Preparation method of plant cell exosome, plant cell exosome and application of plant cell exosome

Abstract

The invention relates to the technical field of plant extraction, in particular to a preparation method of a plant cell exosome, the plant cell exosome and application of the plant cell exosome. The preparation method comprises the following steps: after the surface of a plant leaf is scratched, inoculating the plant leaf into a first culture medium for induction culture, then transferring the plant leaf into a second culture medium for subculture to obtain a callus, and then adding the callus into a buffer solution for grinding to obtain a filtrate; centrifuging the filtrate again to obtain supernate; and adding an exosome complexing agent into the supernate, incubating overnight, centrifuging, and freeze-drying to obtain the exosome. The first culture medium comprises an MS culture medium and the following components added into the MS culture medium: pullulan, 2, 4-D, agar and NAA; the second culture medium comprises an MS culture medium and the following components added into the MS culture medium: pullulan, 2, 4-D, agar, NAA and sodium phytate. The preparation method can increase the protein content in the exosome.

Description

Technical Field

[0001] The present invention relates to the technical field of plant extraction, and particularly relates to a preparation method of plant extracellular vesicles, plant extracellular vesicles and their applications. Background Art

[0002] Narrowly defined, plant extracellular vesicles refer to extracellular vesicles secreted by plant cells within a specified size range. Due to many advantages such as stable properties, easy reaction, no immunogenicity, easy acquisition and convenient modification of plant extracellular vesicles, they are widely used in pharmacologically active substances.

[0003] Plant extracellular vesicles have a variety of special effects. For example, exosomes from Clinacanthus nutans have significant effects in antioxidant and anti-inflammatory aspects, and exosomes from wheat have a promoting effect on the proliferation and migration of endothelial cells and dermal fibroblasts; some plant extracellular vesicles also have an anti-skin aging effect. For example, Chinese Patent CN115584337A, a plant extracellular vesicle, preparation method and application in anti-skin aging products, discloses a method for obtaining plant extracellular vesicles by enzymolyzing plant raw materials and then incubating with an extracellular vesicle complexing agent, and proves that plant extracellular vesicles can effectively inhibit the decrease in the survival rate of fibroblasts caused by ultraviolet rays and make cells younger.

[0004] Common preparation methods of extracellular vesicles mainly involve plant tissue homogenization and ultra-high speed centrifugation methods, etc., and usually have defects such as insufficient activity of the prepared extracellular vesicles, low extraction yield and low purity. In view of this, the present invention provides a preparation method of plant extracellular vesicles in order to accelerate its application in skin care products. Summary of the Invention

[0005] In order to overcome the deficiencies of the prior art, one of the purposes of the present invention is to provide a preparation method of plant extracellular vesicles, which is simple to prepare and easy to practice.

[0006] Another purpose of the present invention is to provide a plant extracellular vesicle with a high protein content.

[0007] Another purpose of the present invention is to provide an application of plant extracellular vesicles with broad prospects.

[0008] One of the purposes of the present invention is achieved by adopting the following technical scheme: A preparation method of plant extracellular vesicles, comprising the following steps: (1) Scratch the surface of plant leaves and inoculate them in a first culture medium for induction culture, and then transfer them to a second culture medium for subculture to obtain callus; (2) Grind the callus obtained in step (1) in a buffer solution, centrifuge and filter to obtain a filtrate; (3) After centrifuging the filtrate from step (2) again, the supernatant is obtained; an exosome complexing agent is added to the supernatant, incubated overnight, centrifuged, and the precipitate is obtained, and the precipitate is then freeze-dried to obtain the product. The first medium comprises MS medium and the following components added to the MS medium: pullulan, 2,4-D, agar, NAA; The second medium comprises MS medium and the following components added to the MS medium: pullulan, 2,4-D, agar, NAA, sodium phytate.

[0009] Further, based on the final concentration of the first medium, the concentrations of pullulan, 2,4-D, agar, and NAA in the first medium are: pullulan 1 - 5 g / L, 2,4-D 0.5 - 1 mg / L, agar 5 - 8 g / L, NAA 0.1 - 0.3 mg / L.

[0010] Further, based on the final concentration of the second medium, the concentrations of pullulan, 2,4-D, agar, NAA, and sodium phytate in the second medium are: pullulan 1 - 5 g / L, 2,4-D 0.5 - 1 mg / L, agar 5 - 8 g / L, NAA 0.1 - 0.3 mg / L, sodium phytate 0.1 - 0.5 mg / L.

