A plant exosome composition, its preparation method and application

Abstract

The present invention relates to a plant exosome composition, which comprises two or more of the following: exosomes of Spirulina platensis, exosomes of Olea europaea leaf, and exosomes of Paeonia lactiflora Pall. The plant exosome composition of the present invention uses exosomes of Spirulina platensis as the main component, and adds exosomes of Olea europaea leaf, exosomes of Paeonia lactiflora Pall, and a protective agent, and has multiple effects of firming, anti-wrinkle, anti-aging, whitening, and repair. The extracted Spirulina platensis exosomes of the present invention not only contain phycocyanin components, but also contain active factors secreted by Spirulina platensis, etc., and the fishy smell carried by Spirulina platensis is removed in terms of smell, and can have long-term stability after adding a protective agent and freeze-drying.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine technology, and particularly to a plant exosome composition, a preparation method thereof, and an application thereof. Background Art

[0002] The skin is a barrier between the human body and the external environment and is an important organ for protecting the body against external changes; the skin also has multiple functions such as defending against ultraviolet rays, regulating body temperature, and neutralizing external stimuli. With the changes in the surrounding environment and the influence of aging, the functions and state of the skin will be damaged. The mission of skin care cosmetics is to reduce the damage to the skin functions and state caused by the external environment and aging.

[0003] At present, the types of spirulina applied in the cosmetics field are mainly Spirulina platensis Geitler and Spirulina maxima (refer to the comparative patents CN118436581A and CN117959236A), and mainly focus on extracting phycocyanin from them. Compared with Spirulina platensis Geitler and Spirulina maxima which are currently used more, Spirulina maxima has a higher protein content, a higher phycocyanin content, and a higher SOD activity.

[0004] At present, the application of spirulina extract in the cosmetics field mainly focuses on the phycocyanin component. However, the active factors secreted by spirulina also have good effects, and phycocyanin has some specific disadvantages of its own: (1) The fishy smell carried by spirulina results in a poor taste experience when used. (2) Stability. Phycocyanin is a natural protein, and proteins generally 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 applied to the two effects of anti-wrinkle and firming of the skin. However, people also have requirements for other effects of cosmetics such as anti-aging, whitening, and repair. Summary of the Invention

[0006] Problems to be Solved by the Invention

[0007] Aiming at the above problems existing in the prior art, the purpose of the present invention is to provide a spirulina extract product with multiple effects such as firming, anti-wrinkle, anti-aging, whitening, and repair.

[0008] Solutions for Solving the Problems

[0009] The present invention provides a plant exosome composition, which comprises two or more of exosomes of Spirulina maxima, exosomes of Olea europaea leaves, and exosomes of Paeonia lactiflora Pall.

[0010] Preferably, the composition comprises exosomes of Spirulina maxima, exosomes of Olea europaea leaves, and exosomes of Paeonia lactiflora Pall.

[0011] Preferably, the mass ratio of the Spirulina platensis exosomes, Olea europaea leaf exosomes, and Paeonia lactiflora exosomes is 1-10:1-5:1-5.

[0012] Preferably, the mass ratio of the Spirulina platensis exosomes, Olea europaea leaf exosomes, and Paeonia lactiflora exosomes is 2-5:1-3:1-3.

[0013] Preferably, the mass ratio of the Spirulina platensis exosomes, Olea europaea leaf exosomes, and Paeonia lactiflora exosomes is 2:1:1.

[0014] Preferably, the preparation method of the composition includes: separately extracting Spirulina platensis exosomes, Olea europaea leaf exosomes, and Paeonia lactiflora exosomes, and then compounding them in proportion.

[0015] Preferably, the extraction of the Spirulina platensis exosomes, Olea europaea leaf exosomes, and Paeonia lactiflora exosomes includes: respectively taking Spirulina platensis, Olea europaea leaves, and Paeonia lactiflora, adding a solvent to break the cell walls, and collecting the supernatant by gradient centrifugation; diluting the obtained supernatant, filtering, and concentrating and purifying.

[0016] Preferably, the solvent is PB buffer and / or PBS buffer.

[0017] Preferably, the dilution is to a turbidity less than 900 NTU.

[0018] Preferably, the filtration is deep filtration using a tangential flow deep filtration membrane module.

[0019] Preferably, the concentration and purification are carried out using a hollow fiber column.

[0020] The present invention also provides an application of the composition in the preparation of skin care products.

[0021] 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 repair effects.

[0022] Effects of the Invention

[0023] The plant exosome composition of the present invention uses Spirulina platensis exosomes as the main component, and adds Olea europaea leaf exosomes and Paeonia lactiflora exosomes, having multiple effects of firming, anti-wrinkle, anti-aging, whitening, and repair. The extracted Spirulina platensis exosomes of the present invention not only contain phycocyanin components, but also contain active factors secreted by Spirulina platensis, and the fishy smell carried by Spirulina platensis is removed in terms of smell. Specific Embodiments

[0024] To make the technical solutions and beneficial effects of the present invention more obvious and understandable, the following will be described in detail by way of specific examples. Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical and scientific fields to which this application belongs.

[0025] The present invention provides a plant exosome composition, which comprises two or more of Spirulina platensis exosomes, Olea europaea leaf exosomes, and Paeonia lactiflora exosomes.

[0026] In certain embodiments, the composition comprises Spirulina platensis exosomes, Olea europaea leaf exosomes, and Paeonia lactiflora exosomes.

[0027] In certain embodiments, the composition consists of Spirulina platensis exosomes, Olea europaea leaf exosomes, and Paeonia lactiflora exosomes.

[0028] In certain embodiments, the composition further comprises a cryoprotectant.

[0029] In certain embodiments, the composition consists of Spirulina platensis exosomes, Olea europaea leaf exosomes, Paeonia lactiflora exosomes, and a cryoprotectant.

[0030] In certain embodiments, the mass ratio of the Spirulina platensis exosomes, Olea europaea leaf exosomes, and Paeonia lactiflora exosomes is 1 to 10: 1 to 5: 1 to 5.

[0031] In some embodiments, the mass ratio of the Spirulina platensis exosomes, Olea europaea leaf exosomes, and Paeonia lactiflora 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 2:2:1, 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 7:1:1or 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.

[0032] In some embodiments, the mass ratio of the Spirulina platensis exosomes, Olea europaea leaf exosomes and Paeonia lactiflora Pall. exosomes is 2 - 5:1 - 3:1 - 3.

[0033] In some embodiments, the mass ratio of the Spirulina platensis exosomes, Olea europaea leaf exosomes and Paeonia lactiflora Pall. exosomes is 2:1:1.

[0034] In some embodiments, the preparation method of the composition includes: separately extracting Spirulina platensis exosomes, Olea europaea leaf exosomes and Paeonia lactiflora Pall. exosomes, and then compounding them in proportion.

[0035] In some embodiments, the extraction of the Spirulina platensis exosomes, Olea europaea leaf exosomes and Paeonia lactiflora Pall. exosomes includes: separately taking Spirulina platensis, Olea europaea leaves and Paeonia lactiflora Pall., adding a solvent to break the cell walls, and collecting the supernatant by gradient centrifugation; diluting the obtained supernatant, filtering, and concentrating and purifying.

[0036] In some embodiments, the solvent is PB buffer and / or PBS buffer.

[0037] In some embodiments, the solvent is PB buffer.

