Application of tea exosome in preparation of medicine for improving skin pigmentation

Abstract

The invention relates to application of a tea exosome in preparation of a medicine for improving skin pigmentation, the tea exosome is creatively extracted and purified, and the tea exosome can effectively inhibit proliferation of melanoma cells and inhibit expression of melanin generation related protein by scavenging free radicals and inhibiting tyrosinase activity, so that the effect of improving skin pigmentation is achieved. The intracellular melanin removal is promoted. In a mouse in-vivo experiment, after the tea exosome is applied, it is observed that the skin epidermis thickness of a mouse is reduced, the dermis thickness is increased, collagen arrangement is tighter and more ordered, the content is increased, and meanwhile generation of inflammatory factors is reduced. The tea exosome can significantly increase the content of a melanin autophagy key factor LC3B and reduce the content of P62 to promote melanin autophagy, and has good percutaneous infiltration capacity, thereby playing a role in improving skin pigmentation.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to the application of tea exosomes in preparing a medicine for improving skin pigmentation. Background Art

[0002] Skin pigmentation is a common skin problem, usually caused by excessive melanin production, and is a sign of a variety of skin pathological conditions, including melasma, freckles, and post-inflammatory pigmentation. These symptoms not only affect the patient's appearance, but may also have a negative impact on the patient's mental health. The pigmentation process is regulated by a variety of internal and external factors, including ultraviolet radiation, inflammatory response, hormone levels, genetic factors, and dietary habits. In modern society, with the increasing demand for skin health and beauty, finding effective and safe natural substances for whitening and anti-pigmentation has become an important research direction in dermatology and cosmetic medicine.

[0003] Tea (Camellia sinensis) has been widely studied and applied in the field of skin care due to its rich bioactive ingredients, such as polyphenols, flavonoids and catechins. The antioxidant and anti-inflammatory properties of tea extracts have been confirmed in numerous studies. These active ingredients can reduce the production of melanin by scavenging free radicals, inhibiting the release of inflammatory factors and inhibiting the activity of tyrosinase. However, tea extracts face the problem of limited bioavailability and skin penetration in practical applications, which restricts their full effect.

[0004] In recent years, exosomes, as a new type of nanoscale bioactive carrier, have attracted widespread attention due to their unique structure and function. Exosomes have a natural cell membrane structure and can carry and deliver a variety of bioactive substances such as proteins, nucleic acids, lipids, etc. They have efficient intracellular delivery ability and good biocompatibility. Based on their potential application prospects in drug delivery, gene therapy and tissue repair, exosomes are considered to be a natural medicine and nanocarrier with broad prospects. Tea-derived exosomes (TLNVs), as a new type of natural nanocarrier, combine the bioactive ingredients of tea and the delivery advantages of exosomes, and may have significant advantages in skin whitening and anti-pigmentation. Preliminary studies have shown that tea exosomes can effectively deliver active ingredients in tea and improve their bioavailability and stability, but their specific effects in reducing melanin production and improving skin pigmentation have not been systematically studied and verified. Summary of the invention

[0005] In view of the deficiencies of the prior art, the object of the present invention is to provide an application of tea exosomes in the preparation of a drug for improving skin pigmentation.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides the use of tea exosomes in the preparation of a medicament for improving skin pigmentation.

[0008] Tea exosomes can effectively inhibit the proliferation of B16-F10 mouse melanoma cells by scavenging free radicals and inhibiting the activity of tyrosinase, reduce the expression of proteins related to melanin production, and promote the removal of intracellular melanin. After using tea exosomes, it was observed that the epidermal thickness of the mouse skin decreased, the dermal thickness increased, the collagen fibers were arranged more tightly and orderly, and the content increased, while the production of inflammatory factors TNF-α and IL-1β decreased. In addition, tea exosomes significantly increased the level of LC3B, a key factor in autophagy, and reduced the content of P62, and had good transdermal absorption ability, thereby playing a role in improving skin pigmentation.

[0009] Preferably, the tea exosomes are prepared by a method comprising the following steps:

[0010] (1) mixing tea homogenate with enzyme, performing enzymolysis, centrifuging and collecting supernatant;

[0011] (2) After ultracentrifugation of the supernatant, the precipitate is collected and resuspended to obtain the product.

