A preparation method of plant cell exosomes, plant cell exosomes and applications thereof

Abstract

The present invention relates to the field of plant extraction technology, and in particular to a method for preparing plant cell exosomes, plant cell exosomes, and their applications. The preparation method comprises the following steps: scratching the surface of a plant leaf, inoculating it in a first culture medium for induction culture, then transferring it to a second culture medium for subculture to obtain callus tissue, which is then added to a buffer solution for grinding to obtain a filtrate; the filtrate is centrifuged again to obtain a supernatant; an exosome complexing agent is added to the supernatant, incubated overnight, centrifuged, and freeze-dried to obtain the exosome complexing agent; the first culture medium comprises MS culture medium and the following components added to the MS culture medium: pullulan, 2,4-D, agar, and NAA; the second culture medium comprises MS culture medium and the following components added to the MS culture medium: pullulan, 2,4-D, agar, NAA, and sodium phytate. The preparation method can increase the protein content in exosomes.

Description

Technical Field

[0001] The present invention relates to the field of plant extraction technology, and in particular to a method for preparing plant cell exosomes, plant cell exosomes and applications thereof. Background Art

[0002] In a narrow sense, plant exosomes refer to extracellular vesicles of a specified size range secreted by plant cells. Due to their many advantages, such as stability, ease of reaction, lack of immunogenicity, ease of acquisition, and amenability to modification, plant exosomes are widely used in the development of pharmacologically active substances.

[0003] Plant exosomes have many special functions. For example, parsnip exosomes have significant effects in anti-oxidation and anti-inflammatory aspects, and wheat exosomes have a proliferation and migration-promoting effect on endothelial cells and dermal fibroblasts. Some plant exosomes also have anti-skin aging effects. For example, Chinese patent CN115584337A, a plant exosome, a preparation method and its application in anti-skin aging products, discloses a method for obtaining plant exosomes by enzymatically hydrolyzing plant raw materials and then adding exosome complexing agents for incubation. It also proves that plant exosomes can effectively inhibit the decline in fibroblast survival rate caused by ultraviolet rays, thereby rejuvenating cells.

[0004] Common methods for preparing exosomes primarily involve homogenization of plant tissues and ultracentrifugation. These methods often result in insufficient exosome activity, low extraction yield, and low purity. In light of this, the present invention provides a method for preparing plant cell exosomes, with the goal of accelerating their application in skincare products. Summary of the Invention

[0005] In order to overcome the deficiencies of the prior art, one of the objectives of the present invention is to provide a method for preparing plant cell exosomes, which is simple to prepare and easy to practice.

[0006] A second object of the present invention is to provide a plant cell exosome containing a high protein content.

[0007] The third purpose of the present invention is to provide an application of plant cell exosomes, which has broad prospects.

[0008] One of the purposes of the present invention is achieved by the following technical solution:

[0009] A method for preparing plant cell exosomes, comprising the following steps:

[0010] (1) scratching the surface of the plant leaf and inoculating it into the first culture medium for induction culture, and then transferring it into the second culture medium for subculture to obtain callus tissue;

[0011] (2) adding the callus tissue obtained in step (1) into a buffer solution and grinding the same, followed by centrifugation and filtration to obtain a filtrate;

[0012] (3) The filtrate of step (2) is centrifuged again to obtain a supernatant; an exosome complexing agent is added to the supernatant, incubated overnight, centrifuged, and a precipitate is obtained, which is then freeze-dried to obtain the product;

[0013] The first culture medium includes MS culture medium and the following components added to the MS culture medium: pullulan, 2,4-D, agar, and NAA;

[0014] The second culture medium includes MS culture medium and the following components added to the MS culture medium: pullulan, 2,4-D, agar, NAA, and sodium phytate.

[0015] Furthermore, based on the final concentration of the first culture medium, the concentrations of the pullulan, 2,4-D, agar, and NAA in the first culture medium are: pullulan 1-5 g / L, 2,4-D 0.5-1 mg / L, agar 5-8 g / L, and NAA 0.1-0.3 mg / L.