[0038] In some embodiments, the solvent is PBS buffer.

[0039] In some embodiments, the specific steps of the gradient centrifugation include:

[0040] (1) Centrifuge at 400 - 800 g for 5 - 20 min;

[0041] (2) Centrifuge at 1000 - 3000 g for 10 - 30 min;

[0042] (3) Centrifuge at 3000 - 5000 g for 20 - 40 min;

[0043] (4) Centrifuge at 8000 - 12000 g for 40 - 70 min.

[0044] In some embodiments, the specific steps of the gradient centrifugation include:

[0045] (1) Centrifuge at 500 g for 10 min;

[0046] (2) Centrifuge at 2000 g for 20 min;

[0047] (3) Centrifuge at 4000 g for 30 min;

[0048] (4) Centrifuge at 10000 g for 60 min.

[0049] In some embodiments, the dilution is to a turbidity less than 900 NTU.

[0050] In some embodiments, the filtration is deep filtration using a tangential flow deep filtration membrane module.

[0051] In some embodiments, the concentration and purification are carried out using a hollow fiber column.

[0052] The present invention also provides an application of the composition in the preparation of skin care products.

[0053] In some 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 repair effects.

[0054] Example 1: Extraction of exosomes from Spirulina platensis

[0055] 1. Cell wall breaking treatment: Transfer the powder of Spirulina platensis to a cell wall breaker, add an appropriate amount of PB buffer solution, and perform cell wall breaking treatment until it is completely mixed.

[0056] 2. Pretreatment centrifugation: Transfer the cell wall broken solution of Spirulina platensis in step 1 to a centrifuge tube, and use a tabletop centrifuge to set the temperature at 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] 3. Supernatant dilution: Calibrate the turbidimeter using a standard product with a turbidity of 800 NTU, take the supernatant obtained in step 2 for turbidity measurement, if the turbidity is greater than 900 NTU, dilute it with PB buffer solution until the turbidity is less than 900 NTU.

[0058] 4. Depth filtration: Pass the diluted supernatant in step 3 through a tangential flow depth filtration membrane module for depth filtration, and collect the liquid after depth filtration.

[0059] 5. Concentration and purification: Pass the depth filtrate obtained in step 4 through a hollow fiber column for concentration and purification, and the suspension obtained after concentration is the exosomes of Spirulina platensis.

[0060] Example 2: Extraction of olive leaf exosomes

[0061] 1. Raw material cleaning: Clean the olive leaf raw materials 3 times with purified water.

[0062] 2. Cell wall breaking treatment: Transfer the olive leaves cleaned in step 1 to a cell wall breaker, add an appropriate amount of PB buffer solution, and perform cell wall breaking treatment until it is completely mixed.

[0063] 3. Pretreatment centrifugation: Transfer the cell wall broken solution of olive leaves obtained in step 2 to a centrifuge tube, and use a tabletop centrifuge to set the temperature at 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.

[0064] 4. Supernatant dilution: Calibrate the turbidimeter using a standard product with a turbidity of 800 NTU, take the supernatant obtained in step 3 for turbidity measurement, if the turbidity is greater than 900 NTU, dilute it with PB buffer solution until the turbidity is less than 900 NTU.

[0065] 5. Depth filtration: Pass the diluted supernatant in step 4 through a tangential flow depth filtration membrane module for depth filtration, and collect the liquid after depth filtration.

[0066] 6. Concentration and purification: Concentrate and purify the deep filtration liquid obtained in step 5 through a hollow fiber column. The suspension obtained after concentration is the olive leaf exosomes.

[0067] Example 3: Extraction of Paeonia lactiflora exosomes

[0068] 1. Raw material cleaning: Clean the sliced raw material of Paeonia lactiflora rhizome 3 times with purified water.

[0069] 2. Cell wall breaking treatment: Transfer the cleaned sliced Paeonia lactiflora rhizome obtained in step 1 to a cell wall breaker, add an appropriate amount of PB buffer for cell wall breaking treatment until completely mixed.

[0070] 3. Pretreatment centrifugation: Transfer the cell wall broken liquid of the sliced Paeonia lactiflora rhizome obtained in step 2 to a centrifuge tube, and use a tabletop centrifuge to set the temperature at 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.

[0071] 4. Supernatant dilution: Calibrate the turbidimeter with a standard product with a turbidity of 800 NTU, take the supernatant obtained in step 3 for turbidity measurement, and if the turbidity is greater than 900 NTU, dilute it with PB buffer until the turbidity is less than 900 NTU.

[0072] 5. Depth filtration: Deeply filter the diluted supernatant obtained in step 4 through a tangential flow depth filtration membrane module, and collect the liquid after depth filtration.

[0073] 6. Concentration and purification: Concentrate and purify the deep filtration liquid obtained in step 5 through a hollow fiber column. The suspension obtained after concentration is the Paeonia lactiflora exosomes.

[0074] Example 4: Preparation of exosome composition

[0075] Compound the Spirulina platensis exosomes, olive leaf exosomes, and Paeonia lactiflora exosomes extracted in Examples 1-3 in a ratio of 2:1:1 to prepare an exosome composition.

[0076] Example 5: Determination of TIMP1 protein content in vitro dermal fibroblasts

[0077] Tissue inhibitor of metalloproteinase-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. By irradiating cells with UVA to simulate the reduction of TIMP1 levels caused by photoaging, the upregulation rate of TIMP1 protein content was measured after administering the test substance, and whether the test substance has an effect in promoting the synthesis of TIMP1 protein was evaluated. On the basis that the test meets the validation of effectiveness, compared with the blank control, the content of TIMP1 protein was upregulated in the test substance, and there was a significant difference (P<0.05), indicating that the test substance has the ability to promote the synthesis of TIMP1 protein at the tested concentration, which can be used as one of the evidence supports for the appellation of anti-aging cosmetic raw materials.

[0078] Determination method:

[0079] 1. Cell seeding:

[0080] Take out the cells from the carbon dioxide incubator, observe the cell growth under the microscope, and randomly select 2 fields of view to take pictures using a low-power microscope (4X) and a high-power microscope (10X) respectively. When the cell density reaches 80%-90%, it is confirmed that harvesting can be carried out. Wipe the outer surface of the culture flask with a dust-free cloth containing alcohol and transfer it into the biosafety cabinet. Gently shake the culture flask back and forth, and use a pipette to remove the old culture medium. Wash the cell surface with an appropriate amount of PBS once, add an appropriate amount of digestive enzyme to the culture flask, and digest at room temperature for 3-5 min. After gently shaking, observe that the cells are in a flowing sand shape with the naked eye, and under the microscope, when the cells are spherical, it indicates that the digestion is complete. Add an appropriate amount of PBS to terminate the digestion, pipette and blow the bottom of the culture flask, and transfer the liquid into a centrifuge tube. The centrifugation parameters are 400 g for 5 min. 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 in each group, and culture the cells for 24 h.

