Arisaema cummingii exocyst vesicle compound loaded with arbutin, preparation method and application thereof

Abstract

This invention relates to the field of plant exovesicles, specifically to a Bletilla striata exovesicle complex loaded with arbutin, its preparation method, and its application. The preparation method includes the following steps: preparing a supernatant of Bletilla striata exovesicles; preparing an aqueous solution of arbutin; mixing the exovesicle supernatant with the arbutin aqueous solution, performing a cyclic freeze-thaw treatment, mixing thoroughly, allowing it to stand at room temperature for 30-40 minutes, centrifuging, and obtaining the precipitate, which is the complex. The exovesicle complex and its preparation method provided by this invention can reduce the melanin content of A375 cells by inhibiting melanin production-related genes while exerting a weak inhibitory effect on cells. Moreover, the inhibitory effect of the drug-loaded exovesicles is stronger than that of arbutin monomer at the same concentration, providing a certain research basis for the development of low-dose, high-efficiency skin whitening products.

Description

Technical Field

[0001] This invention relates to the field of plant exovesicles, and more specifically, to a Bletilla striata exovesicle complex loaded with arbutin, its preparation method, and its application. Background Technology

[0002] Arbutin, a classic whitening ingredient, has a relatively mature application in whitening cosmetics. Within safe concentration ranges, it exhibits low irritation and high safety, making it suitable for long-term use. However, arbutin also has certain limitations. Its aqueous solution is unstable in acidic environments and is prone to degradation and inactivation. Furthermore, due to its molecular characteristics and skin barrier limitations, it often struggles to effectively penetrate the stratum corneum, thus affecting its full whitening effect. In recent years, plant exovesicles, as an emerging biological delivery system, have attracted widespread attention due to their good biocompatibility, low immunogenicity, and high delivery potential. Studies have shown that plant exovesicles can load monomeric active ingredients, enhancing the efficiency of cellular uptake of these ingredients, and possess good skin permeability, facilitating the crossing of the stratum corneum barrier to reach the target site. Based on these characteristics, plant exovesicles hold promise for more efficient delivery of ingredients such as arbutin, thereby achieving the same or even better whitening effects while reducing the required concentration of active ingredients and minimizing potential side effects from high-dose use. Currently, research on applying plant exovesicles to melanin decomposition is still in its early stages, and its potential for enhancing whitening efficacy, improving stability, and improving skin delivery requires further exploration. Future research is expected to provide new technological pathways and solutions for the development of whitening cosmetics. Summary of the Invention

[0003] The purpose of this invention is to provide a Bletilla striata exovesicle complex loaded with arbutin. The drug-loaded exovesicles can reduce the melanin content of A375 cells by inhibiting melanin production-related genes while exerting a weak inhibitory effect on cells. Moreover, the inhibitory effect of the drug-loaded exovesicles at the same concentration is stronger than that of arbutin monomer.

[0004] Another objective of this invention is to provide a method for preparing a Bletilla striata exovesicle complex loaded with arbutin, wherein arbutin is loaded onto Bletilla striata exovesicles through a freeze-thaw cycle, and the encapsulation rate can reach more than 78%.

[0005] The third objective of this invention is to provide an application of a Bletilla striata exovesicle complex loaded with arbutin in a melanin-decomposing product, which can be used to develop low-dose, high-efficiency skin whitening products.

[0006] The technical problem solved by this invention is achieved by the following technical solution.

[0007] On one hand, embodiments of the present invention provide a method for preparing a Bletilla striata exovesicle complex loaded with arbutin, comprising the following steps: Preparation of supernatant from Bletilla striata exovesicles; Prepare an aqueous solution of arbutin; Mix the supernatant of the outer vesicles with an aqueous solution of arbutin, perform a cyclic freeze-thaw treatment, mix well, let stand at room temperature for 30-40 minutes, centrifuge, and the precipitate obtained is the complex.

[0008] In some embodiments of the present invention, the preparation of the supernatant of Bletilla striata exovesicles includes the following steps: S1: Take fresh Bletilla striata and rinse it clean; S2: Cut the Bletilla striata into pieces, crush it, filter it, and extract the juice to obtain the original Bletilla striata juice; S3: Centrifuge the original juice of Bletilla striata to obtain supernatant A; centrifuge supernatant A to obtain supernatant B; mix supernatant B with extraction reagent A, mix well and let stand at 4℃ for 10 min, centrifuge after standing to obtain supernatant C; mix supernatant C with extraction reagent B, mix well and let stand at 4℃ for 1 h, centrifuge after standing, discard the supernatant and collect the precipitate; S4: Add physiological saline to the precipitate to resuspend the precipitate, centrifuge at 2000 g for 10 min to obtain supernatant D, which is the supernatant of Bletilla striata vesicles.