[0012] Preferably, the centrifugation and collection of the supernatant in step (1) are performed three times in total, the speed of the first centrifugation is 800-1200g (e.g., 800g, 850g, 900g, 950g, 1000g, 1050g, 1100g, 1150g, 1200g, etc.), and the time is 20-30min (e.g., 20min, 22min, 24min, 26min, 28min, 30min, etc.); the speed of the second centrifugation is 4000-6000g (e.g., 4000g, 4200g, 4400g, 4600g, 4800g, 5000g, 5200g , 5400g, 5600g, 5800g, 6000g, etc.), and the time is 30-40min (30min, 32min, 34min, 36min, 38min, 40min, etc.); the speed of the third centrifugation is 8000-12000g (for example, 8000g, 8500g, 9000g, 9500g, 10000g, 10500g, 11000g, 11500g, 12000g, etc.), and the time is 60-70min (for example, 60min, 62min, 64min, 66min, 68min, 70min, etc.).

[0013] Other specific point values ​​within the above numerical range can be selected and will not be described in detail here.

[0014] Preferably, the speed of ultracentrifugation is 90000-110000 g, and the time is 60-70 min.

[0015] The speed can be selected as 90000g, 92000g, 95000g, 98000g, 100000g, 102000g, 105000g, 108000g, 110000g, etc. The time is 60min, 62min, 64min, 66min, 68min, 70min, etc. Other specific point values ​​within the above numerical range can be selected, so they will not be elaborated here.

[0016] Preferably, the enzymes include cellulase and pectinase.

[0017] Preferably, the mass of cellulase is 1-1.5% of the mass of tea homogenate, for example 1%, 1.05%, 1.1%, 1.15%, 1.2%, 1.25%, 1.3%, 1.35%, 1.4%, 1.45%, 1.5%, etc. Other specific points within the above numerical range can be selected and will not be described here one by one.

[0018] Preferably, the mass of pectinase is 0.5-0.8% of the mass of tea homogenate, for example 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, etc. Other specific points within the above numerical range can be selected and will not be described here one by one.

[0019] Preferably, the enzymatic hydrolysis temperature is 20-30°C and the time is 10-12 hours.

[0020] The temperature can be selected as 20℃, 22℃, 24℃, 26℃, 28℃, 30℃, etc., and the time can be selected as 10h, 10.2h, 10.4h, 10.6h, 10.8h, 11h, 11.2h, 11.4h, 11.6h, 11.8h, 12h, etc. Other specific point values ​​within the above numerical range can be selected, so they will not be elaborated here.

[0021] Preferably, the ultracentrifugation further comprises filtering the supernatant using a 0.22 μm filter.

[0022] Preferably, the dosage form of the drug includes any one of tablets, capsules, pills, solutions, aerosols, sprays, ointments or films.

[0023] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0024] In a second aspect, the present invention provides the use of tea exosomes in the preparation of cell proliferation inhibitors.

[0025] According to the research results of the present invention, tea exosomes can inhibit cell proliferation at the cellular level (in vitro level), that is, tea exosomes can be made into a simple experimental preparation for exploring the physiological processes of cells. The cell proliferation inhibitor claimed by the present invention is not used to eliminate the cause or lesion, that is, it is an application in the preparation of cell proliferation inhibitors for non-therapeutic purposes.

[0026] In a third aspect, the present invention provides the use of tea exosomes in the preparation of a cell melanin scavenger.

[0027] According to the research results of the present invention, tea exosomes can remove melanin in cells at the cellular level (in vitro level), that is, tea exosomes can be made into a simple experimental preparation for exploring the metabolic process of cells. The cell melanin scavenger claimed by the present invention is not used to eliminate the cause or lesion, that is, it is an application in the preparation of a cell melanin scavenger for non-therapeutic purposes.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] Tea exosomes can effectively inhibit the proliferation of B16-F10 mouse melanoma cells by scavenging free radicals and inhibiting the activity of tyrosinase, reduce the expression of proteins related to melanin production, and promote the removal of intracellular melanin. After using tea exosomes, it was observed that the epidermal thickness of the mouse skin decreased, the dermal thickness increased, the collagen fibers were arranged more tightly and orderly, and the content increased, while the production of inflammatory factors TNF-α and IL-1β decreased. In addition, tea exosomes significantly increased the level of LC3B, a key factor in autophagy, and reduced the content of P62, and had good transdermal absorption ability, thereby playing a role in improving skin pigmentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1A This is a flow chart of the preparation method of tea exosomes.

