A plant exosome composition and its preparation method and application

Abstract

The present invention relates to a plant exosome composition, the composition comprising: two or more of: platen Spirulina exosomes, olive leaf exosomes, and white peony exosomes. The plant exosome composition of the present invention is mainly composed of platen Spirulina exosomes, olive leaf exosomes, white peony exosomes and protective agents, and has multiple effects of firming, anti-wrinkle, anti-aging, whitening and repairing. The spirulina exosomes extracted by the present invention not only contain phycocyanin components, but also contain active factors secreted by spirulina, etc., and the fishy smell carried by spirulina is removed in terms of odor, and can have long-term stability after adding a protective agent for freeze-drying.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a plant exosome composition and a preparation method and application thereof. Background Art

[0002] The skin is a barrier between the human body and the external environment, and is an important organ that protects the body against external changes. The skin also has many functions such as defending against ultraviolet rays, regulating body temperature, and neutralizing external stimuli. With changes in the environment and the influence of aging, the function and condition of the skin will be damaged. The mission of skin care cosmetics is to reduce the damage to the skin's function and condition caused by the external environment and aging.

[0003] At present, the types of spirulina used in the cosmetics field are mainly Spirulina Bonatus and Spirulina Maxima (reference and comparison patents CN118436581A, CN117959236A), and the main focus is on extracting its phycocyanin. Compared with Spirulina Bonatus and Spirulina Maxima, which are currently used more, Spirulina Platensis has higher protein content, more phycocyanin content, and higher SOD activity.

[0004] Spirulina extracts are currently used in the cosmetics field mainly focusing on phycocyanin ingredients. However, the active factors secreted by spirulina also have good efficacy, and phycocyanin has some specific disadvantages: (1) Spirulina carries a fishy smell, which makes the taste experience bad when used. (2) Stability. Phycocyanin is a natural protein, and proteins have the disadvantage of poor stability. When the temperature reaches above 37°C, the activity and color of phycocyanin will be affected.

[0005] In the existing invention patents, spirulina extract is mainly used for anti-wrinkle and firming effects on the skin. However, people also need cosmetics with anti-aging, whitening, and repair effects. Summary of the invention

[0006] Problem that the invention aims to solve In view of the above problems existing in the prior art, the purpose of the present invention is to provide a spirulina extract product with multiple functions such as firming, anti-wrinkle, anti-aging, whitening and repairing.

[0007] Solutions for solving problems The present invention provides a plant exosome composition, comprising: two or more of Spirulina platensis exosomes, olive leaf exosomes, and white peony root exosomes.

[0008] Preferably, the composition comprises: Spirulina platensis exosomes, olive leaf exosomes and white peony root exosomes.

[0009] Preferably, the mass ratio of the Spirulina platensis exosomes, the olive leaf exosomes and the white peony root exosomes is 1-10:1-5:1-5.

[0010] Preferably, the mass ratio of the Spirulina platensis exosomes, the olive leaf exosomes and the white peony root exosomes is 2-5:1-3:1-3.

[0011] Preferably, the mass ratio of the Spirulina platensis exosomes, the olive leaf exosomes and the white peony root exosomes is 2:1:1.

[0012] Preferably, the preparation method of the composition comprises: extracting exosomes from Spirulina platensis, exosomes from olive leaves, and exosomes from white peony root respectively, and then compounding them according to proportions.

[0013] Preferably, the extraction of the exosomes from Spirulina platensis, olive leaf exosomes and white peony root exosomes comprises: taking Spirulina platensis, olive leaf and white peony root respectively, breaking the wall after adding a solvent, and collecting the supernatant by gradient centrifugation; diluting the obtained supernatant, filtering, concentrating and purifying.

[0014] Preferably, the solvent is PB buffer and / or PBS buffer.

[0015] Preferably, the dilution is to a turbidity of less than 900 NTU.

[0016] Preferably, the filtration is performed by deep filtration using a tangential flow depth filtration membrane package.

[0017] Preferably, the concentration and purification is performed using a hollow fiber column.

[0018] The present invention also provides an application of the composition in preparing skin care products.

[0019] Preferably, the skin care products include products with anti-aging effects, products with firming effects, products with anti-wrinkle effects, products with whitening effects, and products with repairing effects.

[0020] Effects of the Invention The plant exosome composition of the present invention has Spirulina platensis exosomes as the main component, and is added with olive leaf exosomes and white peony root exosomes, and has multiple functions of firming, anti-wrinkle, anti-aging, whitening and repairing. The Spirulina exosomes extracted by the present invention not only contain phycocyanin components, but also contain active factors secreted by Spirulina, and the fishy smell carried by Spirulina is removed in terms of odor. DETAILED DESCRIPTION

[0021] In order to make the technical solutions and beneficial effects of the present invention more clearly understood, the following is a detailed description by listing specific embodiments. Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application belongs.

[0022] The present invention provides a plant exosome composition, comprising: two or more of Spirulina platensis exosomes, olive leaf exosomes, and white peony root exosomes.

[0023] In certain embodiments, the composition comprises: Spirulina platensis exosomes, olive leaf exosomes, and white peony root exosomes.

[0024] In certain embodiments, the composition consists of Spirulina platensis exosomes, olive leaf exosomes, and white peony root exosomes.

[0025] In certain embodiments, the composition further comprises a lyoprotectant.

[0026] In certain embodiments, the composition consists of Spirulina platensis exosomes, olive leaf exosomes, white peony root exosomes, and a lyoprotectant.

[0027] In certain embodiments, the mass ratio of the Spirulina platensis exosomes, the olive leaf exosomes and the white peony root exosomes is 1-10:1-5:1-5.

[0028] In certain embodiments, the mass ratio of the Spirulina platensis exosomes, the olive leaf exosomes and the white peony root exosomes is 1:1:1, or 1:1:2, or 1:1:3, or 1:1:4, or 1:1:5, or 1:2:1, or 1:2:2, or 1:2:3, or 1:2:4, or 1:2:5, or 1:3:1, or 1:3:2, or 1:3:3, or 1:3:4, or 1:3:5, or 1:4:1, or 1:4:2, or 1:4:3, or 1:4:4, or 1:4:5, or 1:5:1, or 1:5:2, or 1:5:3, or 1:5:4, or 1:5:5, or 2:1:1, or 2:1:2, or 2:1:3, or 2:1:4, or 2:1:5, or 2:1:6. :2:1, or 2:2:3, or 2:2:5, or 2:3:1, or 2:3:2, or 2:3:3, or 2:3:4, or 2:3:5, or 2:4:1, or 2:4:2, or 2:4:3, or 2:4:5, or 2:5:1, or 2:5:2, or 2:5:3, or 2:5:4, or 2:5:5, or 3:1:1, or 3:1:2, or 3:1:3, or 3:1:4, or 3:1:5, or 3:2:1, or 3:2:2, or 3:2:3, or 3:2:4, or 3:2:5, or 3:3:1, or 3:3:2, or 3:3:4, or 3:3:5, or 3:4:1, or 3:4:2, or 3:4:3, or 3:4:4, or 3:4:5 , or 3:5:1, or 3:5:2, or 3:5:3, or 3:5:4, or 3:5:5, or 4:1:1, or 4:1:2, or 4:1:3, or 4:1:4, or 4:1:5, or 4:2:1, or 4:2:3, or 4:2:5, or 4:3:1, or 4:3:2, or 4:3:3, or 4:3:4, or 4:3 :5, or 4:4:1, or 4:4:3, or 4:4:5, or 4:5:1, or 4:5:2, or 4:5:3, or 4:5:4, or 4:5:5, or 5:1:1, or 5:1:2, or 5:1:3, or 5:1:4, or 5:1:5, or 5:2:1, or 5:2:2, or 5:2:3, or 5:2:4, or 5: 2:5, or 5:3:1, or 5:3:2, or 5:3:3, or 5:3:4, or 5:3:5, or 5:4:1, or 5:4:2, or 5:4:3, or 5:4:4, or 5:4:5, or 5:5:1, or 5:5:2, or 5:5:3, or 5:5:4, or 6:1:1, or 6:1:2, or 6:1:3, or 6:1:4, or 6:1:5, or 6:2:1, or 6:2:3, or 6:2:5, or 6:3:1, or 6:3:2, or 6:3:4, or 6:3:5, or 6:4:1, or 6:4:3, or 6:4:5, or 6:5:1, or 6:5:2, or 6:5:3, or 6:5:4, or 6:5:5, or 7:1:1,or 7:1:2, or 7:1:3, or 7:1:4, or 7:1:5, or 7:2:1, or 7:2:2, or 7:2:3, or 7:2:4, or 7:2:5, or 7:3:1, or 7:3:2, or 7:3:3, or 7:3:4, or 7:3:5, or 7:4:1, or 7:4:2, or 7:4:3, or 7:4:4, or 7:4:5, or 7:5:1, or 7:5:2, or 7:5:3, or 7:5:4 , or 7:5:5, or 8:1:1, or 8:1:2, or 8:1:3, or 8:1:4, or 8:1:5, or 8:2:1, or 8:2:3, or 8:2:5, or 8:3:1, or 8:3:2, or 8:3:3, or 8:3:4, or 8:3:5, or 8:4:1, or 8:4:3, or 8:4:5, or 8:5:1, or 8:5:2, or 8:5:3, or 8:5:4, or 8:5:5, or 9:1:1 , or 9:1:2, or 9:1:3, or 9:1:4, or 9:1:5, or 9:2:1, or 9:2:2, or 9:2:3, or 9:2:4, or 9:2:5, or 9:3:1, or 9:3:2, or 9:3:4, or 9:3:5, or 9:4:1, or 9:4:2, or 9:4:3, or 9:4:4, or 9:4:5, or 9:5:1, or 9:5:2, or 9:5:3, or 9:5:4, or 9:5:5 , or 10:1:1, or 10:1:2, or 10:1:3, or 10:1:4, or 10:1:5, or 10:2:1, or 10:2:3, or 10:2:5, or 10:3:1, or 10:3:2, or 10:3:3, or 10:3:4, or 10:3:5, or 10:4:1, or 10:4:3, or 10:4:5, or 10:5:1, or 10:5:2, or 10:5:3, or 10:5:4. ,

