzymatic hydrolysate supernatant.

[0064] (4) The supernatant of the enzyme digestion filtered in (3) is put into a 200kDa dialysis bag. The dialysis bag is placed in an electrophoresis tank for electrophoresis. The electrophoresis current is 200mA. The electrophoresis direction is changed and the electrophoresis buffer is replaced every hour. The electrophoresis buffer is prepared with deionized water and contains 7.2g / L glycine and 1.5g / L Tris hydrochloride.

[0065] (5) The dialysis liquid obtained in (4) was centrifuged at 4°C and 150,000g for 2 hours. The supernatant was discarded, and the precipitate was resuspended in a phosphate buffer solution with a pH of 7.2 and a concentration of 0.01 mol / L to obtain plant nanovesicles derived from peony.

[0066] Example 4

[0067] Unlike Example 1, the compound enzyme preparation used in (2) is a mixture of 50% cellulase, 45% hemicellulase and 5% ligninase. The remaining steps and operations are the same as in Example 1.

[0068] Example 5

[0069] Unlike Example 1, the compound enzyme preparation used in (2) is a mixture of 70% cellulase, 10% pectinase, 10% cell wall protease and 10% β-1,3-glucanase. The remaining steps and operations are the same as in Example 1.

[0070] Example 6

[0071] Unlike Example 1, the current during horizontal electrophoresis dialysis in (4) is 250mA, while the other steps and operations are the same as in Example 1.

[0072] Example 7

[0073] Unlike Example 1, the current during horizontal electrophoresis dialysis in (4) is 300mA, while the other steps and operations are the same as in Example 1.

[0074] Comparative Example 1

[0075] The biggest difference from Example 1 is that (2) no compound enzyme is added for enzymatic hydrolysis, while the other steps and operations are the same as in Example 1.

[0076] Comparative Example 2

[0077] The biggest difference from Example 1 is that only pectinase is added for enzymatic hydrolysis, while the other steps are the same as in Example 1.

[0078] Comparative Example 3

[0079] The biggest difference from Example 1 is that the horizontal electrophoresis dialysis step (4) is omitted, while the other steps and operations are the same as in Example 1.

[0080] Comparative Example 4

[0081] The biggest difference from Example 1 is that the current during horizontal electrophoresis dialysis in (4) is 500mA, while the other steps and operations are the same as in Example 1.

[0082] Comparative Example 5

[0083] The biggest difference from Example 1 is that the temperature of the ultra-high speed centrifugation in (5) is 26°C, while the other steps and operations are the same as in Example 1.

[0084] Comparative Example 6

[0085] The biggest difference from Example 1 is that the ultra-high speed centrifugal force in (5) is 50000g, while the other steps and operations are the same as in Example 1.

[0086] The quality of the plant nanovesicles derived from peony obtained in the above embodiments and comparative examples is shown in Table 1 below.

[0087] Table 1. Quality of peony-derived plant nanovesicles prepared in each experimental group.

[0088]

[0089]

[0090] The data in Table 1 show that the plant nanovesicles derived from peony prepared in the various embodiments of the present invention have high protein and miRNA content. That is, after enzymatic hydrolysis of peony petal raw materials by using a variety of compound enzyme preparations, the protein and nucleic acid components in peony petals can be released to the maximum extent by horizontal electrophoresis dialysis and ultra-high speed centrifugation.

[0091] In Comparative Examples 1-2, when the peony petal raw materials were not enzymatically hydrolyzed or only a single enzyme preparation was used for enzymatic hydrolysis, the release of active ingredients was obviously insufficient. For example, in Comparative Example 2, the protein content in the peony plant nanovesicles was only 33.83 mg / mL, and the miRNA content was 63.23 ng / mL.

[0092] In Comparative Example 3, no horizontal electrophoresis dialysis was used for extraction. Even though the same compound enzyme preparation as in Example 1 was used for enzymatic hydrolysis, the content of miRNA and protein in the obtained peony nanovesicles was very low. The reason for this phenomenon may be that although the enzymatic hydrolysis method can fully hydrolyze the plant cell wall and release peony nanovesicles, it is impossible to enrich and separate the released nanovesicles without using horizontal electrophoresis dialysis.

[0093] In Comparative Examples 4-6, the parameters for preparing peony nanovesicles were adjusted. Obviously, when the preparation conditions are more stringent, the content of proteins and small RNAs may decrease to some extent due to the destruction or loss of active ingredients in peony petal cells.

[0094] Experimental Example 1

[0095] The efficacy of the plant nanovesicles derived from peony prepared in each embodiment and comparative example was investigated.

[0096] 1.1 Effects of peony-derived plant nanovesicles obtained by different methods on cell viability. Specific experimental procedures are as follows:

[0097] Human fibroblasts were cultured in vitro and divided into four groups: a blank group (normal culture group), a modeling group (UV irradiation group), a common extract group (boiling water extraction sample treatment group), and an example group (plant nanovesicle sample treatment group). After UV irradiation, the cells were treated with different samples for 48 hours. The cell viability of each group was detected by the MTT assay. The results are shown in Table 2 and Appendix. Figure 4 As shown.