[0031] Figure 1B This is the observation result of tea exosomes under transmission electron microscope.

[0032] Figure 1C This is the particle size distribution diagram of tea exosomes.

[0033] Figure 1D This is the zeta potential distribution diagram of tea exosomes.

[0034] Figure 1E This is a composition analysis chart of secondary metabolites of tea exosomes.

[0035] Figure 1F It is a bar graph of miRNA GO analysis.

[0036] Figure 1G It is a bar graph of miRNA KEGG analysis.

[0037] Figure 2A It is the statistical result of PITO clearance rate.

[0038] Figure 2B It is the statistical result of ABTS clearance rate.

[0039] Figure 3 It is the result of tyrosinase activity test.

[0040] Figure 4A These are the results of transmission electron microscopy observation of the control group.

[0041] Figure 4B These are the results of transmission electron microscopy observations of the tea extract group.

[0042] Figure 4C This is the result of transmission electron microscopy observation of tea exosomes.

[0043] Figure 4D It is the statistical result of cell proliferation rate.

[0044] Figure 5A These are the results of transmission electron microscopy observation of mouse melanoma cells.

[0045] Figure 5B It is the statistical result of intracellular melanin content in mouse melanoma cells.

[0046] Fig. 6A It is the statistical result of the relative expression of MITF.

[0047] Figure 6B It is the statistical result of the relative expression of TYR.

[0048] Figure 6C It is the statistical result of the relative expression of TRP-1.

[0049] Fig.6D It is the statistical result of the relative expression of TRP-2.

[0050] Fig. 6E This is the result of the effect of tea exosomes on the expression of melanin production-related proteins.

[0051] Fig. 7A These are the results of melanin, HE and Masson staining observations after tea exosomes intervened in pigmented mice.

[0052] Figure 7B It is the statistical result of the percentage of melanin in the epidermis.

[0053] Figure 7C It is the statistical result of epidermal thickness.

[0054] Fig.7D It is the statistical result of dermis thickness.

[0055] Fig. 7E It is the statistical result of collagen volume fraction.

[0056] Fig. 8A These are the results of immunofluorescence analysis of the effects of tea exosomes on the production of TNF-α and IL-1β.

[0057] Figure 8B It is the statistical result of relative fluorescence intensity of TNF-α.

[0058] Figure 8C It is the statistical result of relative fluorescence intensity of IL-1β.

[0059] Fig. 9A These are the results of immunofluorescence analysis of the effects of tea exosomes on LC3B and P62.

[0060] Fig. 9B It is the relative area coverage statistics of LC3B.

[0061] Fig. 9C It is the relative area coverage statistics of P62.

[0062] Fig. 10A This is the result of heat map analysis of differential proteins before and after drug administration.

[0063] Fig. 10B This is the result of the volcano plot analysis of the differential proteins before and after drug administration.

[0064] Fig. 10C This is the result of GO enrichment analysis of differentially expressed proteins.

[0065] Fig. 10D This is the result of KEGG enrichment analysis of differentially expressed proteins.

[0066] Fig. 10E It is the result of protein interaction analysis of up-regulated differentially expressed molecules.

[0067] Fig.10F It is the result of down-regulated differential molecular protein interaction analysis.

[0068] Figure 10G This is the result of MITF fluorescence quantitative expression analysis.

[0069] Fig. 10H This is the result of TYR fluorescence quantitative expression analysis.

[0070] Fig.11 This is the result of transdermal permeability analysis of tea exosomes. DETAILED DESCRIPTION

[0071] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0072] The sources of the functional ingredients contained in the products involved in the following embodiments and comparative examples are as follows (only the functional ingredients are reflected, and the necessary auxiliary ingredients contained in other commercially available raw materials are not repeated):

[0073] Tea extract (TET) was purchased from Nanjing Puyi Biotechnology Co., Ltd. and is a cosmetic grade raw material.

[0074] Example 1

[0075] Isolation, characterization and determination of tea exosomes

[0076] Isolation of tea exosomes: Figure 1A As shown, the specific methods include:

[0077] Fresh tea leaves were picked and homogenized using a wall breaking machine. After adding 1% cellulase and 0.5% pectinase for overnight digestion for 12 hours, the mixture was centrifuged at 1000g for 20 minutes to remove protoplasts, the supernatant was centrifuged at 5000g for 40 minutes to remove dead cells, the supernatant was centrifuged at 10000g for 1 hour to remove cell debris, and the resulting supernatant was filtered through a 0.22μm filter membrane. After filtration, the mixture was separated and purified by centrifugation at 100000g for 1 hour in an ultra-high speed centrifuge, and then resuspended in PBS to obtain tea exosomes.