[0029] In certain embodiments, the mass ratio of the Spirulina platensis exosomes, the olive leaf exosomes and the white peony root exosomes is 2-5:1-3:1-3.

[0030] In certain embodiments, the mass ratio of the Spirulina platensis exosomes, the olive leaf exosomes and the white peony root exosomes is 2:1:1.

[0031] In certain embodiments, the preparation method of the composition comprises: extracting exosomes from Spirulina platensis, exosomes from olive leaves, and exosomes from white peony root respectively, and then compounding them according to proportions.

[0032] In certain embodiments, the extraction of Spirulina platensis exosomes, olive leaf exosomes, and white peony exosomes comprises: taking Spirulina platensis, olive leaf, and white peony respectively, breaking the cell walls after adding a solvent, and collecting the supernatant by gradient centrifugation; diluting the obtained supernatant, filtering, and concentrating and purifying.

[0033] In certain embodiments, the solvent is PB buffer and / or PBS buffer.

[0034] In certain embodiments, the solvent is PB buffer.

[0035] In certain embodiments, the solvent is PBS buffer.

[0036] In certain embodiments, the specific steps of the gradient centrifugation include: (1) Centrifuge at 400-800 g for 5-20 min; (2) Centrifuge at 1000-3000 g for 10-30 min; (3) Centrifuge at 3000-5000 g for 20-40 min; (4) Centrifuge at 8000-12000g for 40-70 min.

[0037] In certain embodiments, the specific steps of the gradient centrifugation include: (1) Centrifuge at 500 g for 10 min; (2) Centrifugation at 2000 g for 20 min; (3) Centrifugation at 4000 g for 30 min; (4) Centrifuge at 10,000 g for 60 min.

[0038] In certain embodiments, the diluting is to a turbidity of less than 900 NTU.

[0039] In certain embodiments, the filtration is depth filtration using a tangential flow depth filtration membrane package.

[0040] In certain embodiments, the concentration and purification is performed using a hollow fiber column.

[0041] The present invention also provides an application of the composition in preparing skin care products.

[0042] In certain embodiments, the skin care products include products with anti-aging effects, products with firming effects, products with anti-wrinkle effects, products with whitening effects, and products with repairing effects.

[0043] Example 1: Extraction of exosomes from Spirulina platensis 1. Cell wall breaking: Transfer the Spirulina platensis powder to a cell wall breaking machine and add an appropriate amount of PB buffer for cell wall breaking until completely mixed.

[0044] 2. Pretreatment centrifugation: Transfer the wall-breaking liquid of Spirulina platensis powder in step 1 to a centrifuge tube and use a desktop centrifuge set to 4°C for gradient centrifugation: 500g for 10 min, 2000g for 20 min, 4000g for 30 min, and 10000g for 60 min; after centrifugation, collect the supernatant and discard the precipitate.

[0045] 3. Dilution of supernatant: Use a standard with a turbidity of 800 NTU to calibrate the turbidity meter. Take the supernatant obtained in step 2 for turbidity measurement. If the turbidity is greater than 900 NTU, dilute it with PB buffer until the turbidity is less than 900 NTU.

[0046] 4. Deep filtration: The supernatant diluted in step 3 is deep filtered through a tangential flow deep filtration membrane package, and the deep filtered liquid is collected.

[0047] 5. Concentration and purification: The deep filtrate obtained in step 4 is concentrated and purified through a hollow fiber column. The suspension obtained after the concentration is completed is the exosomes of Spirulina platensis.

[0048] Example 2: Extraction of exosomes from olive leaves 1. Raw material cleaning: Use purified water to clean the olive leaf raw material 3 times.

[0049] 2. Wall breaking treatment: Transfer the olive leaves washed in step 1 to a wall breaking machine, add an appropriate amount of PB buffer for wall breaking treatment until completely mixed.

[0050] 3. Pretreatment centrifugation: Transfer the olive leaf wall-breaking liquid obtained in step 2 to a centrifuge tube and use a desktop centrifuge set to 4°C for gradient centrifugation: 500g for 10 min, 2000g for 20 min, 4000g for 30 min, and 10000g for 60 min; after centrifugation, collect the supernatant and discard the precipitate.

[0051] 4. Dilution of supernatant: Use a standard with a turbidity of 800 NTU to calibrate the turbidity meter. Take the supernatant obtained in step 3 for turbidity measurement. If the turbidity is greater than 900 NTU, dilute it with PB buffer until the turbidity is less than 900 NTU.

[0052] 5. Deep filtration: The supernatant diluted in step 4 is deep filtered through a tangential flow deep filtration membrane package, and the deep filtered liquid is collected.

[0053] 6. Concentration and purification: The deep filtrate obtained in step 5 is concentrated and purified through a hollow fiber column. The suspension obtained after concentration is olive leaf exosomes.

[0054] Example 3: Extraction of exosomes from white peony root 1. Raw material cleaning: Use purified water to clean the white peony root and rhizome slices three times.

[0055] 2. Wall breaking treatment: Transfer the white peony root and rhizome slices washed in step 1 to a wall breaking machine, add an appropriate amount of PB buffer for wall breaking treatment until completely mixed.

[0056] 3. Pretreatment centrifugation: Transfer the broken wall liquid of white peony rhizome slices obtained in step 2 to a centrifuge tube and use a desktop centrifuge set to 4°C for gradient centrifugation: 500g for 10 min, 2000g for 20 min, 4000g for 30 min, 10000g for 60 min; after centrifugation, collect the supernatant and discard the precipitate.

[0057] 4. Dilution of supernatant: Use a standard with a turbidity of 800 NTU to calibrate the turbidity meter. Take the supernatant obtained in step 3 for turbidity measurement. If the turbidity is greater than 900 NTU, dilute it with PB buffer until the turbidity is less than 900 NTU.

[0058] 5. Deep filtration: The supernatant diluted in step 4 is deep filtered through a tangential flow deep filtration membrane package, and the deep filtered liquid is collected.

[0059] 6. Concentration and purification: The deep filtrate obtained in step 5 is concentrated and purified through a hollow fiber column. The suspension obtained after concentration is the white peony exosomes.

[0060] Example 4: Preparation of exosome composition The exosomes extracted from Spirulina platensis, olive leaf exosomes, and white peony root exosomes in the ratio of 2:1:1 were mixed to prepare an exosome composition.