[0098] Table 2. Survival rate of fibroblasts in each experimental component

[0099] experimental group Fibroblast survival rate (%) experimental group Fibroblast survival rate (%) Blank group 100 Example 6 65.21 Modeling Group 49.40 Example 7 67.08 Common extract group 53.97 Comparative Example 1 54.37 Example 1 69.73 Comparative Example 2 52.14 Example 2 66.58 Comparative Example 3 52.51 Example 3 66.78 Comparative Example 4 49.64 Example 4 64.88 Comparative Example 5 50.48 Example 5 65.23 Comparative Example 6 54.52

[0100] Clearly, the MTT assay showed that the cell survival rate in Examples 1-7 was significantly higher than that in Comparative Examples 1-6. The main reason for this phenomenon is that miRNA is the main active substance in plant nanovesicles, and the protein and miRNA content in peony-derived plant nanovesicles prepared by different methods varies significantly. The protein and miRNA content in peony-derived plant nanovesicles in Examples 1-7 were much higher than those in Comparative Examples 1-6, so the cell survival rate in each example was significantly higher.

[0101] 1.2 The effect of ELISA on the levels of inflammatory factors IL-6 and type I collagen in mouse skin homogenate was detected. The specific experimental procedure is as follows:

[0102] Female ICR mice (5-6 weeks old, 18-22g) were randomly divided into four groups: a control group (normal feeding group), a modeling group (UV irradiation group), a common extract group (boiling water extraction sample treatment group), and an example group (plant nanovesicle sample treatment group). The mice underwent hair removal on their backs, followed by UVB irradiation 1-2 hours later. Hair was shaved every two to three days. The irradiation dose was 200 mJ / cm² on days 1-3. 2 Subsequently, the amount increased by 50 mJ / cm² every two days. 2Samples were applied before each irradiation. After 14 days of modeling, the skin on the backs of the mice was homogenized, and ELISA was used to detect changes in inflammatory factors and collagen content.

[0103] The levels of inflammatory factors IL-6 and type I collagen in mouse skin homogenates are shown in Table 3 below and appendix. Figure 5-6 As shown.

[0104] Table 3. Changes in inflammatory factors and collagen content in mouse skin homogenate.

[0105]

[0106]

[0107] The results above show that Examples 1-7 can more effectively reduce the inflammatory response of the skin on the back of mice caused by ultraviolet radiation and better prevent the loss of type I collagen compared with Comparative Examples 1-6.

Claims

1. A method for preparing plant nanovesicles derived from peony, characterized in that, Includes the following steps: (1) After washing the peony petals, dry them at a low temperature of 40~60℃, then crush them to 80~120 mesh, and then add 0.01 M phosphate buffer solution with pH 7.

2. Use a tissue homogenizer to homogenize the solution to obtain a slurry; the mass-volume ratio of peony petals to phosphate buffer solution is 1 g: 5~10 mL. (2) Add a compound enzyme preparation to the slurry obtained in (1) for enzymatic hydrolysis to obtain the hydrolysate; The compound enzyme preparation is added at a mass ratio of 0.1% to 0.4% of the peony petals. The aforementioned compound enzyme preparation is a mixed enzyme preparation of cellulase, pectinase, hemicellulase, β-1,3-glucanase, ligninase, and cell wall protease. The mass fraction of each enzyme in the compound enzyme preparation is as follows: cellulase 60%, pectinase 10%, hemicellulase 10%, β-1,3-glucanase 10%, ligninase 5%, and cell wall protease 5%. Alternatively, the compound enzyme preparation may be cellulase, hemicellulase, and β-1,3-glucanase, wherein the mass fraction of each enzyme in the compound enzyme preparation is: 70% cellulase, 15% hemicellulase, and 15% β-1,3-glucanase. (3) Centrifuge and filter the enzymatic hydrolysate obtained in (2) to obtain the enzymatic hydrolysate supernatant; (4) Perform horizontal electrophoresis dialysis on the enzymatic hydrolysis supernatant obtained in (3) to obtain dialysis solution. Specifically, the enzymatic hydrolysis solution is placed into a dialysis bag of 100~300 kDa, the dialysis bag is placed in the electrophoresis tank, and electrophoresis separation is performed using a current of 200~300 mA. The electrophoresis direction is changed and the electrophoresis buffer is replaced every hour. The electrophoresis buffer is prepared with deionized water and contains 7-9 g / L glycine and 1-3 g / L tris(hydroxymethyl)aminomethane hydrochloride. (5) The dialysate obtained in (4) is subjected to ultra-high speed centrifugation. The ultra-high speed centrifugation temperature is 0~10℃, the centrifugation force is 100000~180000 g, the centrifugation time is 1~3 h, the supernatant is discarded, and the obtained precipitate is resuspended in phosphate buffer solution to obtain plant nanovesicles derived from peony.

2. The preparation method according to claim 1, characterized in that, In (3), the enzymatic hydrolysate obtained in (2) is centrifuged at 0~10℃ and centrifugation force of 3000~5000 g for 15~30 min. After centrifugation, it is filtered with a filter membrane with a pore size of 1 μm to obtain the enzymatic hydrolysate supernatant.

3. The application of peony-derived plant nanovesicles prepared by the preparation method described in claim 1 in the preparation of anti-skin aging products.

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