[0009] In some embodiments of the present invention, the volume ratio of supernatant B to extraction reagent A is 2:1; the volume ratio of supernatant C to extraction reagent B is 3:1. In some embodiments of the present invention, in S3, the centrifugation conditions for Bletilla striata juice are: centrifugation at 10°C and 6500 g for 10 min; The centrifugation conditions for supernatant A were: centrifugation at 10℃ and 10000g for 20 min; The centrifugation conditions after mixing supernatant B with extraction reagent A were: centrifugation at 10℃ and 12000 g for 10 min; The centrifugation conditions after mixing supernatant C with extraction reagent B were: centrifugation at 10℃ and 13500 g for 30 min.

[0010] In some embodiments of the present invention, in the cyclic freezing-thawing process, the freezing temperature is -80°C and the thawing temperature is room temperature.

[0011] In some embodiments of the present invention, the concentration of the arbutin aqueous solution is 0.1-1.6 mg / mL; the concentration of the extravesicle supernatant is 1-2 mg / mL.

[0012] Secondly, embodiments of the present invention provide a Bletilla striata exovesicle complex loaded with arbutin, which is prepared by the above-described preparation method.

[0013] Thirdly, embodiments of the present invention provide the application of a Bletilla striata exovesicle complex loaded with arbutin in the preparation of a melanin-decomposing product. The melanin-decomposing product includes pharmaceutically acceptable excipients or cosmetically acceptable excipients.

[0014] Compared with the prior art, the embodiments of the present invention have at least the following advantages or beneficial effects: The extracellular vesicle complex and its preparation method provided by this invention can reduce the melanin content of A375 cells by inhibiting melanin production-related genes while exerting a weak inhibitory effect on cells. Moreover, the inhibitory effect of the extracellular vesicles at the same concentration is stronger than that of arbutin monomer, providing a certain research basis for the development of low-dose and highly effective skin whitening products. Attached Figure Description

[0015] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 The results of transmission electron microscopy (100 nm) of plant exovesicles. Figure 2 A graph showing the tracking and analysis of nanoparticle size in plant exovesicles; Figure 3 The chromatogram of arbutin; Figure 4 This is the HPLC standard curve for arbutin. Figure 5 The results of transmission electron microscopy (100 nm) of the external vesicle complex. Figure 6 This is a particle tracking analysis diagram of the nanoparticle size of the external vesicle complex; Figure 7 A comparative diagram showing the effect of the extravesicular vesicle complex on melanin in various UVB models of A375 cells; Figure 8 A comparison of melanin content in various UVB models of A375 cells by the effect of the extravesicular complex; Figure 9 A comparative diagram showing the effects of the extravesicular vesicle complex on melanin production-related genes in an A375 cell UVB model: where A is MITF; B is TYR; C is TYRP-1; and D is TYRP-2. Figure 10 The image shows the fluorescence spectrum and fluorescence intensity analysis results of the transdermal absorption assay of the external vesicle complex in mouse skin. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0018] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to specific embodiments.

[0019] On one hand, embodiments of the present invention provide a method for preparing a Bletilla striata exovesicle complex loaded with arbutin, comprising the following steps: Prepare the supernatant of Bletilla striata exovesicles with a concentration of 1 mg / mL.

[0020] Prepare an aqueous solution of arbutin with a concentration of 1 mg / mL; The supernatant of the outer vesicles was mixed with an aqueous solution of arbutin, and subjected to a freeze-thaw cycle four times. The freezing temperature was -80°C, and the thawing temperature was room temperature. After mixing, the mixture was allowed to stand at room temperature for 30-40 minutes, centrifuged, and the precipitate obtained was the complex.