[0061] Example 5: Determination of TIMP1 protein content in dermal fibroblasts in vitro Matrix metalloproteinase inhibitor-1 (TIMP1) is a glycoprotein expressed in various tissues of organisms and is a member of the TIMP family. TIMP1 is a natural inhibitor of matrix metalloproteinases (MMPs), which are a group of peptidases involved in the degradation of the extracellular matrix. The decrease in TIMP1 levels caused by photoaging was simulated by irradiating cells with UVA, and the upregulation rate of TIMP1 protein content was determined after administration of the test substance to evaluate whether the test substance was effective in promoting TIMP1 protein synthesis. On the basis that the test met the validity verification, the content of TIMP1 protein was upregulated compared with the blank control, and there was a significant difference (P<0.05), indicating that the test substance has the ability to promote TIMP1 protein synthesis at the tested concentration, which can be used as one of the evidences to support the title of anti-aging cosmetic raw materials.

[0062] Determination method: 1. Cell plating: Remove the cells from the carbon dioxide incubator, observe the cell growth under a microscope, and randomly select two fields of view to take pictures using a low-power microscope (4X) and a high-power microscope (10X). When the cell density reaches 80%-90%, it is confirmed that it can be harvested. Wipe the outer surface of the culture bottle with a dust-free cloth containing alcohol and transfer it to the biosafety cabinet. Gently shake the culture bottle back and forth and use a pipette to remove the old culture medium. Use an appropriate amount of PBS to wash the cell surface once, add an appropriate amount of digestive enzyme to the culture bottle, and digest at room temperature for 3-5 min. After gently shaking, the cells are observed to be quicksand-like by naked eye. Under the microscope, the cells are spherical, indicating that the digestion is complete. Add an appropriate amount of PBS to stop the digestion. After the pipette blows the bottom of the culture bottle, the liquid is transferred to a centrifuge tube, and the centrifugation parameters are 400 g for 5min. Resuspend with complete culture medium and take 20 μL for cell counting. Inoculate into a 96-well plate at a density of 5000 cells / 100 μL / well, with 3 replicates per group, and culture the cells for 24 h.

[0063] 2. UVA Induction: Place an ice pack in the biosafety cabinet, cover the ice pack with four layers of kraft paper, and then place the cell culture plate on the kraft paper. Adjust the position of the UVA lamp so that the distance between the lamp and the cells is 5 cm. Remove the cell culture plate, turn on the lamp to balance for 5 minutes, then place the cell culture plate under the lamp, open the lid, and irradiate for 1 hour. The radiation energy is about 5 J / cm 2 .

[0064] 3. Drug treatment: Treatment drugs were prepared, 400 μL for each group, blank control group: T4 basal medium, ergothioneine (purchased from MERCK) experimental group: 40 μL ergothioneine (1 mg / ml storage mother solution) + 400 μL T4 basal medium, Spirulina platensis exosomes experimental group: 40 μL Spirulina platensis exosomes (1 mg / ml storage mother solution) + 400 μL T4 basal medium, olive leaf exosomes experimental group: 40 μL olive leaf exosomes (1 mg / ml storage mother solution) + 400 μL T4 basal medium, white peony exosomes experimental group: 40 μL white peony exosomes (1 mg / ml storage mother solution) + 400 μL T4 basal medium, combination experimental group: 40 μL combination (1 mg / ml storage mother solution) + 400 μL T4 basal medium.

[0065] After UVA irradiation, the PBS was discarded, and 100 μL of the prepared blank control, ergothioneine, Spirulina platensis exosomes, olive leaf exosomes, white peony exosomes and the combination were added respectively and cultured for 24 h.

[0066] 4. TIMP1 protein detection (refer to the instructions of Lianke Human TIMP1 ELISA Kit) Collect the culture supernatant, centrifuge at 3000 g for 10 min to remove impurities, and test according to the TIMP1 kit. If the test cannot be carried out in time, the supernatant should be aliquoted into 50 μL tubes and stored at -20 °C. Prepare all the necessary reagents and working concentration standards. Remove the unnecessary strips, put them back into the aluminum foil bag with desiccant, and reseal the seal.

[0067] 1) Soak the microplate: Add 300 μL of 1× washing solution and let it soak for 30 seconds. After discarding the washing solution, pat the microplate dry on absorbent paper. After washing, use the microplate immediately and do not let it dry.

[0068] 2) Add standard: Add 100 μL of 2-fold diluted standard to the standard wells. Add 100 μL of standard dilution to the blank wells.

[0069] 3) Add sample: Add 50 μL of cell culture supernatant to the sample well, and add 50 μL of PBS to each well for 2-fold dilution.

[0070] 4) Incubation: Seal the plate with a sealing film and place on a horizontal shaker for 2 hours at room temperature (25°C ± 3°C).

[0071] 5) Washing: Discard the liquid, add 300 μL of washing solution to each well, wash three times, and pat the microplate dry on absorbent paper.

[0072] 6) Add detection antibody: Add 100 μL of detection antibody working solution (1:100 dilution) to each well.

[0073] 7) Incubation: Seal the plate with a sealing film and place on a horizontal shaker for 1 hour at room temperature (25°C ± 3°C).

[0074] 8) Washing: Repeat step 5.

[0075] 9) Add enzyme: Add 100 μL of streptavidin working solution (1:100 dilution) to each well.

[0076] 10) Incubation: Seal the plate with a sealing film and place on a horizontal shaker at room temperature (25℃±3℃) for 45 minutes.

[0077] 11) Washing: Repeat step 5.

[0078] 12) Add substrate for color development: Add 100 μL of color development substrate to each well and incubate at room temperature (25℃±3℃) for 20 minutes in the dark.

[0079] 13) Add stop solution: Add 100 μL of stop solution to each well. Tap gently to mix until the color changes from blue to yellow.

[0080] 14) Detection reading: Within 30 minutes, measure the maximum absorbance wavelength of 450 nm.

[0081] 15) A calibration curve was made based on the A450 value of the standard, with the horizontal axis representing the TIMP1 protein concentration and the vertical axis representing the A450 value. The amount of TIMP1 protein in the sample was calculated according to the formula of the calibration curve. The results are shown in Table 1.

[0082] Table 1: Results of in vitro TIMP1 protein assay in dermal fibroblasts

[0083] The results showed that the composition of Example 4 could significantly promote the secretion of TIMP1, an inhibitor of matrix metalloproteinases, and the effect was better than that of ergothioneine and the raw materials of each component in the composition.

[0084] Example 6: Determination of MMP1 protein content in dermal fibroblasts in vitro MMP1 is a member of the matrix metalloproteinase (MMP) family. The degradation of the extracellular matrix mainly relies on proteolytic enzymes, and MMP is the most important group of proteolytic enzymes, among which MMP-1 is the main enzyme for degrading type I and type III collagen. When MMP-1 is overexpressed, it specifically degrades extracellular matrix components, destroys the normal structure of collagen fibers and elastic fibers, and causes wrinkles and other aging manifestations in the skin. Human dermal fibroblasts can be used as a cell model to study the inhibition of MMP-1 content by cosmetics. UVA irradiation cells simulate the increase of MMP-1 caused by photoaging, and the downregulation rate of MMP-1 protein content after administration of the test substance is determined to evaluate whether the test substance has the effect of inhibiting MMP-1 protein synthesis. On the basis of meeting the validity verification of the test, the content of MMP-1 protein in the test substance is downregulated compared with the blank control, and there is a significant difference (P<0.05), indicating that the test substance has the ability to inhibit MMP-1 protein synthesis at the test concentration, which can be used as one of the evidences to support the title of anti-aging cosmetic raw materials.

[0085] Determination method: 1. Cell plating: Remove the cells from the carbon dioxide incubator, observe the cell growth under a microscope, and randomly select two fields of view to take pictures using a low-power microscope (4X) and a high-power microscope (10X). When the cell density reaches 80%-90%, it is confirmed that it can be harvested. Wipe the outer surface of the culture bottle with a dust-free cloth containing alcohol and transfer it to the biosafety cabinet. Gently shake the culture bottle back and forth and use a pipette to remove the old culture medium. Use an appropriate amount of PBS to wash the cell surface once, add an appropriate amount of digestive enzyme to the culture bottle, and digest at room temperature for 3-5 min. After gently shaking, the cells are observed to be quicksand-like by naked eye. Under the microscope, the cells are spherical, indicating that the digestion is complete. Add an appropriate amount of PBS to stop the digestion. After the pipette blows the bottom of the culture bottle, the liquid is transferred to a centrifuge tube, and the centrifugation parameters are 400 g for 5min. Resuspend with complete culture medium and take 20 μL for cell counting. Inoculate into a 96-well plate at a density of 5000 cells / 100 μL / well, with 3 replicates per group, and culture the cells for 24 h.