[0016] Furthermore, based on the final concentration of the second culture medium, the concentrations of the pullulan, 2,4-D, agar, NAA, and sodium phytate in the second culture 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, and sodium phytate 0.1-0.5 mg / L.

[0017] Furthermore, the induction culture conditions in step (1) are: dark culture, temperature of 25±2°C, and time of 10-15 days.

[0018] Furthermore, the conditions for the subculture in step (1) are: dark culture at a temperature of 25±2°C for 14-20 days.

[0019] Furthermore, the plant in step (1) is Camellia.

[0020] Furthermore, the buffer solution in step (2) is PBS buffer solution.

[0021] Furthermore, the exosome complexing agent in step (3) consists of 10-15wt% PEG8000 and the balance water.

[0022] The second object of the present invention is achieved by adopting the following technical solution:

[0023] A plant cell exosome is prepared using the above-mentioned plant cell exosome preparation method.

[0024] The third object of the present invention is achieved by adopting the following technical solution:

[0025] Application of the above plant cell exosomes in the preparation of anti-aging cosmetics.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. The present invention provides a method for preparing plant cell exosomes, which involves using a first culture medium for induction culture and a second culture medium for subculture. The first culture medium includes pullulan and other ingredients, and the second culture medium includes pullulan, sodium phytate and other ingredients. The protein content in the exosomes obtained by the above preparation method was detected and it was found that the combination of pullulan and sodium phytate can significantly increase the protein content in the exosomes. Specifically, in the induction culture stage, pullulan significantly increases the protein content in the exosomes through mechanisms such as osmotic stress, physical protection and cell viscosity. In the subculture stage, sodium phytate can ensure the continuity of exosome secretion through mechanisms such as anti-oxidation, phosphorus metabolism and inhibition of protein degradation, while also reducing protein degradation.

[0028] 2. The anti-aging effect of the skin care products obtained by adding the plant cell exosomes of the present invention was tested. The results showed that the plant cell exosomes prepared by the present invention have a higher protein content, thereby improving the anti-aging effect of the skin care products.

[0029] 3. The plant cell exosomes of the present invention have broad application prospects in the preparation of skin care products with anti-aging effects. DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with specific embodiments. It should be noted that, under the premise of no conflict, the various embodiments described below or the various technical features can be arbitrarily combined to form new embodiments. Specific conditions not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. All reagents or instruments used, unless otherwise specified, are conventional products obtained through commercial channels.

[0031] (1) Implementation

[0032] Example 1

[0033] This embodiment provides a method for preparing plant cell exosomes. The specific preparation process is as follows:

[0034] (1) Camellia sinensis leaves were first disinfected with 75% alcohol for 40 seconds, then rinsed with sterile water three times, and then disinfected with 2% sodium hypochlorite solution for 15 minutes, and then rinsed with sterile water four times and placed on sterile filter paper to absorb surface moisture. The surface of the leaves was then scratched with an inoculation knife and inoculated into a first culture medium. The leaves were induced and cultured at 25°C in the dark for 12 days, and then transferred to a second culture medium. The leaves were subcultured at 25°C in the dark for 17 days to obtain callus tissue. The first culture medium consisted 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, and sodium phytate 0.4 mg / L, based on the final concentration of the culture medium.

[0035] (2) adding the callus tissue obtained in step (1) into PBS buffer solution and grinding the solution, centrifuging and filtering the solution to obtain a filtrate;

[0036] (3) The filtrate of step (2) was centrifuged again to obtain a supernatant; an exosome complexing agent (composed of 12 wt% PEG8000 and the remainder water) was added to the supernatant, incubated overnight, and centrifuged to obtain a precipitate, which was freeze-dried at -30°C and a vacuum of 200 Pa for 32 h.

[0037] This embodiment also provides a plant cell exosome, which is prepared using the above preparation method.