[0011] Further, the conditions for the induction culture in step (1) are: dark culture, temperature 25 ± 2 °C, time 10 - 15 d.

[0012] Further, the conditions for the subculture in step (1) are: dark culture, temperature 25 ± 2 °C, time 14 - 20 d.

[0013] Further, the plant in step (1) is Camellia.

[0014] Further, the buffer in step (2) is PBS buffer.

[0015] Further, the exosome complexing agent in step (3) consists of 10 - 15 wt% PEG8000 and the balance of water.

[0016] The second object of the present invention is achieved by the following technical solution: A plant cell exosome is prepared by the preparation method of the above plant cell exosome.

[0017] The third object of the present invention is achieved by the following technical solution: Application of the above plant cell exosome in the preparation of an anti-aging cosmetic.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The present invention provides a method for preparing plant extracellular vesicles, which involves induction culture using a first medium and subculture using a second medium. The first medium includes components such as pullulan, and the second medium includes components such as pullulan and sodium phytate. Detection of the protein content in the extracellular vesicles obtained by the above preparation method reveals that the combined use of pullulan and sodium phytate can significantly increase the protein content in the extracellular vesicles. Specifically, in the induction culture stage, pullulan significantly increases the protein content in the extracellular vesicles through mechanisms such as osmotic stress, physical protection, and cell viscosity. In the subculture stage, sodium phytate can ensure the continuous secretion of extracellular vesicles and reduce protein degradation through mechanisms such as antioxidant, phosphorus metabolism, and inhibition of protein degradation.

[0019] 2. Detection of the anti-aging effect of skin care products added with the plant extracellular vesicles of the present invention shows that: due to the high protein content, the plant extracellular vesicles prepared by the present invention enhance their anti-aging effect in skin care products.

[0020] 3. The plant extracellular vesicles of the present invention have broad application prospects in the preparation of skin care products with anti-aging effects. Specific Embodiments

[0021] The following further describes the present invention in conjunction with specific embodiments. It should be noted that, on the premise of no conflict, the following-described embodiments or technical features can be combined arbitrarily to form new embodiments. Specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are conventional products obtained through commercial channels unless otherwise specified.

[0022] (I) Embodiments Example 1 This example provides a method for preparing plant extracellular vesicles. The following is the specific preparation process: (1) Disinfect the camellia leaves with 75% alcohol for 40 s, then rinse them 3 times with sterile water, disinfect them with 2% sodium hypochlorite solution for 15 min, then rinse them 4 times with sterile water and place them on sterile filter paper to absorb the surface moisture. Then, scratch the surface of the leaves with an inoculation knife and inoculate them into the first medium, and induce and culture them for 12 d under dark conditions at 25 °C. Then transfer them to the second medium and subculture them for 17 d under dark conditions at 25 °C to obtain callus. Among them, based on the final concentration of the medium, the first medium consists of MS medium and the following components added to the MS medium: pullulan 3 g / L, 2,4-D 0.6 mg / L, agar 7 g / L, NAA 0.2 mg / L; the second medium consists of MS medium and the following components added to the MS medium: pullulan 3 g / L, 2,4-D 0.6 mg / L, agar 7 g / L, NAA 0.2 mg / L, sodium phytate 0.4 mg / L.

[0023] (2) Add the callus obtained in step (1) to PBS buffer for grinding, and after centrifugation and filtration, obtain a filtrate. (3) After centrifuging the filtrate in step (2) again, obtain the supernatant. Add an exosome complexing agent (consisting of 12 wt% PEG8000 and the balance of water) to the supernatant, incubate overnight and centrifuge to obtain a precipitate, and freeze-dry the precipitate at -30 °C and a vacuum of 200 Pa for 32 h to obtain the product.

[0024] This example also provides a plant extracellular exosome, which is prepared by the above preparation method.

[0025] Example 2 This example provides a preparation method of a plant extracellular exosome. The following is the specific preparation process: (1) Disinfect the camellia leaves with 75% alcohol for 40 s, then rinse them 3 times with sterile water, disinfect them with 2% sodium hypochlorite solution for 15 min, then rinse them 4 times with sterile water and place them on sterile filter paper to absorb the surface moisture. Then, scratch the surface of the leaves with an inoculation knife and inoculate them into the first medium, and induce and culture them for 10 d under dark conditions at 27 °C. Then transfer them to the second medium and subculture them for 14 d under dark conditions at 27 °C to obtain callus. Among them, based on the final concentration of the medium, the first medium consists of MS medium and the following components added to the MS medium: pullulan 5 g / L, 2,4-D 1 mg / L, agar 8 g / L, NAA 0.3 mg / L; the second medium consists of MS medium and the following components added to the MS medium: pullulan 5 g / L, 2,4-D 1 mg / L, agar 8 g / L, NAA 0.3 mg / L, sodium phytate 0.5 mg / L.