[0078] 10 μL of tea exosome sample was added to the copper grid for precipitation, and the floating liquid was removed by filter paper; 10 μL of uranyl acetate was added for negative staining, and the floating liquid was removed by filter paper; after natural drying at room temperature, the sample was imaged with high resolution using a Hitachi cryogenic transmission electron microscope (model HT-7700). The concentration of the number of particles in the exosomes was as follows: Figure 1C The zeta potential results are shown in Figure 1D shown.

[0079] The composition of secondary metabolites of tea exosomes was analyzed. Figure 1E As shown in Figure 2, the main secondary metabolites contained were flavonoids 47.17%, phenolic acids 12.42%, tannins 2.99%, alkaloids 8.81%, lignans and coumarins 8.18%, terpenes 6.92% and quinones 1.10%. GO and KEGG enrichment analysis of tea exosome miRNA was performed by miRNA sequencing, and the results are shown in Figure 2. Figure 1F and Figure 1G As shown, the results showed that miRNAs were mainly enriched in the glucosinolate biosynthesis pathway.

[0080] Example 2

[0081] Test of scavenging ability of PTIO oxygen free radical and ABTS nitrogen free radical

[0082] PTIO free radical scavenging activity test method: prepare a PTIO working solution with a concentration of 0.2 mg / mL, add 40 μL of tea exosomes (TLNVS) or tea extract (TET) with concentrations of 10 μg / mL, 50 μg / mL and 100 μg / mL to a 96-well plate, then add 160 μL of PTIO working solution to mix, place the sample in a constant temperature incubator at 37°C for 30 minutes, and measure the sample absorbance value (A1) at a wavelength of 557 nm. The absorbance after mixing the PTIO working solution with PBS is set as (A0), and the absorbance after mixing the tea exosome or tea extract solution with PBS is set as (A2). Three replicate wells are measured for each concentration, and the experiment is repeated three times. Arbutin is used as a positive control. The PITO clearance rate results are as follows: Figure 2A As shown by Figure 2A It can be seen that tea exosomes have the best PITO clearing effect, which is better than arbutin and tea extract.

[0083]

[0084] ABTS free radical scavenging activity test method: Prepare ABTS solution with a concentration of 3.84 mg / mL and potassium persulfate solution with a concentration of 0.67 mg / mL, and mix them in equal volumes to obtain ABTS working solution. Before use, control the absorbance of the ABTS working solution between 0.2 and 0.7 with distilled water. Add 50 μL of tea exosomes (TLNVS) or tea extract (TET) with a concentration of 10 μg / mL, 50 μg / mL and 100 μg / mL to a 96-well plate, and then add 150 μL of ABTS working solution to mix with it. After reacting at room temperature for 10 minutes, the sample absorbance value (B1) was measured at a wavelength of 737 nm. The absorbance of the ABTS working solution after mixing with PBS was set as (B0), and the absorbance of the tea exosome or tea extract solution after mixing with PBS was set as (B2). For each concentration, three replicate wells were set up to ensure the accuracy of the results, and the overall experimental process was repeated three times. In the experiment, arbutin was used as a positive control, and the ABTS clearance rate results were as follows: Figure 2B As shown by Figure 2B It can be seen that tea exosomes have the best ABTS clearance effect, which is better than arbutin and tea extract.

[0085]

[0086] Example 3

[0087] Inhibitory activity against tyrosinase

[0088] The tyrosinase inhibitory activity of tea exosomes was determined using the BOXBIO tyrosinase activity detection kit. Preheat the microplate reader for 30 minutes, adjust the wavelength to 475nm, and set it to zero with distilled water. Add 20μL of tea exosome solution and 180μL of reagent 1 (dissolved from the extract) in the kit to the 96-well plate in sequence, mix thoroughly and start timing immediately, measure the absorbance at 475nm at 10s (total time), recorded as A1; after an accurate reaction of 180s, measure the absorbance at 475 at 190s (total time), recorded as A2, ΔA=A2-A1, the test results are as follows Figure 3 shown.