[0038] Example 2

[0039] This embodiment provides a method for preparing plant cell exosomes. The specific preparation process is as follows:

[0040] (1) Camellia sinensis leaves were first disinfected with 75% alcohol for 40 seconds, then rinsed with sterile water three times, and then disinfected with 2% sodium hypochlorite solution for 15 minutes, and then rinsed with sterile water four times and placed on sterile filter paper to absorb surface moisture. The surface of the leaves was then scratched with an inoculation knife and inoculated into a first culture medium. The leaves were induced and cultured at 27°C in the dark for 10 days, and then transferred to a second culture medium. The leaves were subcultured at 27°C in the dark for 14 days to obtain callus tissue. The first culture medium consisted of MS culture medium and the following components added to the MS culture medium: pullulan 5 g / L, 2,4-D 1 mg / L, agar 8 g / L, NAA 0.3 mg / L, and sodium phytate 0.5 mg / L, based on the final concentration of the culture medium.

[0041] (2) adding the callus tissue obtained in step (1) into PBS buffer solution and grinding the solution, centrifuging and filtering the solution to obtain a filtrate;

[0042] (3) The filtrate of step (2) was centrifuged again to obtain a supernatant; an exosome complexing agent (composed of 15 wt% PEG8000 and the remainder water) was added to the supernatant, incubated overnight, and centrifuged to obtain a precipitate, which was freeze-dried at -30°C and a vacuum of 200 Pa for 32 h.

[0043] This embodiment also provides a plant cell exosome, which is prepared using the above preparation method.

[0044] Example 3

[0045] This embodiment provides a method for preparing plant cell exosomes. The specific preparation process is as follows:

[0046] (1) Camellia sinensis leaves were first disinfected with 75% alcohol for 40 seconds, then rinsed with sterile water for 3 times, and then disinfected with 2% sodium hypochlorite solution for 15 minutes, and then rinsed with sterile water for 4 times and placed on sterile filter paper to absorb surface moisture. The surface of the leaves was then scratched with an inoculation knife and inoculated into a first culture medium. The leaves were induced and cultured at 23°C in the dark for 15 days, and then transferred to a second culture medium. The leaves were subcultured at 23°C in the dark for 20 days to obtain callus tissue. The first culture medium consisted of MS culture medium and the following components added to the MS culture medium: pullulan 1 g / L, 2,4-D 0.5 mg / L, agar 5 g / L, NAA 0.1 mg / L, and sodium phytate 0.1 mg / L, based on the final concentration of the culture medium. The second culture medium consisted of MS culture medium and the following components added to the MS culture medium: pullulan 1 g / L, 2,4-D 0.5 mg / L, agar 5 g / L, NAA 0.1 mg / L, and sodium phytate 0.1 mg / L.

[0047] (2) adding the callus tissue obtained in step (1) into PBS buffer solution and grinding the solution, centrifuging and filtering the solution to obtain a filtrate;

[0048] (3) The filtrate of step (2) was centrifuged again to obtain a supernatant; an exosome complexing agent (composed of 10 wt% PEG8000 and the remainder water) was added to the supernatant, incubated overnight, and centrifuged to obtain a precipitate, which was freeze-dried at -30°C and a vacuum of 200 Pa for 32 h.

[0049] This embodiment also provides a plant cell exosome, which is prepared using the above preparation method.

[0050] (2) Comparative Example

[0051] Comparative Example 1

[0052] The difference between this comparative example and Example 1 is that, 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: 0.6 mg / L 2,4-D, 7 g / L agar, and 0.2 mg / L NAA; the second culture medium consists of MS culture medium and the following components added to the MS culture medium: 0.6 mg / L 2,4-D, 7 g / L agar, 0.2 mg / L NAA, and 0.4 mg / L sodium phytate. The rest is the same as in Example 1.

[0053] Comparative Example 2

[0054] The difference between this comparative example and Example 1 is that, 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, and 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, and sodium phytate 0.4 mg / L.

[0055] Comparative Example 3

[0056] The difference between this comparative example and Example 1 is that, 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, and 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, and NAA 0.2 mg / L.