[0026] (2) Add the callus obtained in step (1) to PBS buffer for grinding, and after centrifugation and filtration, obtain a filtrate; (3) After centrifuging the filtrate of step (2) again, obtain a supernatant; add an exosome complexing agent (composed of 15 wt% PEG8000 and the balance water) to the supernatant, incubate overnight, and centrifuge to obtain a precipitate. The precipitate is freeze-dried at -30°C and a vacuum of 200 Pa for 32 h to obtain the product.

[0027] This example also provides a plant cell exosome, which is prepared by the above preparation method.

[0028] Example 3 This example provides a preparation method of a plant cell exosome. The following is the specific preparation process: (1) First, disinfect the camellia leaves with 75% alcohol for 40 s, then rinse them 3 times with sterile water, then disinfect them with 2% sodium hypochlorite solution for 15 min, then rinse them 4 times with sterile water and place them on sterile filter paper to absorb the surface moisture. Then, scratch the surface of the leaves with an inoculation knife and inoculate them into the first medium, and induce and culture them under dark conditions at 23°C for 15 d. Then transfer them to the second medium and subculture them under dark conditions at 23°C for 20 d to obtain callus; wherein, based on the final concentration of the medium, the first medium consists of MS medium and the following components added to the MS medium: pullulan 1 g / L, 2,4-D 0.5 mg / L, agar 5 g / L, NAA 0.1 mg / L; the second medium consists of MS medium and the following components added to the MS medium: pullulan 1 g / L, 2,4-D 0.5 mg / L, agar 5 g / L, NAA 0.1 mg / L, phytic acid sodium 0.1 mg / L.

[0029] (2) Add the callus obtained in step (1) to PBS buffer for grinding, and after centrifugation and filtration, obtain a filtrate; (3) After centrifuging the filtrate of step (2) again, obtain a supernatant; add an exosome complexing agent (composed of 10 wt% PEG8000 and the balance water) to the supernatant, incubate overnight, and centrifuge to obtain a precipitate. The precipitate is freeze-dried at -30°C and a vacuum of 200 Pa for 32 h to obtain the product.

[0030] This example also provides a plant cell exosome, which is prepared by the above preparation method.

[0031] (II) Comparative Example Comparative Example 1 The difference between this comparative example and Example 1 is as follows: Based on the final concentration of the culture medium, the first culture medium consists of MS culture medium and the following components added to the MS culture medium: 2,4-D 0.6 mg / L, agar 7 g / L, NAA 0.2 mg / L; the second culture medium consists of MS culture medium and the following components added to the MS culture medium: 2,4-D 0.6 mg / L, agar 7 g / L, NAA 0.2 mg / L, phytic acid sodium 0.4 mg / L, and the rest is the same as in Example 1.

[0032] Comparative Example 2 The difference between this comparative example and Example 1 is as follows: Based on the final concentration of the culture medium, the first culture medium consists of MS culture medium and the following components added to the MS culture medium: sucrose 3 g / L, 2,4-D 0.6 mg / L, agar 7 g / L, NAA 0.2 mg / L; the second culture medium consists of MS culture medium and the following components added to the MS culture medium: sucrose 3 g / L, 2,4-D 0.6 mg / L, agar 7 g / L, NAA 0.2 mg / L, phytic acid sodium 0.4 mg / L.

[0033] Comparative Example 3 The difference between this comparative example and Example 1 is as follows: Based on the final concentration of the culture medium, the first culture medium consists of MS culture medium and the following components added to the MS culture medium: pullulan 3 g / L, 2,4-D 0.6 mg / L, agar 7 g / L, NAA 0.2 mg / L; the second culture medium consists of MS culture medium and the following components added to the MS culture medium: pullulan 3 g / L, 2,4-D 0.6 mg / L, agar 7 g / L, NAA 0.2 mg / L.