[0081] 2. UVA induction:

[0082] Place ice packs in the biosafety cabinet, lay four layers of kraft paper on the ice packs, and then place the cell culture plate on the kraft paper. Adjust the position of the UVA lamp tube so that the distance from the lamp tube to the cells is 5 cm. Remove the cell culture plate, turn on the lamp tube and balance for 5 min, then place the cell culture plate under the lamp tube, open the lid, and irradiate for 1 h. The radiation energy is about 5 J / cm 2 。

[0083] 3. Drug treatment:

[0084] Prepare treatment drugs, 400 μL for each group. Blank control group: T4 basal medium, ergothioneine (purchased from MERCK). Experimental groups: 40 μL ergothioneine (1 mg / ml stock solution) + 400 μL T4 basal medium; Experimental group of Spirulina platensis exosomes: 40 μL Spirulina platensis exosomes (1 mg / ml stock solution) + 400 μL T4 basal medium; Experimental group of Olea europaea leaf exosomes: 40 μL Olea europaea leaf exosomes (1 mg / ml stock solution) + 400 μL T4 basal medium; Experimental group of Paeonia lactiflora Pall. exosomes: 40 μL Paeonia lactiflora Pall. exosomes (1 mg / ml stock solution) + 400 μL T4 basal medium; Experimental group of composition: 40 μL composition (1 mg / ml stock solution) + 400 μL T4 basal medium.

[0085] After UVA irradiation, discard the PBS, and add 100 μL of the prepared blank control, ergothioneine, Spirulina platensis exosomes, Olea europaea leaf exosomes, Paeonia lactiflora Pall. exosomes, and composition respectively, and culture for 24 h.

[0086] 4. Detection of TIMP1 protein (refer to the instruction manual of the United Science Human TIMP1 ELISA Kit detection kit)

[0087] Collect the culture supernatant, centrifuge at 3000 g for 10 min to remove impurities, and detect according to the TIMP1 kit. If detection cannot be carried out in time, aliquot the supernatant into 50 μL per tube and store at -20 °C. Prepare all the required reagents and working concentration standards. Remove the unnecessary strips, put them back into the aluminum foil bag containing desiccant, and reseal the bag.

[0088] 1) Soak the ELISA plate: Add 300 μL of 1× washing solution and let it stand for 30 seconds. After discarding the washing solution, pat the well plate dry on the absorbent paper. After washing the plate, immediately use the microplate without allowing it to dry.

[0089] 2) Add standards: Add 100 μL of 2-fold serial dilution standards to the standard wells. Add 100 μL of standard diluent to the blank well.

[0090] 3) Add samples: Add 50 μL of cell culture supernatant to the sample wells, and supplement each well with 50 μL of PBS for 2-fold dilution.

[0091] 4) Incubation: Seal the plate with a sealing film. Place it on a horizontal shaker and incubate at room temperature (25 °C ± 3 °C) for 2 hours.

[0092] 5) Washing: Discard the liquid, add 300 μL of washing solution to each well to wash the plate, and wash 3 times. Pat the microplate dry on the absorbent paper.

[0093] 6) Add detection antibody: Add 100 μL of the detection antibody working solution (diluted 1:100) to each well.

[0094] 7) Incubation: Seal the plate with a sealing film. Incubate on a horizontal shaker at room temperature (25°C ± 3°C) for 1 hour.

[0095] 8) Washing: Repeat step 5.

[0096] 9) Add enzyme: Add 100 μL of the streptavidin working solution (diluted 1:100) to each well.

[0097] 10) Incubation: Seal the plate with a sealing film. Incubate on a horizontal shaker at room temperature (25°C ± 3°C) for 45 minutes.

[0098] 11) Washing: Repeat step 5.

[0099] 12) Add substrate for color development: Add 100 μL of the colorimetric substrate to each well. Incubate at room temperature (25°C ± 3°C) in the dark for 20 minutes.

[0100] 13) Add stop solution: Add 100 μL of the stop solution to each well. Gently tap to mix evenly, and the color changes from blue to yellow.

[0101] 14) Detection and reading: Measure the maximum absorption wavelength at 450 nm within 30 minutes.

[0102] 15) Make a standard curve based on the A450 value of the standard product. The abscissa is the TIMP1 protein concentration, and the ordinate is the A450 value. Calculate the amount of TIMP1 protein in the sample according to the formula of the standard curve. The results are shown in Table 1.

[0103] Table 1: Results of the determination of TIMP1 protein content in vitro in dermal fibroblasts

[0104]

[0105] The results show that the composition of Example 4 can significantly promote the secretion of the matrix metalloproteinase inhibitor TIMP1, and the effect is better than that of ergothioneine and each component raw material in the composition.

[0106] Example 6: Determination of MMP1 protein content in vitro in dermal fibroblasts

[0107] 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.

[0108] Determination method:

[0109] 1. Cell plating:

[0110] 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.

[0111] 2. UVA Induction:

[0112] 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 .

[0113] 3. Drug treatment:

[0114] 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.

[0115] 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.

[0116] 4. MMP1 protein detection (refer to the instructions of Lianke Human MMP1 ELISA Kit):

[0117] 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 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 4) Incubation: Seal the plate with a sealing film and place in a mixer at room temperature (25℃±3℃) for 2 hours.

[0122] 5) Washing: Discard the liquid. Add 300 μL of washing solution to each well and wash the plate 3 times. Pat the microplate dry on absorbent paper.

[0123] 6) Adding detection antibody: Add 100 μL of the detection antibody working solution (diluted 1:100) to each well.

[0124] 7) Incubation: Seal the plate with a sealing film. Place it on a shaker and incubate at room temperature (25°C ± 3°C) for 1 hour.

[0125] 8) Washing: Repeat step 5.

[0126] 9) Adding enzyme: Add 100 μL of the streptavidin working solution (diluted 1:100) to each well.

[0127] 10) Incubation: Seal the plate with a sealing film. Place it on a shaker and incubate at room temperature (25°C ± 3°C) for 45 minutes.

[0128] 11) Washing: Repeat step 5.

[0129] 12) Adding substrate for color development: Add 100 μL of the color development substrate to each well and incubate in the dark at room temperature (25°C ± 3°C) for 20 minutes.

[0130] 13) Adding stop solution: Add 100 μL of the stop solution to each well. Gently tap to mix, and the color changes from blue to yellow.

[0131] 14) Detection and reading: Measure the maximum absorption wavelength at 450 nm within 30 minutes.

[0132] 15) Make a standard curve based on the A450 values of the standards. The abscissa is the MMP1 protein concentration, and the ordinate is the A450 value. Calculate the amount of MMP1 protein in the samples according to the formula of the standard curve. The results are shown in Table 2.

[0133] Table 2: Results of the determination of MMP1 protein content in vitro in dermal fibroblasts

[0134]

[0135] The results show that the composition of Example 4 can significantly inhibit the secretion of matrix metalloproteinase MMP1 in dermal fibroblasts, and the effect is better than that of hydroxyproline and each component raw material in the composition.

[0136] Example 7: Determination of SA-β-Gal staining in vitro in dermal fibroblasts

[0137] SA-β-gal staining is a commonly used method for detecting cell senescence, which evaluates the cell senescence status by detecting the activity of acidic β-galactosidase in cells. The activity of acidic β-galactosidase increases in senescent cells. The β-galactosidase staining kit uses X-Gal as a substrate, and under the catalysis of senescence-specific β-galactosidase, a dark blue product will be generated. UVA irradiation of cells is used to simulate photoaging, resulting in an increase in intracellular acidic β-galactosidase. The test substance is administered to inhibit the upregulation of acidic β-galactosidase. The efficacy of the test substance in inhibiting cell senescence is evaluated by the number of SA-β-gal staining positive cells. On the basis that the test meets the validation of effectiveness, compared with the blank control, the number of SA-β-gal staining positive cells is downregulated, and there is a significant difference (P<0.05), indicating that the test substance has the ability to inhibit cell senescence at the tested concentration, which can be used as one of the evidence supports for the claim of anti-aging cosmetic raw materials.