[0021] In some embodiments of the present invention, the preparation of the supernatant of Bletilla striata exovesicles includes the following steps: S1: Take fresh Bletilla striata and rinse it clean; S2: Cut the Bletilla striata into pieces, crush it, filter it, and extract the juice to obtain the original Bletilla striata juice; S3: Centrifuge the original juice of Bletilla striata to obtain supernatant A; centrifuge supernatant A to obtain supernatant B; mix supernatant B with extraction reagent A, mix well, and let stand at 4℃ for 10 min. After standing, centrifuge to obtain supernatant C; mix supernatant C with extraction reagent B, mix well, and let stand at 4℃ for 1 h. After standing, centrifuge, discard the supernatant and collect the precipitate; the centrifugation conditions for the original juice of Bletilla striata are: centrifuge at 10℃ and 6500 g for 10 min; the centrifugation conditions for supernatant A are: centrifuge at 10℃ and 10000 g for 20 min; the centrifugation conditions for supernatant B after mixing with extraction reagent A are: centrifuge at 10℃ and 12000 g for 10 min; the centrifugation conditions for supernatant C after mixing with extraction reagent B are: centrifuge at 10℃ and 13500 g for 30 min.

[0022] S4: Add physiological saline to the precipitate to resuspend the precipitate, centrifuge at 2000 g for 10 min to obtain supernatant D, which is the supernatant of Bletilla striata vesicles.

[0023] The volume ratio of supernatant B to extraction reagent A is 2:1; the volume ratio of supernatant C to extraction reagent B is 3:1.

[0024] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0025] Example 1. Preparation of external vesicles S1: Take 1kg of fresh Bletilla striata, put it in a water tank and rinse it with drinking water, then rinse it with ultrapure water.

[0026] S2: Cut the washed Bletilla striata into pieces, put them into a juicer to mix and grind, filter, and extract the juice to obtain Bletilla striata juice.

[0027] S3: Centrifuge the original juice of Bletilla striata at 10℃ and 6500 g for 10 min to obtain supernatant A; centrifuge supernatant A at 10℃ and 10000 g for 20 min to obtain supernatant B; add extraction reagent A at a volume ratio of supernatant B to extraction reagent A = 2:1, mix well, and let stand at 4℃ for 10 min. After standing, centrifuge at 10℃ and 12000 g for 10 min to obtain supernatant C; add extraction reagent B at a volume ratio of supernatant C to extraction reagent B = 3:1, mix well, and let stand at 4℃ for 1 h. After standing, centrifuge at 10℃ and 13500 g for 30 min, discard the supernatant and collect the precipitate.

[0028] S4: Add sterile physiological saline to resuspend the precipitate (the amount of physiological saline added should be based on the concentration of Bletilla striata exovesicant supernatant of 1 mg / mL), centrifuge at 2000 g for 10 min to obtain supernatant D, which is the Bletilla striata exovesicant supernatant (Exo).

[0029] The extraction reagents A and B mentioned above correspond to Isolation Reagent A and Isolation Reagent B in the Plant Tissue Exosome Extraction Kit (Catalog No.: TW4001) sold by Shenzhen Shifangjie Technology Co., Ltd.

[0030] 2. Characterization and detection of external vesicles The extracted vesicle supernatant was examined by transmission electron microscopy (TEM). 10 μL of the supernatant was added to a copper grid and allowed to precipitate for 1 min; the supernatant was then removed with filter paper. 10 μL of uranium acetate was added to the copper grid and allowed to precipitate for 1 min; the supernatant was then removed with filter paper. The mixture was dried at room temperature for several minutes and then examined and imaged using an electron microscope at 100 kV. The results are as follows: Figure 1 As shown, from Figure 1 As can be seen, a distinct cup-shaped double-layered vesicle structure can be observed under a transmission electron microscope.

[0031] Using a nanoparticle size analyzer (NTA), after adjusting the instrument to its optimal state with ultrapure water, samples were injected for particle size analysis. The particle size distribution and density of the exovesicles were determined. The purity was initially assessed based on the peak distribution; fewer peaks indicate higher purity. The yield of exovesicles was determined by the number of exovesicles per milliliter; a higher number of exovesicles indicates a higher yield. Results are as follows: Figure 2 As shown, the NTA results indicate that the average particle size of the separated extravesicles was 166.3 ± 43.7 nm, and the concentration was 2.18 × 10⁻⁶. 10 The particle size per mL is within the range of 50-200 nm for external vesicles, and the purity and yield are high.