[0034] (III) Test Examples The protein content in the plant cell exosomes prepared in Examples 1-3 and Comparative Examples 1-3 and its effect on the proliferation ability of human skin fibroblasts were detected. The following are the specific detection methods: Protein content: The total protein content of the plant cell exosomes prepared in Examples 1-3 and Comparative Examples 1-3 was detected using a BCA protein concentration detection kit, and the results are shown in Table 1; Relative cell viability: Human skin fibroblasts in the logarithmic growth phase were taken, at 5×10 per well 3Cells were seeded on 96-well plates, with 5 replicate wells in each group. After they adhered, they were respectively replaced with 100 μL of DMEM medium containing the exosomes obtained in Examples 1-3 or Comparative Examples 1-3 (which also contained 5% FBS, and the concentration of exosomes in each group was 10 mg / L). The blank group was DMEM medium containing 5% FBS without added exosomes. After continued culture for 48 h, 10 μL of CCK-8 reagent was added to each well and incubated for 2 h. The absorbance at a wavelength of 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader. Taking the blank group as 100%, the relative cell viability was calculated. The results are shown in Table 1.

[0035] Relative cell viability (%) = OD value of the experimental group / OD value of the blank group × 100%.

[0036] Table 1 It can be seen from observing Table 1 that the plant exosomes obtained in Examples 1-3 of the present invention have a higher protein content and a more obvious effect on the proliferation ability of human skin fibroblasts.

[0037] Compared with Example 1, Pullulan was omitted in Comparative Example 1, sucrose was used instead of Pullulan in Comparative Example 2, and Sodium phytate was omitted in Comparative Example 3. The protein content in the obtained plant exosomes decreased, and the influence on the proliferation ability of human skin fibroblasts decreased. The above results show that the combined use of Pullulan and Sodium phytate can significantly increase the total protein content of exosomes, thereby promoting the proliferation ability of human skin fibroblasts.

[0038] Furthermore, through further analysis, Pullulan is a natural polysaccharide with adhesiveness and film-forming properties. It can form a microenvironment barrier to reduce mechanical damage to callus during culture and maintain the integrity of cell membranes, thereby reducing the leakage of intracellular proteins and improving the enrichment efficiency of proteins in exosomes. Moreover, Pullulan regulates the osmotic pressure of the culture medium, causes mild osmotic stress in plant cells, activates the cell stress signal pathway, prompts cells to secrete more exosomes to transmit stress signals, and increases the total protein content of exosomes. In addition, the adhesive properties of Pullulan can also enhance the physical connection between callus cells, form a more stable three-dimensional structure, facilitate the directional secretion of exosomes, and reduce protein loss. The addition of Sodium phytate can scavenge the reactive oxygen species accumulated during subculture, reduce the damage of oxidative stress to cells, maintain the continuous ability of cells to secrete exosomes, and avoid the decrease in protein content caused by cell senescence. Moreover, the phosphate ions released after the decomposition of Sodium phytate can participate in cell energy metabolism and signal transduction, activate the pathways related to exosome biosynthesis, and promote the packaging of specific functional proteins. In addition, Sodium phytate can also inhibit the activity of polyphenol oxidase, reduce protein denaturation caused by the oxidation of phenolic substances, and protect the integrity of exosome proteins.

[0039] (IV) Application Example An essence, which is composed of raw materials with the following mass percentages: 4% exosomes, 0.5% p - hydroxyacetophenone, 1% hyaluronic acid, 6% grape seed oil, 0.05% sodium carboxymethyl cellulose, 6% squalane, and the balance is water; the above - mentioned exosomes are respectively the exosomes prepared in Examples 1 - 3 or Comparative Examples 1 - 3.

[0040] Application Example 1 According to the method of the human skin occlusive patch test in Part 5 of the "Technical Specifications for Cosmetics Safety" (2015 Edition), the safety of the essence added with the plant cell exosomes of Examples 1 - 3 and Comparative Examples 1 - 3 was detected. The following is the specific detection method: 70 subjects aged 30 - 50 were selected and randomly divided into 7 groups, with a male - to - female ratio of 1:1 in each group. The essence of each group and deionized water (blank control group) were taken, and the occlusive patch test was carried out simultaneously for each group. The number of people with allergic phenomena such as skin redness or erythema was counted. The results are shown in Table 2.

[0041] Table 2 It can be seen from observing Table 2 that none of the volunteers had allergic phenomena such as skin redness or erythema. The above results indicate that the addition of the plant cell exosomes of the present invention and the obtained essence have high safety.