[0138] Determination method:

[0139] 1. Cell seeding:

[0140] Take out the cells from the carbon dioxide incubator, observe the cell growth under the microscope, and randomly select 2 fields of view to take pictures using a low-power microscope (4X) and a high-power microscope (10X) respectively. When the cell density reaches 80%-90%, it is confirmed that harvesting can be carried out. Wipe the outer surface of the culture flask with a dust-free cloth containing alcohol and transfer it into the biosafety cabinet. Gently shake the culture flask back and forth, and use a pipette to remove the old medium. Wash the cell surface with an appropriate amount of PBS once, add an appropriate amount of digestive enzyme to the culture flask, and digest at room temperature for 3-5 min. After gently shaking, observe with the naked eye that the cells are in a flowing sand state. Under the microscope, when the cells are spherical, it indicates that the digestion is complete. Add an appropriate amount of PBS to terminate the digestion. After pipetting the bottom of the culture flask, transfer the liquid into a centrifuge tube, and the centrifugation parameters are 400 g for 5 min. Resuspend with complete medium, and take 20 μL for cell counting. Seed into a 24-well plate at a density of 10,000 cells / 500 μL / well, with 3 replicates in each group. Culture the cells for 24 h.

[0141] 2. UVA induction:

[0142] Place ice packs in the biosafety cabinet, lay four layers of kraft paper on the ice packs, and then place the cell culture plate on the kraft paper. Adjust the position of the UVA lamp tube so that the distance from the lamp tube to the cells is 5 cm. Remove the cell culture plate, turn on the lamp tube and balance for 5 min, discard the medium, add 200 μL of pre-cooled PBS, and then place the cell culture plate under the lamp tube, open the lid, and irradiate for 1 h. The radiation energy is about 5 J / cm 2 .

[0143] 3. Drug treatment:

[0144] Prepare treatment drugs, 2 mL for each group. Blank control group: T4 basal medium, TGF-β (purchased from MERCK). Experimental groups: 200 μL TGF-β (1 mg / ml stock solution) + 2 mL T4 basal medium; Experimental group of Spirulina platensis exosomes: 200 μL Spirulina platensis exosomes (1 mg / ml stock solution) + 2 mL T4 basal medium; Experimental group of Olea europaea leaf exosomes: 200 μL Olea europaea leaf exosomes (1 mg / ml stock solution) + 2 mL T4 basal medium; Experimental group of Paeonia lactiflora Pall. exosomes: 200 μL Paeonia lactiflora Pall. exosomes (1 mg / ml stock solution) + 2 mL T4 basal medium; Experimental group of the composition: 200 μL composition (1 mg / ml stock solution) + 2 mL T4 basal medium.

[0145] After UVA irradiation, discard the PBS, and add 500 μL of the prepared blank control, TGF-β, Spirulina platensis exosomes, Olea europaea leaf exosomes, Paeonia lactiflora Pall. exosomes, and the composition respectively, and culture for 24 h.

[0146] 4. Re-induce with UVA and drug treatment:

[0147] Repeat the operation steps of 2 and 3 in Example 7.

[0148] 5. SA-β-gal staining (refer to the instruction manual of the β-galactosidase staining kit from Shangbao Biotechnology):

[0149] 1) Aspirate the cell culture medium, wash once with PBS, add 200 μL of β-galactosidase staining fixative, and fix at room temperature for 15 minutes.

[0150] 2) Aspirate the cell fixative, wash the cells 3 times with PBS, 3 minutes each time.

[0151] 3) Aspirate the PBS, and add 300 μL of staining working solution to each well.

[0152] 4) Place in a biochemical incubator and incubate overnight at 37 °C. The well plate can be sealed with a sealing film to prevent evaporation.

[0153] 5) Wash the cells 3 times with PBS, 3 minutes each time, and observe under an ordinary optical microscope. If counting cannot be performed in time, it can be stored at 4 °C for one week; or after adding a cover slip mounting solution, it can be stored at 4 °C for a longer time.

[0154] 6) Data analysis: Use Image J to count the number of SA-β-Gal+ cells and total cells, SA-β-Gal + (%) = SA-β-Gal +Cell / total cell, the experimental group was compared with the blank control group, and the results are shown in Table 3.

[0155] Table 3: Results of SA-β-Gal staining assay of in vitro dermal fibroblasts

[0156]

[0157] The results showed that the composition of Example 4 could significantly down-regulate the amount of SA-β-gal staining positive cells, and the effect was better than that of TGF-β and each component raw material in the composition.

[0158] Example 8: Proliferation assay of in vitro dermal fibroblasts

[0159] Cell proliferation is one of the important physiological functions of living cells and an important vital feature of organisms. In addition to secreting collagen fibers and elastic fibers to maintain skin elasticity, fibroblasts also have adhesiveness, which plays an important role in maintaining the elasticity and toughness of the skin. The decrease in the proliferation activity of fibroblasts is one of the important reasons for the formation of wrinkles. The CCK-8 method is based on the highly water-soluble tetrazolium salt WST-8 to measure cell proliferation / toxicity. Under the action of an electron mediator, WST-8 can be reduced to generate a water-soluble orange-yellow formazan product, producing a color reaction. The more and faster the cells proliferate, the darker the color; the greater the cell toxicity, the lighter the color. For the same cells, the depth of the color is proportional to the number of living cells. Dermal fibroblasts can be used as a cell model for studying the proliferation of in vitro dermal fibroblasts in cosmetics. On the basis of meeting the validity verification of the experiment, compared with the blank control group, the cell proliferation in the experimental group was up-regulated, and there was a significant difference (P < 0.05), indicating that the experimental sample had the ability to promote cell proliferation at the tested concentration, which could be used as one of the evidence supports for the title of raw materials for firming cosmetics.

[0160] Determination method:

[0161] 1. Cell seeding:

[0162] Remove the cells from the carbon dioxide incubator, observe the cell growth under the microscope, and randomly select 2 fields of view to take pictures using a low-power microscope (4X) and a high-power microscope (10X) respectively. When the cell density reaches 80%-90%, confirm that harvesting can be carried out. Wipe the outer surface of the culture flask with a lint-free cloth containing alcohol and transfer it into the biosafety cabinet. Gently shake the culture flask back and forth, and use a pipette to remove the old culture medium. Wash the cell surface once with an appropriate amount of PBS, add an appropriate amount of digestive enzyme to the culture flask, and digest at room temperature for 3-5 min. After gently shaking, observe with the naked eye that the cells are in a flowing sand state. Under the microscope, when the cells are spherical, it indicates complete digestion. Add an appropriate amount of PBS to terminate the digestion. After pipetting the bottom of the culture flask, transfer the liquid into a centrifuge tube, and centrifuge at 400 g for 5 min. 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 in each group, and culture the cells for 24 h.