[0032] 3. Preparation of EV-Arbutin (Bletilla striata exovesicle-arbutin complex) Arbutin was dissolved in pure water to prepare a 1 mg / mL standard. An equal volume of Exo (1 mg / mL) was mixed with the Arbutin solution to obtain a mixture. This mixture was rapidly frozen in liquid nitrogen at -80°C, then allowed to dissolve at room temperature, followed by rapid freezing. This process was repeated four times, with each mixture thoroughly mixed and allowed to equilibrate at room temperature for 30 min. The mixture was then centrifuged at 13500g for 40 min, the supernatant was removed, and the precipitate was resuspended in pure water. The concentration of free Arbutin in the precipitate was determined to calculate the drug loading rate. The resulting precipitate was identified as the Bletilla striata exovesicle-arbutin complex (EV-Arbutin).

[0033] 4. Arbutin loading determination The loading of Arbutin was determined by HPLC and the encapsulation efficiency was calculated using the following formula: ; .

[0034] Chromatographic conditions: Supersil ODS2-C18 column (4.6 mm × 250 mm, 5 μm); mobile phase: methanol-water (10:90). Flow rate: 1 mL / min; column temperature: 25 °C; detection wavelength: 282 nm; injection volume: 20 μL.

[0035] Arbutin standards were determined by HPLC, such as... Figure 3 and Figure 4 As shown, where, Figure 3 The chromatogram of arbutin is shown below. Figure 4 This is the standard curve for arbutin; the linear regression equation for the standard curve of arbutin is Y = 9711X - 16.376 (R²). 2=1), and arbutin showed good linearity in the mass concentration range of 0.05-0.8 mg / mL (corresponding to an aqueous arbutin concentration of 0.1-1.6 mg / mL). The encapsulation efficiency of Exo for Arbutin was 78.75% by measuring the change in arbutin concentration in the precipitate after centrifugation, as shown in Table 1.

[0036] Table 1

[0037] 5. Characterization and detection of EV-Arbutin Transmission electron microscopy was performed on the drug-loaded exovesicle complex, and the results are as follows: Figure 5 As shown, a distinct cup-shaped double-layered vesicle structure can be observed under a transmission electron microscope.

[0038] The particle size after drug loading was detected using a nanoparticle tracking analyzer (NTA). Figure 6 As shown, the particle size increased after drug loading, with an average particle size of 174 ± 28.6 nm and a concentration of 3.04 × 10⁻⁶. 10 particle / mL indicates that drug loading has little effect on the morphology of external vesicles.

[0039] 6. Cell Culture and Model Construction 6.1 A375 cell culture A375 cells were cultured in DMEM high-glucose medium containing 10% FBS and 1% penicillin-streptomycin at 37°C in a 5% CO2 incubator.

[0040] 6.2 UVB Model Use 20J / cm 2 Irradiate for 50 seconds, then administer medication according to the group.

[0041] 6.3 Cell Experiment Grouping: The cell proliferation experiment was divided into four groups: CK group, Arbutin group, EV-Arbutin group, and EV group. The treatments for each group were as follows: CK group: cells that grow normally and are not treated.

[0042] Arbutin group: Arbutin was added and diluted with cell culture medium to a final concentration of 0.2 mg / mL.

[0043] EV-Arbutin group: EV-Arbutin was added and diluted with cell culture medium to a final concentration of 0.2 mg / mL.

[0044] EV group: Add EV (Bletilla striata exovesicle supernatant), dilute with cell culture medium to a final concentration of 0.2 mg / mL.

[0045] The melanin content and qPCR experiments were divided into five groups: CK group, UVB group, UVB+Arbutin group, UVB+EV-Arbutin group, and UVB+EV group. The treatments for each group were as follows: CK group: cells that grow normally and are not treated.

[0046] UVB group: using 20J / cm 2 A UVB model was established after irradiation for 50 seconds.

[0047] UVB + Arbutin group: using 20J / cm 2 After establishing a UVB model by irradiation for 50 seconds, arbutin was added and diluted with cell culture medium to a final concentration of 0.2 mg / mL.

[0048] UVB+EV-Arbutin group: using 20J / cm 2 After establishing a UVB model by irradiation for 50 seconds, EV-Arbutin was added and diluted with cell culture medium to a final concentration of 0.2 mg / mL.

[0049] UVB+EV group: using 20J / cm 2 After establishing a UVB model by irradiation for 50 seconds, EV-Arbutin was added and diluted with cell culture medium to a final concentration of 0.2 mg / mL.