[0042] Application Example 2 The anti - aging effects of the essence added with the plant cell exosomes of Examples 1 - 3 and Comparative Examples 1 - 3 were detected. The following is the specific detection method: 60 volunteers aged 30 - 50 were selected and randomly divided into 6 groups, with a male - to - female ratio of 1:1 in each group. The volunteers used the essence added with the plant cell exosomes of Examples 1 - 3 and Comparative Examples 1 - 3 once in the morning and once in the evening every day, with the dosage... (It seems there is something missing here about the dosage). Before use (T0) and after 28 days of use (T28), each volunteer was followed up at the same time during the day. During the experiment, other products of the same type were not allowed to be used. Before the test, the volunteers sat still in a constant - temperature and constant - humidity waiting area at a temperature of 22°C and a relative humidity of 50% RH for 30 minutes; the tester used a skin elasticity measuring instrument MPA580 to measure the skin elasticity of the stratum corneum in the cheek area of the subjects, and selected the parameter R2 as the comparison index (R2: the ratio of the skin rebound amount without negative pressure to the maximum stretching amount with negative pressure. The closer the ratio is to 1, the better the skin elasticity). The improvement effect of the product on the skin elasticity of the test area was evaluated by measuring the change in the skin elasticity value R2 before and after using the product.

[0043] The change rate of R2 = (the R2 parameter value at T28 - the R2 parameter value at T0) / the R2 parameter value at T0×100%. The results are shown in Table 3.

[0044] Table 3 As can be seen from Table 3, the essence obtained by adding the exosomes of plant cells in Examples 1-3 has good anti-aging effects.

[0045] Compared with the essence obtained by adding the exosomes of plant cells in Example 1, the change rate of R2 of the essence obtained by adding the exosomes of plant cells in Comparative Examples 1-3 is reduced. By analyzing the protein content in the plant cell exosomes and the effect on the proliferation ability of human fibroblasts, it is known that the preparation method of the plant exosomes provided by the present invention can significantly increase the activity of the exosomes, and thus effectively improve their anti-aging effects in skin care products.

[0046] The above embodiments are only the preferred embodiments of the present invention, and the scope of protection of the present invention cannot be limited thereby. Any non-substantive changes and substitutions made by those skilled in the art on the basis of the present invention fall within the scope of protection required by the present invention.

Claims

1. A method for preparing plant extracellular vesicles, characterized in that, It includes the following steps: (1) Scratch the surface of plant leaves and inoculate them into the first medium for induction culture, and then transfer them to the second medium for subculture to obtain callus; (2) Add the callus obtained in step (1) to a buffer for grinding, and after centrifugation and filtration, obtain a filtrate; (3) After centrifuging the filtrate obtained in step (2) again, obtain a supernatant; add an exosome complexing agent to the supernatant, incubate overnight and centrifuge to obtain a precipitate, and then freeze-dry the precipitate to obtain the product; The first medium includes MS medium and the following components added to the MS medium: pullulan, 2,4-D, agar, NAA; The second medium includes MS medium and the following components added to the MS medium: pullulan, 2,4-D, agar, NAA, sodium phytate.

2. The preparation method of the plant extracellular vesicles according to claim 1, characterized in that, Calculated based on the final concentration of the first medium, the concentrations of pullulan, 2,4-D, agar, and NAA in the first medium are: pullulan 1-5 g / L, 2,4-D 0.5-1 mg / L, agar 5-8 g / L, NAA 0.1-0.3 mg / L.

3. The preparation method of the plant extracellular vesicles according to claim 1, characterized in that, Calculated based on the final concentration of the second medium, the concentrations of pullulan, 2,4-D, agar, NAA, and sodium phytate in the second medium are: pullulan 1-5 g / L, 2,4-D 0.5-1 mg / L, agar 5-8 g / L, NAA 0.1-0.3 mg / L, sodium phytate 0.1-0.5 mg / L.

4. The preparation method of the plant extracellular vesicles according to claim 1, wherein The conditions for the induction culture in step (1) are: dark culture, temperature 25±2°C, time 10-15 d.

5. The preparation method of the plant extracellular vesicles according to claim 1, wherein, The conditions for the subculture in step (1) are: dark culture, temperature 25±2°C, time 14-20 d.

6. The preparation method of the plant extracellular exosomes according to claim 1, wherein, The plant in step (1) is Camellia.

7. The method for preparing extracellular vesicles of plant cells according to claim 1, wherein, The buffer in step (2) is PBS buffer.

8. The preparation method of the plant extracellular vesicles according to claim 1, characterized in that, The exosome complexing agent in step (3) consists of 10-15 wt% PEG8000 and the balance of water.

9. A plant extracellular exosome, characterized in that, It is prepared by using the method for preparing plant extracellular exosomes according to any one of claims 1-8.

10. Use of the plant extracellular exosomes according to claim 9 in the preparation of anti-aging skin care products.

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