[0163] 2. Drug treatment:

[0164] Prepare the treatment drugs, 400 μL for each group. Blank control group: T4 basal medium. Astaxanthin (purchased from Biotopped) experimental group: 40 μL of astaxanthin (1 mg / ml stock solution) + 400 μL of T4 basal medium. Exosome of Spirulina platensis experimental group: 40 μL of exosome of Spirulina platensis (1 mg / ml stock solution) + 400 μL of T4 basal medium. Exosome of Olea europaea leaf experimental group: 40 μL of exosome of Olea europaea leaf (1 mg / ml stock solution) + 400 μL of T4 basal medium. Exosome of Paeonia lactiflora experimental group: 40 μL of exosome of Paeonia lactiflora (1 mg / ml stock solution) + 400 μL of T4 basal medium. Composition experimental group: 40 μL of composition (1 mg / ml stock solution) + 400 μL of T4 basal medium; Discard the cell supernatant, and add 100 μL of negative control reagent, positive control group reagent, and experimental group reagent respectively, and culture for 24 h.

[0165] 3. CCK8 assay (refer to the instruction manual of Abbkine SuperKine™ Ultra-sensitive Cell Proliferation Detection Reagent Kit):

[0166] 1) Prepare a detection solution containing 10% CCK8 reagent using basal medium.

[0167] 2) After removing the culture medium in the well plate, add 100 μL of CCK8 solution to each well and continue to incubate in the cell incubator for 2 hours.

[0168] 3) Measure the absorbance at 450 nm.

[0169] 4) Data analysis: Cell proliferation rate % = OD experimental group / OD blank control group × 100%, where OD experimental group is the absorbance value after treatment with the test drug, and OD blank control group is the absorbance value after treatment with the blank control drug. The results are shown in Table 4.

[0170] Table 4: Results of in vitro dermal fibroblast proliferation assay

[0171]

[0172] The results showed that the composition of Example 4 could significantly promote the cell proliferation of dermal fibroblasts, thus achieving a firming effect, and the effect was better than that of astaxanthin and each component raw material in the composition.

[0173] Example 9: Determination of type I collagen content in in vitro dermal fibroblasts

[0174] Type I collagen is one of the main components of the dermal extracellular matrix. Type I procollagen is synthesized intracellularly by dermal fibroblasts, secreted extracellularly, and polymerized into collagen fibers after the telopeptides are separated under the action of terminal procollagen peptidase. Human dermal fibroblasts can be used as a cell model to study the increase in type I collagen content by cosmetics. By measuring the upregulation rate of type I collagen content after administration of the blank control and the test substance, it is evaluated whether the test substance has an effect in promoting collagen synthesis. On the basis of meeting the validation of effectiveness in the experiment, compared with the blank / solvent control, the content of type I collagen is upregulated in the test substance, and there is a significant difference (P < 0.05), indicating that the test substance has the ability to promote the synthesis of type I collagen at the tested concentration, which can be used as one of the evidence supports for the claim of being a raw material for firming cosmetics.

[0175] Determination method:

[0176] 1. Cell seeding:

[0177] Take out the cells from the carbon dioxide incubator, observe the cell growth under the microscope, and randomly select 2 fields of view to take pictures using a low-power microscope (4X) and a high-power microscope (10X) respectively. When the cell density reaches 80%-90%, confirm that harvesting can be carried out. Wipe the outer surface of the culture flask with a lint-free cloth containing alcohol and transfer it into the biosafety cabinet. Gently shake the culture flask back and forth, and use a pipette to remove the old culture medium. Wash the cell surface once with an appropriate amount of PBS, add an appropriate amount of digestive enzyme to the culture flask, and digest at room temperature for 3-5 min. After gently shaking, observe with the naked eye that the cells are in a flowing sand state. Under the microscope, when the cells are spherical, it indicates complete digestion. Add an appropriate amount of PBS to terminate the digestion. After pipetting the bottom of the culture flask, transfer the liquid into a centrifuge tube, and centrifuge at a parameter of 400 g for 5 min. Resuspend with complete 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 in each group, and culture the cells for 24 h.

[0178] 2. Drug treatment:

[0179] Prepare the treatment drugs, 400 μL for each group. Blank control group: T4 basal medium. Astaxanthin (purchased from Biotopped) experimental group: 40 μL of astaxanthin (1 mg / ml stock solution) + 400 μL of T4 basal medium. Exosome of Spirulina platensis experimental group: 40 μL of exosome of Spirulina platensis (1 mg / ml stock solution) + 400 μL of T4 basal medium. Exosome of Olea europaea leaf experimental group: 40 μL of exosome of Olea europaea leaf (1 mg / ml stock solution) + 400 μL of T4 basal medium. Exosome of Paeonia lactiflora experimental group: 40 μL of exosome of Paeonia lactiflora (1 mg / ml stock solution) + 400 μL of T4 basal medium. Composition experimental group: 40 μL of composition (1 mg / ml stock solution) + 400 μL of T4 basal medium; Discard the cell supernatant, and add 100 μL of negative control reagent, positive control group reagent, and experimental group reagent respectively, and culture for 24 h.

[0180] 3. Detection of type I collagen (refer to the detection instructions of the Lianke Human Pro-Collagen I α1 ELISA Kit):

[0181] 1) Soak the enzyme-labeled plate: Add 300 μL of 1× washing solution and let it stand for 30 seconds. After discarding the washing solution, pat the well plate dry on the absorbent paper. After the plate washing is completed, immediately use the microplate without allowing it to dry.

[0182] 2) Add standard products: Add 100 μL of standard products diluted 2-fold serially diluted to the standard product wells. Add 100 μL of standard product diluent to the blank wells.

[0183] 3) Add sample: Add 5 μL of cell culture supernatant to the sample well, and supplement each well with 95 μL of PBS for 20-fold dilution.

[0184] 4) Incubation: Seal the plate with a sealing film. Place it in a thermostatic mixer and incubate at room temperature (25°C ± 3°C) for 2 h.

[0185] 5) Washing: Discard the liquid, add 300 μL of washing solution to each well to wash the plate, and wash 3 times. Pat the microplate dry on absorbent paper.

[0186] 6) Add detection antibody: Add 100 μL of detection antibody working solution (diluted 1:100) to each well.

[0187] 7) Incubation: Seal the plate with a sealing film, place it in a thermostatic mixer, and incubate at room temperature (25°C ± 3°C) for 1 h.

[0188] 8) Washing: Repeat step 5.

[0189] 9) Add enzyme: Add 100 μL of streptavidin working solution (diluted 1:100) to each well.

[0190] 10) Incubation: Seal the plate with a sealing film. Place it in a thermostatic mixer and incubate at room temperature (25°C ± 3°C) for 45 min.

[0191] 11) Washing: Repeat step 5.

[0192] 12) Add substrate for color development: Add 100 μL of colorimetric substrate to each well and incubate at room temperature (25°C ± 3°C) in the dark for 20 min.

[0193] 13) Add stop solution: Add 100 μL of stop solution to each well. Gently tap to mix, and the color changes from blue to yellow.

[0194] 14) Detection and reading: Measure the maximum absorption wavelength at 450 nm within 30 minutes.

[0195] 15) Make a standard curve based on the A450 value of the standard product. The abscissa is the concentration of type I collagen, and the ordinate is the A450 value. Calculate the amount of type I collagen in the sample according to the formula of the standard curve, and compare the experimental group with the blank control group. The results are shown in Table 5.