[0050] 6.4 Data Processing Statistical analysis was performed using Prism 6.0 (GraphPad) software. Two-way ANOVA was used for comparisons among multiple groups. Results are expressed as mean ± standard deviation. ).

[0051] 7. Effects of EV-Arbutin on Cell Proliferation The effect of EV-Arbutin on cell proliferation was evaluated using the CCK8 assay. Logarithmic phase cells were collected and seeded into 96-well plates at a rate of 5000 cells / well. After cell adhesion, the corresponding reagents were added according to the grouping and the cells were incubated in an incubator for 24 h. Then, 10 μL of CCK8 solution was added and the cells were incubated at 37°C for 2 h. The absorbance at 450 nm was measured using a microplate reader, and the cell viability was calculated.

[0052] The results are as follows Figure 7 As shown, compared with the CK group, Figure 7 In the diagram, "*" represents "P<0.05" and "**" represents "P<0.01"; compared with the UVB model group, Figure 7 In this context, "#" represents "P<0.05", and "##" represents "P<0.01".

[0053] from Figure 7 The results showed that, compared with the control group (CK group), arbutin and external vesicles had almost no significant effect on cell viability, while the drug-loaded external vesicle complex had a weak inhibitory effect.

[0054] 8. Effects of EV-Arbutin on cellular melanin content Log-phase cells were collected and seeded into 96-well plates at a rate of 5000 cells / well. After cell adhesion, cells were treated according to their grouping and incubated for 24 hours. The cells were washed twice with PBS, digested with trypsin, and then the digestion was stopped by adding complete culture medium. The cells were pipetted into a cell suspension, centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. 100 μL of 1M NaOH was added, and the cells were incubated at 37°C for 1 hour. Then, 400 μL of deionized water was added and mixed thoroughly. 100 μL of the mixture was then added to each well of the 96-well plate, and the OD value of each well was measured at 490 nm. Based on the relative ratio, the changes in melanin content were further analyzed and calculated.

[0055] The results are as follows Figure 8 As shown, compared with the CK group, Figure 8 In the diagram, "*" represents "P<0.05" and "**" represents "P<0.01"; compared with the UVB model group, Figure 8 In the text, "#" represents "P<0.05", and "##" represents "P<0.01".

[0056] from Figure 8 The results show that UVB treatment alone significantly increased the melanin content in cells, while treatment with external vesicles, arbutin, and drug-loaded external vesicle complexes significantly inhibited the high melanin expression induced by UVB. Among them, the drug-loaded external vesicle complex had the strongest inhibitory effect, which was basically the same as the expression level in the CK group.

[0057] 9. Effects of EV-Arbutin on the melanin production pathway Collect cells in the logarithmic growth phase and arrange the cells at a ratio of 4 × 10⁻⁶. 5 Cells were seeded into 12-well plates and placed in a cell culture incubator. After cell adhesion, cells were treated according to their assigned groups and incubated for another 24 hours. Cells were collected, and the expression of MITF, TYP, TYRP-1, and TYRP-2 was detected according to the instructions of the RNA extraction kit, reverse transcription kit, and qPCR assay. Primer sequences are shown in Table 2.

[0058] Table 2

[0059] The results are as follows Figure 9 As shown, compared with the CK group, Figure 9In the diagram, "*" represents "P<0.05" and "**" represents "P<0.01"; compared with the UVB model group, Figure 9 In the text, "#" represents "P<0.05", and "##" represents "P<0.01".

[0060] from Figure 9 The results show that UVB treatment significantly increased the expression of MITF, TYR, TYRP-1, and TYRP-2 genes. After UVB treatment, treatment with arbutin, exovesicles, and drug-loaded exovesicle complex significantly reduced the expression of these genes. Among them, the drug-loaded exovesicle complex showed the strongest inhibitory effect, indicating that the drug-loaded exovesicle complex can inhibit melanin production in A375 cells by inhibiting the expression of melanin-producing genes.