[0057] (3) Test examples

[0058] The protein content in the plant cell exosomes prepared in Examples 1-3 and Comparative Examples 1-3 and their effects on the proliferation ability of human skin fibroblasts were detected. The specific detection method is as follows:

[0059] 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. The results are shown in Table 1.

[0060] Relative cell viability: Human skin fibroblasts in logarithmic growth phase were taken and 5×10 3 Cells were seeded onto 96-well plates, with five replicate wells per group. After attachment, the cells were replaced with 100 μL of DMEM medium containing exosomes obtained from Examples 1-3 or Comparative Examples 1-3 (also containing 5% FBS, with an exosome concentration of 10 mg / L in each group). A blank control group was cultured in DMEM medium containing 5% FBS without exosomes. After 48 hours of continued incubation, 10 μL of CCK-8 reagent was added to each well and incubated for 2 hours. Absorbance at 450 nm was measured using a microplate reader. Relative cell viability was calculated, with the blank control group as 100%. The results are shown in Table 1.

[0061] Relative cell viability (%) = OD value of experimental group / OD value of blank group × 100%.

[0062] Table 1

[0063]

[0064] It can be seen from Table 1 that the plant exosomes obtained in Examples 1-3 of the present invention have higher protein content and more obvious effects on the proliferation ability of human skin fibroblasts.

[0065] Compared with Example 1, the protein content in the plant exosomes obtained by omitting pullulan in Comparative Example 1, replacing pullulan with sucrose in Comparative Example 2, and omitting sodium phytate in Comparative Example 3 was reduced, and the degree of influence on the proliferation ability of human skin fibroblasts was reduced. 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.

[0066] Further analysis revealed that pullulan is a natural polysaccharide with adhesive and film-forming properties. It can form a microenvironmental barrier to reduce mechanical damage to callus tissue during culture, maintain cell membrane integrity, thereby reducing intracellular protein leakage and improving the efficiency of protein enrichment in exosomes. Furthermore, pullulan regulates the osmotic pressure of the culture medium, causing mild osmotic stress in plant cells, activating cellular stress signaling pathways, prompting cells to secrete more exosomes to transmit stress signals, and increasing the total protein content of exosomes. In addition, the adhesive properties of pullulan can also enhance the physical connection between callus cells, forming a more stable three-dimensional structure, which is conducive to the directional secretion of exosomes and reduces protein loss. The addition of sodium phytate can scavenge reactive oxygen species accumulated during subculture, reduce oxidative stress damage to cells, maintain the continuous ability of cells to secrete exosomes, and avoid the decline in protein content due to cell aging. Moreover, the phosphate ions released after the decomposition of sodium phytate can participate in cellular energy metabolism and signal transduction, activate pathways related to exosome biosynthesis, and promote the packaging of specific functional proteins. In addition, sodium phytate can inhibit the activity of polyphenol oxidase, reduce protein denaturation caused by oxidation of phenolic substances, and protect the integrity of exosome proteins.

[0067] (IV) Application Examples

[0068] An essence, which is composed of the following raw materials in percentage by weight:

[0069] 4% exosomes, 0.5% p-hydroxyacetophenone, 1% hyaluronic acid, 6% grape seed oil, 0.05 sodium hydroxymethylcellulose, 6% squalane, and the balance is water; the above exosomes are respectively the exosomes prepared in Examples 1-3 or Comparative Examples 1-3.

[0070] Application Example 1

[0071] The safety of the essences obtained by adding plant cell exosomes from Examples 1-3 and Comparative Examples 1-3 was tested in accordance with the human skin occluded patch test method in Part 5 of the "Technical Specifications for Safety of Cosmetics" (2015 edition). The specific testing method is as follows:

[0072] A total of 70 subjects aged 30-50 years were selected and randomly divided into 7 groups, with a male-to-female ratio of 1:1 in each group. Essence and deionized water (blank control group) were taken from each group, and each group was simultaneously subjected to a closed patch test. The number of subjects in each group who developed allergic symptoms such as skin redness or erythema was counted. The results are shown in Table 2.