[0196] Table 5: Results of the determination of type I collagen content in vitro dermal fibroblasts

[0197]

[0198] The results show that the composition of Example 4 can significantly promote the secretion of type I collagen by dermal fibroblasts, thus achieving a firming effect, and the effect is better than that of astaxanthin and each component raw material in the composition.

[0199] Example 10: Determination of the Ability of Dermal Fibroblasts in Vitro to Scavenge Reactive Oxygen Species

[0200] Reactive oxygen species (ROS) refer to the general term for oxygen-containing free radicals and peroxides that are prone to form free radicals in organisms related to oxygen metabolism, widely referring to oxygen-derived free radicals and non-free radicals, including superoxide anion (O 2- ), hydrogen peroxide (H 2 O 2 ), hydroxyl radical (OH - ), ozone (O 3 ), singlet oxygen (1O 2 ), etc. Normal physiological metabolism and environmental impacts in the human body will lead to the generation of reactive oxygen species (ROS). However, excessive ROS will cause problems such as skin aging. ROS generated by skin exposure to ultraviolet irradiation 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 and causing an increase in intracellular ROS, and detecting the amount of intracellular ROS using the DCFH-DA fluorescent probe, the efficacy of the test substance in inhibiting cellular ROS can be evaluated. On the basis of meeting the validation of effectiveness in the experiment, compared with the blank control, the DCFH-DA fluorescence intensity of the test substance is down-regulated and shows a significant difference (P<0.05), indicating that the test substance has the ability to scavenge cellular ROS at the tested concentration, which can be used as one of the evidence supports for the claim of anti-wrinkle cosmetic raw materials.

[0201] Determination method:

[0202] 1. Cell seeding:

[0203] Take out the cells from the carbon dioxide incubator, observe the cell growth under the microscope, and randomly select 2 fields of view to take pictures using a low-power microscope (4X) and a high-power microscope (10X) respectively. Confirm that harvesting can be carried out when the cell density reaches 80%-90%. Wipe the outer surface of the culture flask with a dust-free cloth containing alcohol and transfer it into the biosafety cabinet. Gently shake the culture flask back and forth, and use a pipette to remove the old culture medium. Wash the cell surface once with an appropriate amount of PBS, add an appropriate amount of digestive enzyme to the culture flask, and digest at room temperature for 3-5 min. After gently shaking, observe with the naked eye that the cells are in a flowing sand state. Under the microscope, when the cells are spherical, it indicates complete digestion. Add an appropriate amount of PBS to terminate digestion. After pipetting the bottom of the culture flask, transfer the liquid into a centrifuge tube, and centrifuge at 400 g for 5 min. Resuspend with complete culture medium, and take 20 μL for cell counting. Seed into a 24-well plate at a density of 50000 cells / 500 μL / well, with 3 replicates in each group, and culture the cells for 24 h.

[0204] 2. Drug treatment:

[0205] Prepare the treatment drugs, 2 mL for each group. Blank control group: T4 basal medium, hydroxyprolisane (purchased from MedChemExpress). Experimental groups: 200 μL hydroxyprolisane (1 mg / ml stock solution) + 2 mL T4 basal medium, exosomes of Spirulina platensis experimental group: 200 μL exosomes of Spirulina platensis (1 mg / ml stock solution) + 2 mL T4 basal medium, exosomes of Olea europaea L. leaf experimental group: 200 μL exosomes of Olea europaea L. leaf (1 mg / ml stock solution) + 2 mL T4 basal medium, exosomes of Paeonia lactiflora Pall. experimental group: 200 μL exosomes of Paeonia lactiflora Pall. (1 mg / ml stock solution) + 2 mL T4 basal medium, composition experimental group: 200 μL composition (1 mg / ml stock solution) + 2 mL T4 basal medium; Discard the cell culture supernatant, add 500 μL of negative control reagent, positive control group reagent, and experimental group reagent respectively, and culture for 24 h.

[0206] 3. UVA induction:

[0207] Place ice packs in the biosafety cabinet, lay four layers of kraft paper on the ice packs, and then place the cell culture plate on the kraft paper. Adjust the position of the UVA lamp tube so that the distance from the lamp tube to the cells is 5 cm. Remove the cell culture plate, turn on the lamp tube and balance for 5 min, discard the culture supernatant, add 100 μL of pre-cooled PBS, then place the cell culture plate under the lamp tube, open the lid, irradiate for 1 h, and the radiation energy is about 5 J / cm 2 .

[0208] 4. Drug treatment:

[0209] Discard the cell culture supernatant, add 500 μL of blank control reagent, positive control group reagent, and experimental group reagent respectively, and culture for 3 - 5 h.

[0210] 5. Sample collection and detection:

[0211] Prepare the DCFH-DA working solution according to a dilution ratio of 1:1000. Dilute DCFH-DA with the basal medium to a final concentration of 10 μmol / L. Discard the PBS in the 24-well plate, add 200 μL of DCFH-DA working solution to each well, and incubate in a CO2 incubator for 30 min. After incubation, wash the cells 3 times with PBS, place them on the detection platform of the fluorescence microplate reader, set the incident light wavelength to 529 nm, the excitation light wavelength to 504 nm, read the values, and the results are shown in Table 6.

[0212] Table 6: Determination results of the ability of dermal fibroblasts to scavenge reactive oxygen species in vitro

[0213]

[0214] The results showed that the composition of Example 4 could significantly reduce the ROS level of dermal fibroblasts, thereby achieving the anti-wrinkle effect, and the effect was better than that of hydroxyprolisone and each component raw material in the composition.

[0215] Example 11: In vitro determination of the inhibitory activity of tyrosinase in melanocytes

[0216] Tyrosinase is a copper ion-containing metalloenzyme belonging to polyphenol oxidase. It is an important rate-limiting enzyme in a series of chemical reactions during the generation of melanin. Tyrosinase catalyzes the formation of dopachrome from L-dopa, which has a characteristic absorption peak at 492 nm, and then the activity of tyrosinase is measured. B16 mouse melanoma cells can be used as a cell model for studying the inhibitory rate of tyrosinase activity in cosmetics. On the basis of meeting the validity verification, compared with the blank control group, the inhibitory rate of tyrosinase activity in the experimental group was significantly up-regulated, and there was a significant difference (P<0.05), indicating that the experimental sample had the ability to inhibit tyrosinase activity at the tested concentration, which could be used as one of the evidence supports for the claim of whitening cosmetic raw materials.

[0217] Determination method:

[0218] 1. Cell seeding:

[0219] B16 mouse melanoma cells were normally cultured in RPMI-1640 complete medium (culturing in RPMI-1640 complete medium will not generate melanin). Take out the cells from the carbon dioxide incubator, observe the cell growth under the microscope, and randomly select 2 fields of view to take pictures using a low-power microscope (4X) and a high-power microscope (10X) respectively. When the cell density reaches 80%-90%, it is confirmed that the cells can be harvested. Wipe the outer surface of the culture flask with a dust-free cloth containing alcohol and transfer it into the biosafety cabinet. Gently shake the culture flask back and forth, remove the old medium using a pipette, wash the cell surface once with an appropriate amount of PBS, digest with 0.25% trypsin for 2 min, observe under the microscope, when most cells become round and are in a suspended state, add about 2-3 times the volume of RPMI-1640 complete medium containing serum to terminate digestion, and collect the cells into a centrifuge tube. Centrifuge at 1500 rpm for 5 min. After centrifugation, discard the supernatant, add a certain volume of DMEM high-glucose complete medium to the centrifuge tube (resuspend the cells and use DMEM high-glucose medium for the following experiments. B16 cells will generate melanin in DMEM medium). After resuspending evenly, use a cell counter to count the cells. Take 20 μL for cell counting. Inoculate the cells into a 24-well plate at a density of 100000 cells / well. The blank control group does not inoculate cells and only adds DMEM high-glucose medium, and culture for 24 h.