[0086] 2. UVA Induction: Place an ice pack in the biosafety cabinet, cover the ice pack with four layers of kraft paper, and then place the cell culture plate on the kraft paper. Adjust the position of the UVA lamp so that the distance from the lamp to the cells is 5 cm. Discard the cell culture supernatant and add 50 μL of pre-cooled PBS to each well. Remove the cell culture plate, turn on the lamp to balance for 5 min, then place the cell culture plate under the lamp, open the lid, and irradiate for 1 h. The radiation energy is about 5 J / cm 2 .

[0087] 3. Drug treatment: Treatment drugs were prepared, 400 μL for each group, blank control group: T4 basal medium, bosone (purchased from MedChemExpress) experimental group: 40 μL bosone (1 mg / ml storage mother solution) + 400 μL T4 basal medium, Spirulina platensis exosomes experimental group: 40 μL Spirulina platensis exosomes (1 mg / ml storage mother solution) + 400 μL T4 basal medium, olive leaf exosomes experimental group: 40 μL olive leaf exosomes (1 mg / ml storage mother solution) + 400 μL T4 basal medium, white peony exosomes experimental group: 40 μL white peony exosomes (1 mg / ml storage mother solution) + 400 μL T4 basal medium, combination experimental group: 40 μL combination (1 mg / ml storage mother solution) + 400 μL T4 basal medium.

[0088] After UVA irradiation, the PBS was discarded, and 100 μL of the prepared blank control, Bossain, Spirulina platensis exosomes, olive leaf exosomes, white peony exosomes and the combination were added respectively and cultured for 24 h.

[0089] 4. MMP1 protein detection (refer to the instructions of Lianke Human MMP1 ELISA Kit): Collect the culture supernatant, centrifuge at 3000 g for 10 min to remove impurities, and test according to the MMP1 kit. If the test cannot be carried out in time, the supernatant should be divided into 50 μL per tube and stored at -20℃. Prepare all the necessary reagents and working concentration standards. Remove the unnecessary strips, put them back into the aluminum foil bag with desiccant, and reseal the seal.

[0090] 1) Soak the microplate: Add 300 μL of 1× washing solution and let it soak for 30 seconds. After discarding the washing solution, pat the microplate dry on absorbent paper. After washing, use the microplate immediately and do not let it dry.

[0091] 2) Add standard: Add 100 μL of 2-fold diluted standard to the standard wells. Add 100 μL of standard dilution to the blank wells.

[0092] 3) Add samples: Add 50 μL of cell culture supernatant to the sample wells, and add 50 μL of PBS to each well for 2-fold dilution.

[0093] 4) Incubation: Seal the plate with a sealing film and place in a mixer at room temperature (25℃±3℃) for 2 hours.

[0094] 5) Washing: Discard the liquid, add 300 μL of washing solution to each well, wash three times, and pat the microplate dry on absorbent paper.

[0095] 6) Add detection antibody: Add 100 μL of detection antibody working solution (1:100 dilution) to each well.

[0096] 7) Incubation: Seal the plate with a sealing film and place in a mixer at room temperature (25℃±3℃) for 1 hour.

[0097] 8) Washing: Repeat step 5.

[0098] 9) Add enzyme: Add 100 μL of streptavidin working solution (1:100 dilution) to each well.

[0099] 10) Incubation: Seal the plate with a sealing film and place in a mixer at room temperature (25℃±3℃) for 45 minutes.

[0100] 11) Washing: Repeat step 5.

[0101] 12) Add substrate for color development: Add 100 μL of color development substrate to each well and incubate at room temperature (25℃±3℃) for 20 minutes in the dark.

[0102] 13) Add stop solution: Add 100 μL of stop solution to each well. Tap gently to mix until the color changes from blue to yellow.

[0103] 14) Detection reading: Within 30 minutes, measure the maximum absorbance wavelength of 450 nm.

[0104] 15) A calibration curve was made based on the A450 value of the standard, with the horizontal axis representing the MMP1 protein concentration and the vertical axis representing the A450 value. The amount of MMP1 protein in the sample was calculated according to the formula of the calibration curve. The results are shown in Table 2.

[0105] Table 2: Results of in vitro dermal fibroblast MMP1 protein content determination

[0106] The results showed that the composition of Example 4 could significantly inhibit the secretion of matrix metalloproteinase MMP1 of dermal fibroblasts, and the effect was better than that of bosine and the raw materials of each component in the composition.

[0107] Example 7: In vitro SA-β-Gal staining assay of dermal fibroblasts SA-β-gal staining is a commonly used method for detecting cell aging. It evaluates the aging state of cells by detecting the activity of intracellular acidic β-galactosidase. The activity of acidic β-galactosidase in aged cells increases. The β-galactosidase staining kit uses X-Gal as a substrate, and generates a dark blue product under the catalysis of aging-specific β-galactosidase. UVA irradiation of cells simulates photoaging, causing an increase in intracellular acidic β-galactosidase. The upregulation of acidic β-galactosidase is inhibited by administering the test substance. The amount of SA-β-gal staining-positive cells is used to evaluate whether the test substance has efficacy in inhibiting cell aging. On the basis that the test meets the validity verification, the amount of SA-β-gal staining-positive cells of the test substance is downregulated compared with the blank control, and there is a significant difference (P<0.05), indicating that the test substance has the ability to inhibit cell aging at the tested concentration, and can be used as one of the evidences to support the title of anti-aging cosmetic raw materials.

[0108] Determination method: 1. Cell plating: Remove the cells from the carbon dioxide incubator, observe the cell growth under a microscope, and randomly select two fields of view to take pictures using a low-power microscope (4X) and a high-power microscope (10X). When the cell density reaches 80%-90%, it is confirmed that it can be harvested. Wipe the outer surface of the culture bottle with a dust-free cloth containing alcohol and transfer it to the biosafety cabinet. Gently shake the culture bottle back and forth and use a pipette to remove the old culture medium. Use an appropriate amount of PBS to wash the cell surface once, add an appropriate amount of digestive enzyme to the culture bottle, and digest at room temperature for 3-5 min. After gently shaking, the cells are observed to be quicksand-like by naked eye. Under the microscope, the cells are spherical, indicating that the digestion is complete. Add an appropriate amount of PBS to stop the digestion. After the pipette blows the bottom of the culture bottle, the liquid is transferred to a centrifuge tube, and the centrifugation parameters are 400 g for 5min. Resuspend with complete culture medium and take 20 μL for cell counting. Inoculate into a 24-well plate at a density of 10,000 cells / 500μL / well, with 3 replicates per group. Culture the cells for 24 h.

[0109] 2. UVA Induction: Place an ice pack in the biosafety cabinet, cover the ice pack with four layers of kraft paper, and then place the cell culture plate on the kraft paper. Adjust the position of the UVA lamp so that the distance between the lamp and the cells is 5 cm. Remove the cell culture plate, turn on the lamp for 5 min, discard the culture medium, add 200 μL of pre-cooled PBS, and then place the cell culture plate under the lamp, open the lid, and irradiate for 1 h. The radiation energy is about 5 J / cm 2 .

[0110] 3. Drug treatment: Treatment drugs were prepared, 2 mL for each group, blank control group: T4 basal medium, TGF-β (purchased from MERCK) experimental group: 200 μL TGF-β (1 mg / ml storage mother solution) + 2 mL T4 basal medium, Spirulina platensis exosome experimental group: 200 μL Spirulina platensis exosome (1 mg / ml storage mother solution) + 2 mL T4 basal medium, olive leaf exosome experimental group: 200 μL olive leaf exosome (1 mg / ml storage mother solution) + 2 mL T4 basal medium, white peony exosome experimental group: 200 μL white peony exosome (1 mg / ml storage mother solution) + 2 mL T4 basal medium, combination experimental group: 200 μL combination (1 mg / ml storage mother solution) + 2 mL T4 basal medium.

[0111] After UVA irradiation, the PBS was discarded, and 500 μL of the prepared blank control, TGF-β, Spirulina platensis exosomes, olive leaf exosomes, white peony exosomes and the combination were added respectively and cultured for 24 h.

[0112] 4. UVA re-induction and drug treatment: Repeat the steps 2 and 3 of Example 7.

[0113] 5. SA-β-gal staining (refer to the instructions of Shangbao Bio β-galactosidase staining kit): 1) Aspirate the cell culture medium, wash once with PBS, add 200 μL β-galactosidase staining fixative, and fix at room temperature for 15 minutes.