[0089] Tyrosinase activity (U / mL) = 180.18 × ΔA

[0090] The tyrosinase activity measured without adding any substance is recorded as B1, and the tyrosinase activity measured after adding the sample to be tested is recorded as B2. The relative enzyme activity (%) is calculated by the following formula:

[0091]

[0092] Example 4

[0093] Effects on the proliferation of B16-F10 mouse melanoma cells

[0094] Using BeyoClick TM The EdU-488 cell proliferation detection kit was used to detect the proliferation of B16-F10 cells. B16-F10 mouse melanoma cells were treated with tea exosomes (TLNVS) or tea extracts (TET) at concentrations of 10μg / mL, 50μg / mL and 100μg / mL, respectively, and labeled with EDU working solution after 48 hours of culture. After labeling, 4% paraformaldehyde was added and fixed at room temperature for 15 minutes, followed by incubation at room temperature for 15 minutes with PBS containing 0.3% TritonX-100. Finally, the Click reaction solution was added and incubated at room temperature in the dark for 30 minutes, and Hoechst 33342 was incubated at room temperature for 10 minutes. The collected images were observed under a fluorescence microscope. The results of the control group are shown in the figure. Figure 4A The results for tea extracts are shown in Figure 4B As shown, the results of tea exosomes are as follows Figure 4C The experiment was repeated three times with tea extract and 100 μg / mL arbutin as controls. The statistical results are shown in Figure 4D As shown in Figure 4, at the same concentration, tea exosomes have a better inhibitory effect on melanoma cell proliferation.

[0095] Example 5

[0096] Determination of the ability of tea exosomes to clear intracellular melanin in B16-F10 mouse melanoma cells

[0097] The experiment used 100 μg / mL arbutin as a positive control and PBS as a negative control. B16-F10 mouse melanoma cells were treated with tea exosomes and tea extracts and incubated for 48 h, and then the cells were fixed by incubation with a mixed solution of 200 mM cacodylate buffer containing 8% glutaraldehyde and 20% paraformaldehyde. After dehydration with ethanol, sections were prepared using a Leica EMUC7 microtome (Leica) and collected on 200-mesh copper grids. The sections were stained with 1% uranyl acetate and lead citrate, and images were obtained using a JEOL JEM 1010 transmission electron microscope (JEOL) at 80 kV. The results are shown in Figure 2. Figure 5A As shown, the control groups in the figure are, from left to right, the untreated group, the arbutin positive control group, and the PBS negative control group.

[0098] The degree of melanin removal of B16-F10 mouse melanoma cells by tea exosomes was determined. After B16-F10 mouse melanoma cells were treated with tea exosomes, tea extracts and arbutin for 48 hours, the 12-well plate cells were washed with PBS, the cells were incubated and separated with 2.5% trypsin, and the cell precipitates were collected. The melanin 99% standard was diluted to 625, 312.5, 156.25, 78.125, 39.063, 19.531 and 9.766 μg / mL, and diluted into standards of different concentrations. The samples and standards were added to 250 μL of 1% DMSO NaOH solution and lysed in an 80°C water bath for 1 hour. The cell lysate was transferred to a 96-well plate, and the OD value at 405 nm was measured by an ELISA reader. Melanin content was determined using a melanin standard curve constructed from 0 to 100 μg / mL synthetic melanin solutions dissolved in NaOH. Figure 5B shown.

[0099] Example 6

[0100] This example determines the effect of tea exosomes on the expression of melanin production-related proteins.

[0101] B16-F10 mouse melanoma cells were treated with tea extract and tea exosomes at concentrations of 10 μg / mL, 50 μg / mL, and 100 μg / mL, respectively. The cells were then collected and proteins were extracted with lysis buffer, and the protein concentration was determined by the BCA method. The proteins were separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. The membrane was blocked with skimmed milk powder and incubated with specific primary and secondary antibodies in sequence. Finally, the target protein was detected using an enhanced chemiluminescent substrate, and the results are shown in Figure 2. Fig. 6EAs shown in the figure, image analysis and statistical tests were used to evaluate the effects of tea exosomes on the expression of melanin production-related proteins MITF, TYR, TRP-1, and TRP-2. The results are shown in Fig. 6A -D, it can be seen from the figure that the tea exosomes of the present invention can significantly reduce the expression of melanin production-related proteins, and the effect of tea exosomes is better than that of tea extracts.

[0102] Example 7

[0103] This example determines the ability of tea exosomes to improve pigmentation in vivo.