[0073] Table 2

[0074]

[0075] As can be seen from Table 2, none of the volunteers experienced skin redness or erythema or other allergic phenomena. The above results indicate that the addition of the plant cell exosomes of the present invention and the resulting essence have high safety.

[0076] Application Example 2

[0077] The anti-aging effects of the essences obtained by adding plant cell exosomes from Examples 1-3 and Comparative Examples 1-3 were tested. The specific testing method is as follows:

[0078] A total of 60 volunteers aged 30-50 were randomly divided into six groups, with a male-to-female ratio of 1:1. The volunteers used the essences prepared from plant cell exosomes from Examples 1-3 and Comparative Examples 1-3, once daily, in the morning and evening. Follow-up visits were conducted before use (T0) and 28 days after use (T28), at the same time of day. No other products of the same type were used during the experiment. Before the test, the volunteers sat in a constant temperature and humidity waiting area at 22°C and 50% relative humidity for 30 minutes. Skin elasticity of the stratum corneum on the cheeks of the subjects was measured using an MPA580 skin elasticity meter. The parameter R2 (R2: the ratio of skin rebound without negative pressure to maximum stretch with negative pressure; the closer the ratio is to 1, the better the skin elasticity) was selected as a comparison indicator. The changes in R2 before and after use were measured to evaluate the improvement in skin elasticity in the test area.

[0079] R2 change rate = (R2 parameter value at T28 - R2 parameter value at T0) / R2 parameter value at T0 × 100%. The results are shown in Table 3.

[0080] Table 3

[0081]

[0082] As can be seen from Table 3, the essence obtained by adding the plant cell exosomes of Examples 1-3 has a good anti-aging effect.

[0083] Compared to Example 1, the R2 change rate of the essences obtained by adding plant cell exosomes from Comparative Examples 1-3 was reduced. Analysis of the protein content in plant cell exosomes and their effect on the proliferation of human fibroblasts indicates that the plant exosome preparation method provided by the present invention can significantly increase the activity of exosomes, thereby effectively enhancing their anti-aging effects in skin care products.

[0084] The above embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and replacements made by technicians in this field 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 cell exosomes, characterized in that: The following steps are involved: (1) scratching the surface of Camellia japonica leaves and inoculating them into a first culture medium for induction culture, and then transferring them into a second culture medium for subculture to obtain callus tissue; (2) adding the callus tissue obtained in step (1) into a buffer solution and grinding the same, followed by centrifugation and filtration to obtain a filtrate; (3) The filtrate of step (2) is centrifuged again to obtain a supernatant; an exosome complexing agent is added to the supernatant, incubated overnight, centrifuged, and a precipitate is obtained, which is then freeze-dried to obtain the product; The first culture medium includes MS culture medium and the following components added to the MS culture medium: pullulan, 2,4-D, agar, and NAA; based on the final concentration of the first culture medium, the concentrations of the pullulan, 2,4-D, agar, and NAA in the first culture medium are: pullulan 1-5 g / L, 2,4-D 0.5-1 mg / L, agar 5-8 g / L, and NAA 0.1-0.3 mg / L; The second culture medium includes MS culture medium and the following components added to the MS culture medium: pullulan, 2,4-D, agar, NAA, and sodium phytate; based on the final concentration of the second culture medium, the concentrations of the pullulan, 2,4-D, agar, NAA, and sodium phytate in the second culture 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, and sodium phytate 0.1-0.5 mg / L.

2. The method for preparing plant cell exosomes according to claim 1, characterized in that: The induction culture conditions in step (1) are: dark culture, temperature of 25±2°C, and time of 10-15 days.

3. The method for preparing plant cell exosomes according to claim 1, characterized in that: The conditions for the subculture in step (1) are: dark culture at a temperature of 25±2°C for 14-20 days.

4. The method for preparing plant cell exosomes according to claim 1, characterized in that: The buffer solution in step (2) is PBS buffer solution.

5. The method for preparing plant cell exosomes according to claim 1, characterized in that: The exosome complexing agent in step (3) consists of 10-15wt% PEG8000 and the balance water.

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