[0220] 2. Drug treatment:

[0221] Prepare treatment drugs, 2 mL for each group. Blank control group: high-glucose DMEM complete medium, nicotinamide (purchased from MERCK). Experimental groups: 200 μL nicotinamide (1 mg / ml stock solution) + 2 mL high-glucose DMEM complete medium, Spirulina platensis exosome experimental group: 200 μL Spirulina platensis exosomes (1 mg / ml stock solution) + 2 mL high-glucose DMEM complete medium, Olea europaea leaf exosome experimental group: 200 μL Olea europaea leaf exosomes (1 mg / ml stock solution) + 2 mL high-glucose DMEM complete medium, Paeonia lactiflora Pall. exosome experimental group: 200 μL Paeonia lactiflora Pall. exosomes (1 mg / ml stock solution) + 2 mL high-glucose DMEM complete medium, composition experimental group: 200 μL composition (1 mg / ml stock solution) + 2 mL high-glucose DMEM complete medium; Discard the cell culture supernatant, add 500 μL of blank control reagent, positive control group reagent, and experimental group reagent respectively, and culture for 72 h. During this period, observe the cell state and the medium situation every day and replace the fresh blank control reagent, positive control group reagent, and experimental group reagent.

[0222] 3. Cell collection:

[0223] 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 pipette the cells. Collect the cells into a 1.5 mL centrifuge tube, centrifuge at 4000 rpm for 5 min, and discard the supernatant.

[0224] 4. Detection:

[0225] Add 100 μl of 1% Triton-x-100 to each tube to lyse the cells, place at -80 °C for 30 min, then place at room temperature for 10 min, centrifuge at 12000 rpm at 4 °C for 20 min to remove cell debris. Take 50 μl of the supernatant, add 150 μl of 2 mg / ml L-DOPA solution, incubate at 37 °C for 1 h, and measure the absorbance at 492 nm with an enzyme-linked immunosorbent assay (ELISA) reader.

[0226] 5. Data analysis:

[0227] Enzyme activity inhibition rate = [1 - (As - Ab) / (Ac - Ab)] × 100%, where 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.

[0228] Table 7: Results of the determination of tyrosinase activity inhibition in vitro in melanocytes

[0229]

[0230] The results showed that the composition of Example 4 could significantly inhibit the tyrosinase activity of melanocytes, thus achieving the whitening effect, and the effect was better than that of niacinamide and each component raw material in the composition.

[0231] Example 12: In vitro determination of melanin synthesis inhibition in melanocytes

[0232] Melanin is a dark brown pigment existing in animals and plants. It is produced and stored in a special type of cell, namely melanocytes (also known as melanin mother cells). It is because of the existence of melanin that the skin has color. The main function of melanocytes is to synthesize melanin. The synthesis of melanin mainly takes tyrosine as the raw material in melanocytes and forms melanin through the catalysis of tyrosinase. α-MSH can bind to the MC1R receptor on melanocytes, activate adenylate cyclase (AC), resulting in 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 for studying the inhibition of melanin synthesis in cosmetics. On the basis of meeting the validity verification, compared with the negative control group, the inhibition rate of melanin synthesis in the experimental group was significantly up-regulated, and there was a significant difference (P < 0.05), indicating that the experimental sample had the ability to inhibit melanin synthesis at the tested concentration, which could be used as one of the evidence supports for the appellation of raw materials for whitening cosmetics.

[0233] Determination method:

[0234] 1. Cell seeding:

[0235] B16 mouse melanoma cells are normally cultured using RPMI-1640 complete medium (culturing with RPMI-1640 complete medium will not generate melanin). Take out the cells from the carbon dioxide incubator, observe the cell growth under a microscope, and randomly select 2 fields of view to take pictures using a low-power microscope (4X) and a high-power microscope (10X) respectively. When the cell density reaches 80%-90%, confirm that harvesting can be carried out. Wipe the outer surface of the culture flask with a dust-free cloth containing alcohol and transfer it into the biosafety cabinet. Gently shake the culture flask back and forth, use a pipette to remove the old medium, wash the cell surface once with an appropriate amount of PBS, digest with 0.25% trypsin for 2 min, observe under the microscope, when most cells become round and are in a suspended state, add about 2-3 times the volume of RPMI-1640 complete medium containing serum to terminate digestion, and collect it into a centrifuge tube, centrifuge at 1500 rpm for 5 min. After centrifugation, discard the supernatant, add a certain volume of DMEM high-glucose complete medium to the centrifuge tube (resuspend the cells and use DMEM high-glucose medium for the following experiments. B16 cells will generate melanin in DMEM medium), resuspend evenly and use a cell counter to count. Take 20 μL for cell counting. Inoculate into a 24-well plate at a density of 100000 cells / well. The blank control group does not inoculate cells and only adds DMEM high-glucose medium, and culture for 24 h.

[0236] 2. Drug treatment:

[0237] Prepare the treatment drugs, 2 mL for each group. Blank control group: PBS + DMEM high-glucose complete medium with the same sample volume. Nicotinamide (purchased from MERCK) experimental group: 200 μL of nicotinamide (1 mg / ml stock solution) + 1 mL of DMEM high-glucose complete medium + 1 mL of PBS. Spirulina platensis exosome experimental group: 200 μL of Spirulina platensis exosomes (1 mg / ml stock solution) + 1 mL of DMEM high-glucose complete medium + 1 mL of PBS. Olea europaea leaf exosome experimental group: 200 μL of Olea europaea leaf exosomes (1 mg / ml stock solution) + 1 mL of DMEM high-glucose complete medium + 1 mL of PBS. Paeonia lactiflora exosome experimental group: 200 μL of Paeonia lactiflora exosomes (1 mg / ml stock solution) + 1 mL of DMEM high-glucose complete medium + 1 mL of PBS. Composition experimental group: 200 μL of composition (1 mg / ml stock solution) + 1 mL of DMEM high-glucose complete medium + 1 mL of PBS; Discard the culture supernatant of the cells, add 500 μL of negative control reagent, positive control group reagent, and experimental group reagent respectively, and culture for 72 h. During this period, observe the cell state and the medium situation every day and replace the fresh blank control reagent, positive control group reagent, and experimental group reagent.

[0238] 3. Cell collection:

[0239] 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 pipette the cells. Collect the cells into a centrifuge tube, centrifuge at 4000 rpm for 5 min, and discard the supernatant.

[0240] 4. Detection:

[0241] Add 100 μl of melanin extraction solution to each tube, shake well, and heat in a water bath at 80 °C for 1 hour. After cooling, centrifuge the droplets on the tube wall moderately, pipette evenly, and pipette 90 μl of the solution from each centrifuge tube into a 96-well plate. Use a microplate reader to measure the absorbance of each well at a wavelength of 405 nm.

[0242] 5. Data analysis:

[0243] Melanin synthesis inhibition rate = [1 - (As - Ab) / (Ac - Ab)] × 100%, where 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.