[0114] 2) Aspirate the cell fixative and wash the cells three times with PBS, 3 minutes each time.

[0115] 3) Aspirate PBS and add 300 μL of staining working solution to each well.

[0116] 4) Place the plate in a biochemical incubator and incubate overnight at 37°C. Seal the plate with sealing film to prevent evaporation.

[0117] 5) Wash the cells 3 times with PBS, 3 minutes each time, and observe under an ordinary optical microscope. If it is not possible to observe and count in time, they can be stored at 4℃ for one week; or after sealing with sealing solution, they can be stored at 4℃ for a longer time.

[0118] 6) Data analysis: Image J was used to count the number of SA-β-Gal+ cells and total cells. + (%)=SA-β-Gal + The experimental group was compared with the blank control group, and the results are shown in Table 3.

[0119] Table 3: Results of in vitro SA-β-Gal staining assay of dermal fibroblasts

[0120] The results showed that the composition of Example 4 could significantly reduce the amount of SA-β-gal positive cells, and the effect was better than that of TGF-β and the raw materials of each component in the composition.

[0121] Example 8: In vitro dermal fibroblast proliferation assay Cell proliferation is one of the important physiological functions of living cells and an important life characteristic of organisms. In addition to secreting collagen fibers and elastic fibers to maintain skin elasticity, fibroblasts also have adhesion, which plays an important role in maintaining skin elasticity and toughness. The reduction of fibroblast proliferation activity is one of the important causes of wrinkles. The CCK-8 method is based on the highly water-soluble tetrazolium salt WST-8 to measure cell proliferation / toxicity. WST-8 can be reduced to a water-soluble orange-yellow formazan product under the action of an electron mediator, producing a color reaction. The more and faster the cell proliferation, the darker the color; the greater the cell toxicity, the lighter the color. For the same cell, the depth of color is proportional to the number of living cells. Dermal fibroblasts can be used as a cell model for studying the proliferation of dermal fibroblasts in vitro in cosmetics. On the basis of the test meeting the validity verification, the experimental group was compared with the blank control group, and the cell proliferation was upregulated, and there was a significant difference (P<0.05), indicating that the experimental sample has the ability to promote cell proliferation at the tested concentration, which can be used as one of the evidences to support the title of firming cosmetic raw materials.

[0122] Determination method: 1. Cell plating: Remove the cells from the carbon dioxide incubator, observe the cell growth under a microscope, and randomly select two fields of view to take pictures using a low-power microscope (4X) and a high-power microscope (10X). When the cell density reaches 80%-90%, it is confirmed that it can be harvested. Wipe the outer surface of the culture bottle with a dust-free cloth containing alcohol and transfer it to the biosafety cabinet. Gently shake the culture bottle back and forth and use a pipette to remove the old culture medium. Use an appropriate amount of PBS to wash the cell surface once, add an appropriate amount of digestive enzyme to the culture bottle, and digest at room temperature for 3-5 min. After gently shaking, the cells are observed to be quicksand-like by naked eye. Under the microscope, the cells are spherical, indicating that the digestion is complete. Add an appropriate amount of PBS to stop the digestion. After the pipette blows the bottom of the culture bottle, the liquid is transferred to a centrifuge tube, and the centrifugation parameters are 400 g for 5min. Resuspend with complete culture medium and take 20 μL for cell counting. Inoculate into a 96-well plate at a density of 5000 cells / 100 μL / well, with 3 replicates per group, and culture the cells for 24 h.

[0123] 2. Drug treatment: Treatment drugs were prepared, 400 μL for each group, blank control group: T4 basal medium, astaxanthin (purchased from Biotopped) experimental group: 40 μL astaxanthin (1 mg / ml storage mother solution) + 400 μL T4 basal medium, Spirulina platensis exosomes experimental group: 40 μL Spirulina platensis exosomes (1 mg / ml storage mother solution) + 400 μL T4 basal medium, olive leaf exosomes experimental group: 40 μL olive leaf exosomes (1 mg / ml storage mother solution) + 400 μL T4 basal medium, white peony exosomes experimental group: 40 μL white peony exosomes (1 mg / ml storage mother solution) + 400 μL T4 basal medium, combination experimental group: 40 μL combination (1 mg / ml storage mother solution) + 400 μL T4 basal medium; discard the cell supernatant, add 100 μL negative control reagent, positive control group reagent, and experimental group reagent, respectively, and culture for 24 h.

[0124] 3. CCK8 detection (refer to the instructions of Abbkine SuperKine™ Ultrasensitive Cell Proliferation Detection Reagent Kit): 1) Prepare the detection solution containing 10% CCK8 reagent using basal culture medium.

[0125] 2) After removing the culture medium from the well plate, add 100 μL of CCK8 solution to each well and continue incubating in the cell culture incubator for 2 hours.

[0126] 3) Measure the absorbance at 450 nm.

[0127] 4) Data analysis: Cell proliferation rate % = OD experimental group / OD blank control group × 100%, OD experimental group: absorbance value after treatment with the test drug, OD blank control group: absorbance value of the blank control drug treatment. The results are shown in Table 4.

[0128] Table 4: In vitro dermal fibroblast proliferation assay results

[0129] The results show that the composition of Example 4 can significantly promote the proliferation of dermal fibroblasts to achieve a firming effect, and the effect is better than astaxanthin and the raw materials of each component in the composition.

[0130] Example 9: Determination of collagen type I content in dermal fibroblasts in vitro Type I collagen is one of the main components of the extracellular matrix of dermal cells. Type I procollagen is synthesized intracellularly by dermal fibroblasts and secreted extracellularly. Under the action of terminal procollagen peptidase, the telopeptide is separated and polymerized to form collagen fibers. Human dermal fibroblasts can be used as a cell model to study the improvement of type I collagen content in cosmetics. By measuring the increase rate of type I collagen content after administration of blank control and test substance, the efficacy of the test substance in promoting collagen synthesis was evaluated. On the basis that the test meets the validity verification, the content of type I collagen in the test substance is increased compared with the blank / solvent control, and there is a significant difference (P<0.05), indicating that the test substance has the ability to promote type I collagen synthesis at the tested concentration, and can be used as one of the evidences to support the title of firming cosmetic raw materials.

[0131] Determination method: 1. Cell plating: Remove the cells from the carbon dioxide incubator, observe the cell growth under a microscope, and randomly select two fields of view to take pictures using a low-power microscope (4X) and a high-power microscope (10X). When the cell density reaches 80%-90%, it is confirmed that it can be harvested. Wipe the outer surface of the culture bottle with a dust-free cloth containing alcohol and transfer it to the biosafety cabinet. Gently shake the culture bottle back and forth and use a pipette to remove the old culture medium. Use an appropriate amount of PBS to wash the cell surface once, add an appropriate amount of digestive enzyme to the culture bottle, and digest at room temperature for 3-5 min. After gently shaking, the cells are observed to be quicksand-like by naked eye. Under the microscope, the cells are spherical, indicating that the digestion is complete. Add an appropriate amount of PBS to stop the digestion. After the pipette blows the bottom of the culture bottle, the liquid is transferred to a centrifuge tube, and the centrifugation parameters are 400 g for 5min. Resuspend with complete culture medium and take 20 μL for cell counting. Inoculate into a 96-well plate at a density of 5000 cells / 100 μL / well, with 3 replicates per group, and culture the cells for 24 h.

[0132] 2. Drug treatment: Treatment drugs were prepared, 400 μL for each group, blank control group: T4 basal medium, astaxanthin (purchased from Biotopped) experimental group: 40 μL astaxanthin (1 mg / ml storage mother solution) + 400 μL T4 basal medium, Spirulina platensis exosomes experimental group: 40 μL Spirulina platensis exosomes (1 mg / ml storage mother solution) + 400 μL T4 basal medium, olive leaf exosomes experimental group: 40 μL olive leaf exosomes (1 mg / ml storage mother solution) + 400 μL T4 basal medium, white peony exosomes experimental group: 40 μL white peony exosomes (1 mg / ml storage mother solution) + 400 μL T4 basal medium, combination experimental group: 40 μL combination (1 mg / ml storage mother solution) + 400 μL T4 basal medium; discard the cell supernatant, add 100 μL negative control reagent, positive control group reagent, and experimental group reagent, respectively, and culture for 24 h.