[0061] 10. Transdermal absorption of external vesicles in mice Experimental materials: 6-week-old Balb / C mice; Experimental groups: ①CK: DIO staining working solution ②EV: DIO-labeled exovesicles ③EV-Arbutin: DIO-labeled drug-loaded exovesicle complex; Labeling exosomes and drug-loaded exosomes using the DIO kit: The DIO green fluorescent probe was diluted with staining buffer at a ratio of 1:250 to prepare the DIO staining working solution. Following a staining working solution:exosome (exosome or drug-loaded exosome) ratio of 10 μL:1 μg, the solution was incubated at 37°C in the dark for 30 min. The exosomes were then labeled, centrifuged at 13500g for 30 min, and resuspended in physiological saline. After removing hair from the mouse abdomen, the skin was cleaned with physiological saline using a sterile cotton swab. After complete absorption of the saline, 200 μL was applied according to the group. After 30 min, the abdominal skin was placed on a slide, and the fluorescence intensity was observed and recorded under a fluorescence microscope.

[0062] The results are as follows Figure 10 As shown, compared with the CK group, Figure 10 In the text, "*" represents "P<0.05" and "**" represents "P<0.01".

[0063] from Figure 10 The results show that, compared with the CK group, the fluorescence of the external vesicles (EV group) and the drug-loaded external vesicle complex (EV-Arbutin group) is stronger, indicating that both the external vesicles and the drug-loaded external vesicle complex can significantly penetrate the abdominal skin of mice, and the transdermal effect of the drug-loaded external vesicle complex is slightly stronger than that of the external vesicles.

[0064] The embodiments described above are some, but not all, embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A method for preparing a Bletilla striata exovesicle complex loaded with arbutin, characterized in that, Includes the following steps: Preparation of supernatant from Bletilla striata exovesicles; Prepare an aqueous solution of arbutin; Mix the supernatant of the outer vesicles with an aqueous solution of arbutin, perform a cyclic freeze-thaw treatment, mix well, let stand at room temperature for 30-40 minutes, centrifuge, and the precipitate obtained is the complex.

2. The method for preparing the arbutin-loaded Bletilla striata exovesicle complex according to claim 1, characterized in that, The preparation of the supernatant from the exovesicles of Bletilla striata includes the following steps: S1: Take fresh Bletilla striata and rinse it clean; S2: Cut the Bletilla striata into pieces, crush it, filter it, and extract the juice to obtain the original Bletilla striata juice; S3: Centrifuge the original juice of Bletilla striata to obtain supernatant A; centrifuge supernatant A to obtain supernatant B; mix supernatant B with extraction reagent A, mix well and let stand at 4℃ for 10 min, centrifuge after standing to obtain supernatant C; mix supernatant C with extraction reagent B, mix well and let stand at 4℃ for 1 h, centrifuge after standing, discard the supernatant and collect the precipitate; S4: Add physiological saline to the precipitate to resuspend the precipitate, centrifuge at 2000 g for 10 min to obtain supernatant D, which is the supernatant of Bletilla striata vesicles.

3. The method for preparing the arbutin-loaded Bletilla striata exovesicle complex according to claim 2, characterized in that, The volume ratio of supernatant B to extraction reagent A is 2:1; the volume ratio of supernatant C to extraction reagent B is 3:

1.

4. The method for preparing the arbutin-loaded Bletilla striata exovesicle complex according to claim 1, characterized in that, In S3, the centrifugation conditions for Bletilla striata juice were: centrifugation at 10℃ and 6500 g for 10 min; The centrifugation conditions for supernatant A were: centrifugation at 10℃ and 10000g for 20 min; The centrifugation conditions after mixing supernatant B with extraction reagent A were: centrifugation at 10℃ and 12000 g for 10 min; The centrifugation conditions after mixing supernatant C with extraction reagent B were: centrifugation at 10℃ and 13500 g for 30 min.

5. The method for preparing the arbutin-loaded Bletilla striata exovesicle complex according to claim 1, characterized in that, In the cyclic freezing-thawing process, the freezing temperature is -80℃ and the thawing temperature is room temperature.

6. The method for preparing the arbutin-loaded Bletilla striata exovesicle complex according to claim 1, characterized in that, The concentration of arbutin in aqueous solution is 0.1-1.6 mg / mL; the concentration of extravesicle supernatant is 1-2 mg / mL.

7. A Bletilla striata exovesicle complex loaded with arbutin, characterized in that, It is prepared by the preparation method according to any one of claims 1-6.

8. Application of a Bletilla striata exovesicle complex loaded with arbutin in the preparation of melanin decomposition products.

9. The application of the arbutin-loaded Bletilla striata exovesicle complex according to claim 8 in the preparation of melanin decomposition products, characterized in that, The melanin decomposition products include pharmaceutically acceptable excipients or cosmetically acceptable excipients.

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