[0244] Table 8: Results of the determination of melanin synthesis inhibition in vitro in melanocytes

[0245]

[0246] The results show that the composition of Example 4 can significantly inhibit the melanin synthesis of melanocytes, thus achieving a whitening effect, and the effect is better than that of niacinamide and each component raw material in the composition.

[0247] Example 13: Determination of the proliferation ability of keratinocytes in vitro

[0248] Cell proliferation is one of the important physiological functions of living cells and an important vital feature of organisms, indicating cell vitality and status. The epidermis is located on the surface of the skin. It is very thin but tough. Most epidermal cells are keratinocytes. Keratinocytes have functions such as proliferation, differentiation, migration, and secretion of extracellular components, and it is a determinant of whether the skin can recover to a healthy state. The CCK-8 method is based on the highly water-soluble tetrazolium salt WST-8 to measure cell proliferation / toxicity. Under the action of an electron mediator, WST-8 can be reduced to form a water-soluble orange-yellow formazan product, 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 cells, the depth of color is proportional to the number of living cells. Hacat human immortalized keratinocytes can be used as a cell model for studying the proliferation of keratinocytes in vitro for cosmetics. On the basis that the experiment meets the validity verification, compared with the blank control group, the cell proliferation in the experimental group is up-regulated and has a significant difference (P<0.05), indicating that the experimental sample has the ability to promote cell proliferation at this tested concentration and can be used as one of the evidence supports for the claim of repair cosmetic raw materials.

[0249] Determination method:

[0250] 1. Cell seeding:

[0251] Take out the cells from the carbon dioxide incubator, observe the cell growth under the microscope, and randomly select 2 fields of view to take pictures using a low-power microscope and a high-power microscope respectively. When the cell density reaches 80%-90%, confirm that harvesting can be carried out; wipe the outer surface of the culture flask with a dust-free cloth containing alcohol and transfer it into the biosafety cabinet. Gently shake the culture flask back and forth, and use a pipette to remove the old culture medium. Wash the cell surface once with an appropriate amount of PBS, add an appropriate amount of digestive enzyme to the culture flask, and digest at room temperature for 5-8 min. After gently shaking, observe with the naked eye that the cells are in a flowing sand state, and observe under the microscope. When the cells are spherical, it indicates that the digestion is complete. Add an appropriate amount of termination solution to terminate the digestion, transfer the liquid into a centrifuge tube, and centrifuge at 400 g for 5 min; resuspend with DMEM complete culture medium and take 20 μL for counting. Inoculate into a 96-well plate at a density of 2×10 4 / well: Take the required amount of cells and dilute them to 2×10 5 / ml, mix well and add the cell suspension to the 96-well plate, 100 μL / well. Incubate the cells for 24 h for standby.

[0252] 2. Drug treatment:

[0253] Prepare treatment drugs, 400 μL for each group. Blank control group: DMEM basal medium, ceramide (purchased from MedChemExpress). Experimental groups: 40 μL ceramide (1 mg / ml stock solution) + 400 μL DMEM basal medium; Experimental group of Spirulina platensis exosomes: 40 μL Spirulina platensis exosomes (1 mg / ml stock solution) + 400 μL DMEM basal medium; Experimental group of Olea europaea leaf exosomes: 40 μL Olea europaea leaf exosomes (1 mg / ml stock solution) + 400 μL DMEM basal medium; Experimental group of Paeonia lactiflora Pall. exosomes: 40 μL Paeonia lactiflora Pall. exosomes (1 mg / ml stock solution) + 400 μL DMEM basal medium; Experimental group of the composition: 40 μL composition (1 mg / ml stock solution) + 400 μL DMEM basal medium; Discard the cell supernatant, and add 100 μL of blank control reagent, positive control group reagent, and experimental group reagent respectively, and culture for 24 h.

[0254] 3. CCK8 assay (refer to the instruction manual of Abbkine SuperKine™ Ultra-sensitive Cell Proliferation Detection Reagent Kit):

[0255] 1) Prepare a solution containing 10% CCK8 reagent using the medium.

[0256] 2) Add 100 μL of CCK8 solution to each well and continue to incubate in the cell culture incubator for 2 - 4 h.

[0257] 3) Measure the absorbance at 450 nm.

[0258] 4. Data analysis:

[0259] Cell proliferation rate % = OD experimental group / OD blank control group × 100%, OD experimental group: absorbance value after treatment with the experimental group drug, OD blank control group: absorbance value after treatment with the blank control group drug. The results are shown in Table 9.

[0260] Table 9: Results of the determination of the proliferation ability of keratinocytes in vitro

[0261]

[0262] The results show that the composition of Example 4 can significantly improve the cell viability of keratinocytes, thus achieving a repair effect, and the effect is better than that of ceramide and each component raw material in the composition.

[0263] Example 14: Anti-wrinkle test on human face

[0264] To evaluate the test results of the composition provided by this invention patent in anti-wrinkle treatment on the human face, 5 testers over 35 years old were recruited. The composition provided by this invention patent was non-invasively introduced into the faces of the testers with the assistance of a Shu Min Star introducer. The usage interval was once every 3 weeks, and it was used twice in total. The dosage for each time was 6 mL, and the concentration was 0.4 mg / mL. The effects 7 days and 21 days after the first introduction and 7 days and 21 days after the second introduction were evaluated, and static wrinkles and textures such as facial dry lines and wrinkles were photographed using a photographing device (Eagle Eye).

[0265] According to comprehensive data such as the length, depth, and width of the wrinkles, AI simulation scoring was carried out. The higher the proportion, the more serious the wrinkle condition. The statistical results are shown in Table 10. Among them, D0 means before using the composition provided by this invention patent, D7 means 7 days after the first use of the composition provided by this invention patent, D21 means 21 days after the first use of the composition provided by this invention patent, D28 means 7 days after the second use of the composition provided by this invention patent, and D42 means 21 days after the second use of the composition provided by this invention patent.

[0266] Table 10: Statistical results of anti-wrinkle data on the human face

[0267]

[0268] The results showed that the facial wrinkles of the 5 testers were improved to varying degrees after two introductions of the composition provided by this invention patent.

[0269] It should be understood that the above embodiments are all exemplary and do not cover all possible implementation manners included in the claims. Without departing from the scope of the present disclosure, various deformations and changes can also be made based on the above embodiments. Similarly, the various technical features of the above embodiments can also be arbitrarily combined to form additional embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only represent several implementation manners of the present invention and do not limit the protection scope of this invention patent.

Claims

1. A plant exosome composition, characterized in that: The composition comprises: exosomes of Spirulina platensis, exosomes of olive leaves and exosomes of white peony root; Wherein, 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.

2. The composition according to claim 1, 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.

3. 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.

4. The composition according to claim 3, 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.

5. The composition according to claim 4, characterized in that The solvent is PB buffer and / or PBS buffer.

6. The composition according to claim 4, characterized in that The dilution is to dilute to a turbidity of less than 900 NTU.

7. The composition according to claim 4, characterized in that The filtration is performed using a tangential flow depth filtration membrane package.

8. The composition according to claim 4, characterized in that 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 whitening effects, and products with repairing effects.

11. The use according to claim 10, characterized in that: The products with anti-aging effects include products with firming effects and products with anti-wrinkle effects.

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