[0133] 3. Type I collagen detection (refer to the Human Pro-Collagen I α1 ELISA Kit test instructions): 1) Soak the microplate: Add 300μL 1× washing solution and let it soak for 30 seconds. After discarding the washing solution, pat the microplate dry on absorbent paper. After washing, use the microplate immediately and do not let it dry.

[0134] 2) Add standard: Add 100 μL of 2-fold diluted standard to the standard wells. Add 100 μL of standard dilution to the blank wells.

[0135] 3) Add sample: Add 5 μL of cell culture supernatant to the sample well, and add 95 μL of PBS to each well for 20-fold dilution.

[0136] 4) Incubation: Seal the plate with a sealing film and place in a constant temperature mixer at room temperature (25℃±3℃) for 2 h.

[0137] 5) Washing: Discard the liquid, add 300 μL of washing solution to each well, wash three times, and pat the microplate dry on absorbent paper.

[0138] 6) Add detection antibody: Add 100 μL detection antibody working solution (1:100 dilution) to each well.

[0139] 7) Incubation: Seal the plate with a sealing film, place in a constant temperature mixer, and incubate at room temperature (25℃±3℃) for 1 h.

[0140] 8) Washing: Repeat step 5.

[0141] 9) Add enzyme: Add 100 μL of streptavidin working solution (1:100 dilution) to each well.

[0142] 10) Incubation: Seal the plate with a sealing film and place in a constant temperature mixer at room temperature (25℃±3℃) for 45 min.

[0143] 11) Washing: Repeat step 5.

[0144] 12) Add substrate for color development: Add 100 μL of color development substrate to each well and incubate at room temperature (25℃±3℃) for 20 min in the dark.

[0145] 13) Add stop solution: Add 100 μL of stop solution to each well. Tap gently to mix until the color changes from blue to yellow.

[0146] 14) Detection reading: Within 30 minutes, measure the maximum absorbance wavelength of 450 nm.

[0147] 15) A calibration curve was made based on the A450 value of the standard, with the horizontal axis representing the type I collagen concentration and the vertical axis representing the A450 value. The amount of type I collagen in the sample was calculated based on the formula of the calibration curve. The experimental group was compared with the blank control group. The results are shown in Table 5.

[0148] Table 5: Results of in vitro determination of collagen type I content in dermal fibroblasts

[0149] The results showed that the composition of Example 4 can significantly promote the secretion of type I collagen in dermal fibroblasts to achieve a firming effect, and the effect is better than astaxanthin and the raw materials of each component in the composition.

[0150] Example 10: Determination of the reactive oxygen species scavenging ability of dermal fibroblasts in vitro Reactive oxygen species (ROS) refers to the general term for oxygen-containing free radicals and peroxides that are easy to form free radicals related to oxygen metabolism in the body. It broadly refers to free radicals and non-free radicals derived from oxygen, including superoxide anions (O 2- ), hydrogen peroxide (H2O2), hydroxyl radical (OH -), ozone (O3) and singlet oxygen (1O2), etc. The normal physiological metabolism of the human body and the impact of the environment will lead to the generation of reactive oxygen species (ROS), but excessive ROS will lead to problems such as skin aging. The ROS produced when the skin is exposed to ultraviolet radiation can induce the expression of matrix metalloproteinases (MMPs) and increase their activity. In particular, MMP-1 and MMP-3 will accelerate the skin aging process by degrading collagen and elastin. By irradiating cells with UVA to simulate photoaging, the cellular ROS increased, and the amount of intracellular ROS was detected by the DCFH-DA fluorescent probe to evaluate whether the test substance is effective in inhibiting cellular ROS. On the basis that the test meets the validity verification, the DCFH-DA fluorescence intensity of the test substance is downregulated compared with the blank control, and there is a significant difference (P<0.05), indicating that the test substance has the ability to clear cellular ROS at the tested concentration, which can be used as one of the evidences to support the title of anti-wrinkle cosmetic raw materials.

[0151] Determination method: 1. Cell plating: Remove the cells from the carbon dioxide incubator, observe the cell growth under a microscope, and randomly select two fields of view to take pictures using a low-power microscope (4X) and a high-power microscope (10X). When the cell density reaches 80%-90%, it is confirmed that it can be harvested. Wipe the outer surface of the culture bottle with a dust-free cloth containing alcohol and transfer it to the biosafety cabinet. Gently shake the culture bottle back and forth and use a pipette to remove the old culture medium. Use an appropriate amount of PBS to wash the cell surface once, add an appropriate amount of digestive enzyme to the culture bottle, and digest at room temperature for 3-5 min. After gently shaking, the cells are observed to be quicksand-like by naked eye. Under the microscope, the cells are spherical, indicating that the digestion is complete. Add an appropriate amount of PBS to stop the digestion. After the pipette blows the bottom of the culture bottle, the liquid is transferred to a centrifuge tube, and the centrifugation parameters are 400 g for 5min. Resuspend with complete culture medium and take 20 μL for cell counting. Inoculate into a 24-well plate at a density of 50,000 cells / 500 μL / well, with 3 replicates per group, and culture the cells for 24 h.

[0152] 2. Drug treatment: Treatment drugs were prepared, 2 mL for each group, blank control group: T4 basal medium, bosogenin (purchased from MedChemExpress) experimental group: 200 μL bosogenin (1 mg / ml storage mother solution) + 2 mL T4 basal medium, Spirulina platensis exosomes experimental group: 200 μL Spirulina platensis exosomes (1 mg / ml storage mother solution) + 2 mL T4 basal medium, olive leaf exosomes experimental group: 200 μL olive leaf exosomes (1 mg / ml storage mother solution) + 2 mL T4 basal medium, white peony exosomes experimental group: 200 μL white peony exosomes (1 mg / ml storage mother solution) + 2 mL T4 basal medium, combination experimental group: 200 μL combination (1 mg / ml storage mother solution) + 2 mL T4 basal medium; the culture supernatant of the cells was discarded, and 500 μL of negative control reagent, positive control group reagent, and experimental group reagent were added respectively, and cultured for 24 h.

[0153] 3. UVA Induction: Place an ice pack in the biosafety cabinet, cover the ice pack with four layers of kraft paper, and then place the cell culture plate on the kraft paper. Adjust the position of the UVA lamp so that the distance between the lamp and the cells is 5 cm. Remove the cell culture plate, turn on the lamp to balance for 5 min, discard the culture supernatant, add 100 μL of pre-cooled PBS, and then place the cell culture plate under the lamp, open the lid, and irradiate for 1 h. The radiation energy is about 5 J / cm 2 .

[0154] 4. Drug treatment: The cell culture supernatant was discarded, and 500 μL of blank control reagent, positive control group reagent, and experimental group reagent were added respectively and cultured for 3-5 h.

[0155] 5. Sample collection and testing: The DCFH-DA working solution was prepared at a dilution ratio of 1:1000. DCFH-DA was diluted with basal medium to a final concentration of 10 μmol / L. PBS in the 24-well plate was discarded, 200 μL of DCFH-DA working solution was added to each well, and the cells were incubated in a CO2 incubator for 30 min. After the incubation, the cells were washed 3 times with PBS, placed on the fluorescence microplate reader, and the incident light wavelength was set to 529 nm and the excitation light wavelength to 504 nm. The readings were taken and the results are shown in Table 6.

[0156] Table 6: Results of in vitro dermal fibroblast reactive oxygen species scavenging ability assay

[0157] The results show that the composition of Example 4 can significantly reduce the ROS level of dermal fibroblasts to achieve anti-wrinkle effect, and the effect is better than that of bosquine and the raw materials of each component in the composition.

[0158] Example 11: In vitro melanocyte tyrosinase activity inhibition assay Tyrosinase is a copper-containing metal enzyme belonging to the polyphenol oxidase class. It is an important rate-limiting enzyme in a series of chemical reactions during the production of melanin. Tyrosinase catalyzes L-DOPA to generate dopachrome, which has a characteristic absorption peak at 492 nm, and then the activity of tyrosinase is determined. B16 mouse melanoma cells can be used as a cell model to study the inhibition rate of tyrosinase activity in cosmetics. On the basis of meeting the effectiveness verification, the inhibition rate of tyrosinase activity in the experimental group was significantly increased compared with the blank control group, and there was a significant difference (P<0.05), indicating that the experimental sample has the ability to inhibit tyrosinase activity at the tested concentration, which can be used as one of the evidences to support the title of whitening cosmetic raw materials.