[0104] After C57BL / 6 mice were adapted for one week, a small area of ​​hair about 2 cm × 2 cm was shaved off the back of the mice to ensure skin exposure. The mice were irradiated with UVB, with an initial radiation dose of 0.5 J / cm 2 , increase by 0.1J / cm per week 2 , until it reaches 1J / cm 2 . Four times a week for 5-10 weeks to establish pigmentation model mice.

[0105] The pigmentation model mice were randomly divided into 5 groups: blank control group, PBS group (negative control), arbutin group (positive control), tea extract group and tea exosome group. 100 μL of the corresponding agent with a concentration of 100 μg / mL was applied topically every day for 2 weeks.

[0106] After the intervention, skin tissue samples were taken for melanin staining, HE staining, and Masson staining. The results were as follows: Fig. 7A As shown in Figure 2, image analysis software (such as ImageJ) was used to quantitatively analyze the melanin coverage, epidermal thickness, dermal thickness and collagen volume fraction, and the differences between the groups were compared. The results are shown in Figure 2 Figure 7B -E as shown.

[0107] Immunofluorescence analysis was performed on inflammatory factors TNF-α and IL-1 and melanin autophagy marker proteins LC3B and P62. The fluorescence results of inflammatory factors TNF-α and IL-1 are shown in Fig. 8A The statistical results are shown in Figure 8B , Figure 8C The fluorescence results of LC3B and P62 are shown in Fig. 9A The statistical results are shown in Fig. 9B , Fig. 9C shown.

[0108] Proteomics was used to perform GO and KEGG enrichment analysis and key protein fluorescence quantitative expression analysis on the differentially expressed proteins in mouse skin before and after tea exosome administration to evaluate the improvement effect of tea exosomes on pigmentation in vivo. The results are shown in Figure 10.

[0109] Example 8

[0110] Transdermal penetration ability of tea exosomes

[0111] The tea exosomes were labeled with DiI fluorescent dye to observe their transdermal distribution in the skin. The labeled exosome solution was evenly applied to the back skin area of ​​the mouse. After 2 hours, the skin samples of the treated area were taken and fixed immediately. The skin samples were imaged using a laser confocal microscope, and the transdermal depth and distribution of the exosomes in the skin tissue were evaluated by detecting the DiI fluorescent signal. The results are shown in Figure 2. Fig.11 shown.

[0112] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement modes and shall be included in the protection scope of the present invention.

[0113] The applicant declares that the present invention illustrates the application of the tea exosomes of the present invention in the preparation of a drug for improving skin pigmentation through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0114] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0115] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. Application of tea exosomes in the preparation of drugs for improving skin pigmentation.

2. The use according to claim 1, characterized in that: The tea exosomes are prepared by a method comprising the following steps: (1) mixing tea homogenate with enzyme, performing enzymolysis, centrifuging and collecting supernatant; (2) After ultracentrifugation of the supernatant, the precipitate is collected and resuspended to obtain the product.

3. The use according to claim 2, characterized in that: The centrifugation and collection of the supernatant in step (1) are performed three times in total, the first centrifugation is performed at a speed of 800-1200 g for 20-30 min; the second centrifugation is performed at a speed of 4000-6000 g for 30-40 min; and the third centrifugation is performed at a speed of 8000-12000 g for 60-70 min.

4. The use according to claim 2 or 3, characterized in that: The speed of ultracentrifugation is 90000-110000g and the time is 60-70min.

5. The use according to any one of claims 2 to 4, characterized in that: The enzymes include cellulase and pectinase; Preferably, the mass of the cellulase is 1-1.5% of the mass of the tea homogenate; Preferably, the mass of pectinase is 0.5-0.8% of the mass of tea homogenate.

6. The use according to any one of claims 2 to 5, characterized in that: The temperature of enzymatic hydrolysis is 20-30°C and the time is 10-12h; Preferably, the ultracentrifugation further comprises filtering the supernatant using a 0.22 μm filter.

7. The use according to any one of claims 1 to 6, characterized in that: The dosage form of the drug includes any one of tablets, capsules, pills, solutions, aerosols, sprays, ointments or films.

8. The use according to any one of claims 1 to 7, characterized in that: The drug also includes pharmaceutically acceptable excipients.

9. Application of tea exosomes in the preparation of cell proliferation inhibitors.

10. Application of tea exosomes in the preparation of cellular melanin scavengers.

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