[0159] Determination method: 1. Cell plating: B16 mouse melanoma cells are normally cultured using RPMI-1640 complete medium (RPMI-1640 complete medium culture does not produce melanin). Remove the cells from the carbon dioxide incubator, observe the cell growth under a microscope, and randomly select two fields of view to take pictures using a low-power microscope (4X) and a high-power microscope (10X). When the cell density reaches 80%-90%, it is confirmed that it can be harvested. Wipe the outer surface of the culture bottle with a dust-free cloth containing alcohol and transfer it to the biosafety cabinet. Gently shake the culture bottle back and forth, remove the old culture medium with a pipette, wash the cell surface once with an appropriate amount of PBS, and digest with 0.25% trypsin for 2 min. When most of the cells become round and suspended under a microscope, add about 2-3 times the volume of RPMI-1640 complete medium containing serum to terminate the digestion, and collect them in a centrifuge tube, centrifuge at 1500 rpm for 5 min. After centrifugation. Discard the supernatant and add a certain volume of DMEM high-glucose complete medium to the centrifuge tube (resuspend the cells at this time and use DMEM high-glucose medium in the following experiments. B16 cells will produce melanin in DMEM medium). After resuspending evenly, use a cell counter to count. Take 20 μL for cell counting. Inoculate into a 24-well plate at a density of 100,000 cells / well. The blank control group does not inoculate cells but only adds DMEM high-glucose medium and culture for 24 hours.

[0160] 2. Drug treatment: The treatment drugs were prepared, 2 mL for each group, blank control group: DMEM high-glucose complete medium, nicotinamide (purchased from MERCK) experimental group: 200 μL nicotinamide (1 mg / ml storage mother solution) + 2 mL DMEM high-glucose complete medium, Spirulina platensis exosome experimental group: 200 μL Spirulina platensis exosome (1 mg / ml storage mother solution) + 2 mL DMEM high-glucose complete medium, olive leaf exosome experimental group: 200 μL olive leaf exosome (1 mg / ml storage mother solution) + 2 mL DMEM high-glucose complete medium, white peony exosome experimental group: 200 μL white peony exosome (1 mg / ml storage mother solution) + 2 mL DMEM high-glucose complete medium, combination experimental group: 200 μL combination (1 mg / ml storage mother solution) + 2 mL DMEM high-glucose complete medium; the culture supernatant of the cells was discarded and 500 μL of each was added. μL blank control reagent, positive control group reagent, and experimental group reagent, and culture for 72 hours. During this period, the cell status and culture medium were observed every day and fresh blank control reagent, positive control group reagent, and experimental group reagent were replaced.

[0161] 3. Cell collection: After removing the culture supernatant, wash once with an appropriate amount of PBS. Add 200 μl of 0.25% trypsin to each well to digest the cells at 37°C for 2 min. Add 400 μl of PBS containing 10% FBS to terminate the digestion and blow the cells, collect the cells in a 1.5 mL centrifuge tube, centrifuge at 4000 rpm for 5 min, and discard the supernatant.

[0162] 4. Testing: Add 100 μl 1% Triton-x-100 to each tube to lyse the cells, freeze at -80°C for 30 min, place at room temperature for 10 min, and centrifuge at 12,000 rpm at 4°C for 20 min to remove cell debris. Take 50 μl of the supernatant, add 150 μl 2 mg / ml L-DOPA solution, incubate at 37°C for 1 h, and measure the absorbance at 492 nm with an enzyme-labeled instrument.

[0163] 5. Data Analysis: Enzyme activity inhibition rate = [1-(As-Ab) / (Ac-Ab)] × 100%, As: absorbance of the experimental group or positive control group, Ac: absorbance of the negative control well, Ab: absorbance of the blank control group. The results are shown in Table 7.

[0164] Table 7: In vitro melanocyte tyrosinase activity inhibition assay results

[0165] The results show that the composition of Example 4 can significantly inhibit the tyrosinase activity of melanocytes to achieve a whitening effect, and the effect is better than niacinamide and the raw materials of each component in the composition.

[0166] Example 12: In vitro melanocyte melanin synthesis inhibition assay Melanin is a dark brown pigment found in animals and plants. It is produced and stored in a special cell, the melanocyte (also known as the melanin matrix). It is because of the presence of melanin that the skin has color. The main function of melanocytes is to synthesize melanin. The synthesis of melanin is mainly through the use of tyrosine as a raw material in melanocytes and the catalysis of tyrosinase to form melanin. α-MSH can bind to the MC1R receptor on melanocytes and activate adenylate cyclase (AC), leading to an increase in intracellular cAMP. The increased cAMP activates tyrosinase through PKA, thereby promoting melanin production. B16 mouse melanoma cells can be used as a cell model to study the inhibition of melanin synthesis in cosmetics. On the basis of meeting the effectiveness verification, the inhibition rate of melanin synthesis in the experimental group was significantly increased compared with the negative control group, and there was a significant difference (P<0.05), indicating that the experimental sample has the ability to inhibit melanin synthesis at the tested concentration, which can be used as one of the evidences to support the title of whitening cosmetic raw materials.

[0167] Determination method: 1. Cell plating: B16 mouse melanoma cells are normally cultured using RPMI-1640 complete medium (RPMI-1640 complete medium culture does not produce melanin). Remove the cells from the carbon dioxide incubator, observe the cell growth under a microscope, and randomly select two fields of view to take pictures using a low-power microscope (4X) and a high-power microscope (10X). When the cell density reaches 80%-90%, it is confirmed that it can be harvested. Wipe the outer surface of the culture bottle with a dust-free cloth containing alcohol and transfer it to the biosafety cabinet. Gently shake the culture bottle back and forth, remove the old culture medium with a pipette, wash the cell surface once with an appropriate amount of PBS, and digest with 0.25% trypsin for 2 min. When most of the cells become round and suspended under a microscope, add about 2-3 times the volume of RPMI-1640 complete medium containing serum to terminate the digestion, and collect them in a centrifuge tube, centrifuge at 1500 rpm for 5 min. After centrifugation. Discard the supernatant and add a certain volume of DMEM high-glucose complete medium to the centrifuge tube (resuspend the cells at this time and use DMEM high-glucose medium in the following experiments. B16 cells will produce melanin in DMEM medium). After resuspending evenly, use a cell counter to count. Take 20 μL for cell counting. Inoculate into a 24-well plate at a density of 100,000 cells / well. The blank control group does not inoculate cells but only adds DMEM high-glucose medium and culture for 24 hours.

[0168] 2. Drug treatment: Treatment drugs were prepared, 2 mL for each group, blank control group: PBS + DMEM high glucose complete medium of equal sample volume, nicotinamide (purchased from MERCK) experimental group: 200 μL nicotinamide (1 mg / ml storage mother solution) + 1 mL DMEM high glucose complete medium + 1 mL PBS, Spirulina platensis exosome experimental group: 200 μL Spirulina platensis exosome (1 mg / ml storage mother solution) + 1 mL DMEM high glucose complete medium + 1 mL PBS, olive leaf exosome experimental group: 200 μL olive leaf exosome (1 mg / ml storage mother solution) + 1 mL DMEM high glucose complete medium + 1 mL PBS, white peony exosome experimental group: 200 μL white peony exosome (1 mg / ml storage mother solution) + 1 mL DMEM high glucose complete medium + 1 mL PBS, combination experimental group: 200 μL combination (1 mg / ml storage mother solution) + 1 mL DMEM high glucose complete medium + 1 mL PBS; discard the cell culture supernatant, add 500 μL of negative control reagent, positive control group reagent, and experimental group reagent respectively, and culture for 72 hours. During this period, observe the cell status and culture medium every day and replace with fresh blank control reagent, positive control group reagent, and experimental group reagent.

[0169] 3. Cell collection: After removing the culture supernatant, wash once with PBS. Add 200 μl of 0.25% trypsin to each well to digest the cells, and place them in a CO2 incubator for 2 min. Add 400 μl of PBS containing 10% FBS to terminate the digestion and blow the cells, collect the cells in a centrifuge tube, centrifuge at 4000 rpm for 5 min, and discard the supernatant.

[0170] 4. Testing: Add 100 μl of melanin extract to each tube, shake well, and heat in an 80°C water bath for 1 hour. After cooling, centrifuge the tube wall droplets moderately, blow and beat evenly, pipette 90 μl of solution from each centrifuge tube, transfer to a 96-well plate, and use an ELISA reader to measure the absorbance of each well at a wavelength of 405 nm.

[0171] 5. Data Analysis: Melanin synthesis inhibition rate = [1-(As-Ab) / (Ac-Ab)] × 100%, As: absorbance of the experimental group or positive control group, Ac: absorbance of the negative control well, Ab: absorbance of the blank control group. The results are shown in Table 8.

[0172] Table 8: In vitro melanocyte melanin synthesis inhibition assay results

[0173] The results show that the composition of Example 4 can significantly inhibit the melanin synthesis of melanocytes to achieve a whitening effect, and the effect is better than niacinamide and the raw materials of each component in the composition.

[0174] Example 13: In vitro keratinocyte proliferation assay Cell proliferation is one of the important physiological functions of living cells and an important life characteristic of organisms. It indicates cell vitality and status. The epidermis is located on the surface of the skin. It is thin but tough. Most epidermal cells are keratinocytes. Keratinocytes have functions such as proliferation, differentiation, migration and secretion of extracellular components. They are the determining factors for whether the skin can return to a healthy state. The CCK-8 method is based on the highly water-soluble tetrazolium salt WST-8 to measure cell proliferation / toxicity. WST-8 can be reduced to a water-soluble orange-yellow formazan product under the action of an electron mediator, producing a color reaction. The more and faster the cell proliferation, the darker the color; the greater the cell toxicity, the lighter the color. For the same cell, the depth of the color is proportional to the number of living cells. Hacat immortalized human keratinocytes can be used as a cell model for studying the proliferation of keratinocytes in vitro for cosmetics. On the basis that the test met the effectiveness verification, the cell proliferation in the experimental group was upregulated compared with the blank control group, and there was a significant difference (P<0.05), indicating that the experimental sample has the ability to promote cell proliferation at the tested concentration, which can be used as one of the evidences to support the title of repair cosmetic raw material.

[0175] Determination method: 1. Cell plating: Remove the cells from the carbon dioxide incubator, observe the cell growth under a microscope, and randomly select two fields of view to take pictures using a low-power microscope and a high-power microscope. When the cell density reaches 80%-90%, it is confirmed that it can be harvested; wipe the outer surface of the culture bottle with a dust-free cloth containing alcohol and transfer it to the biosafety cabinet. Gently shake the culture bottle back and forth and use a pipette to remove the old culture medium. Use an appropriate amount of PBS to wash the cell surface once, add an appropriate amount of digestive enzyme to the culture bottle, and digest at room temperature for 5-8 min. After gently shaking, observe the cells with the naked eye and they appear to be quicksand-like. Under the microscope, the cells appear to be spherical, indicating that the digestion is complete. Add an appropriate amount of stop solution to stop the digestion, transfer the liquid to a centrifuge tube, and centrifuge at 400 g for 5 min; resuspend with DMEM complete culture medium and take 20 μL for counting. According to the density of 2×10 4 / well inoculated into a 96-well plate: Take the required amount of cells and dilute the cells to 2×10 5 / ml, mix well and add the cell suspension to a 96-well plate, 100 μL / well. Culture the cells for 24 hours before use.

[0176] 2. Drug treatment: Treatment drugs were prepared, 400 μL for each group, blank control group: DMEM basal medium, ceramide (purchased from MedChemExpress) experimental group: 40 μL ceramide (1 mg / ml storage mother solution) + 400 μL DMEM basal medium, Spirulina platensis exosomes experimental group: 40 μL Spirulina platensis exosomes (1 mg / ml storage mother solution) + 400 μL DMEM basal medium, olive leaf exosomes experimental group: 40 μL olive leaf exosomes (1 mg / ml storage mother solution) + 400 μL DMEM basal medium, white peony exosomes experimental group: 40 μL white peony exosomes (1 mg / ml storage mother solution) + 400 μL DMEM basal medium, combination experimental group: 40 μL combination (1 mg / ml storage mother solution) + 400 μL DMEM basal medium; discard the cell supernatant, add 100 μL blank control reagent, positive control group reagent, and experimental group reagent, respectively, and culture for 24 h.

[0177] 3. CCK8 detection (refer to the instructions of Abbkine SuperKine™ Ultrasensitive Cell Proliferation Detection Reagent Kit): 1) Prepare a solution containing 10% CCK8 reagent using culture medium.

[0178] 2) Add 100 μL of CCK8 solution to each well and continue incubating in the cell culture incubator for 2-4 hours.

[0179] 3) Measure the absorbance at 450 nm.

[0180] 4. Data Analysis: Cell proliferation rate %=OD experimental group / OD blank control group×100%, OD experimental group: absorbance value of experimental group after drug treatment, OD blank control group: absorbance value of blank control group after drug treatment. The results are shown in Table 9.

[0181] Table 9: Results of in vitro keratinocyte proliferation assay

[0182] The results show that the composition of Example 4 can significantly improve the cell viability of keratinocytes to achieve a repairing effect, and the effect is better than ceramide and the raw materials of each component in the composition.

[0183] Example 14: Human face anti-wrinkle test In order to evaluate the anti-wrinkle test results of the composition provided by the present invention on the human face, 5 testers aged 35 and above were recruited. The composition provided by the present invention was non-invasively introduced into the testers' faces with the help of the Shumin Star introduction instrument. The use interval was once every 3 weeks, and it was used twice in total. The dosage was 6 mL each time, and the concentration was 0.4 mg / mL. The effects were evaluated 7 days and 21 days after a single introduction, and 7 days and 21 days after the second introduction. The static lines and textures such as dry lines and wrinkles on the face were photographed by a camera (Eagle Eye).

[0184] According to the comprehensive data of the length, depth and width of the lines, AI simulation scoring is performed. The higher the ratio, the more serious the wrinkles. The statistical results are shown in Table 10. Among them, D0 means before using the composition provided by the present invention, D7 means 7 days after the first use of the composition provided by the present invention, D21 means 21 days after the first use of the composition provided by the present invention, D28 means 7 days after the second use of the composition provided by the present invention, and D42 means 21 days after the second use of the composition provided by the present invention.

[0185] Table 10: Statistical results of human face anti-wrinkle data

[0186] The results showed that the facial wrinkles of the five test subjects were improved to varying degrees after twice introducing the composition provided by the present invention.

[0187] It should be understood that the above embodiments are exemplary and are not intended to include all possible implementations included in the claims. Various modifications and changes may be made on the basis of the above embodiments without departing from the scope of the present disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only express several implementations of the present invention and do not limit the scope of protection of the patent of the present invention.

Claims

1. A plant exosome composition, characterized in that: The composition comprises: two or more of the following: exosomes from Spirulina platensis, exosomes from olive leaves, and exosomes from white peony root.

2. The composition according to claim 1, characterized in that The composition comprises: exosomes from Spirulina platensis, exosomes from olive leaves and exosomes from white peony root.

3. The composition according to claim 2, characterized in that The mass ratio of the Spirulina platensis exosomes, the olive leaf exosomes and the white peony root exosomes is 1-10:1-5:1-5.

4. The composition according to claim 3, characterized in that The mass ratio of the Spirulina platensis exosomes, the olive leaf exosomes and the white peony root exosomes is 2-5:1-3:1-3.

5. The composition according to claim 4, characterized in that The mass ratio of the Spirulina platensis exosomes, the olive leaf exosomes and the white peony root exosomes is 2:1:

1.

6. The composition according to claim 1, characterized in that The preparation method of the composition comprises: extracting exosomes from Spirulina platensis, exosomes from olive leaves, and exosomes from white peony root respectively, and then compounding them according to proportions.

7. The composition according to claim 6, characterized in that The extraction of the exosomes from Spirulina platensis, olive leaves and white peony root comprises: taking Spirulina platensis, olive leaves and white peony root respectively, breaking the wall after adding a solvent, collecting the supernatant by gradient centrifugation; diluting the obtained supernatant, filtering, concentrating and purifying.

8. The composition according to claim 7, characterized in that The solvent is PB buffer and / or PBS buffer; Preferably, the dilution is to a turbidity of less than 900 NTU; Preferably, the filtration is performed using a tangential flow depth filtration membrane package; Preferably, the concentration and purification is performed using a hollow fiber column.

9. Use of the composition according to any one of claims 1 to 8 in preparing a skin care product.

10. The use according to claim 9, characterized in that: The skin care products include products with anti-aging effects, products with firming effects, products with anti-wrinkle effects, products with whitening effects, and products with repairing effects.

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