Chlorella exosome freeze-dried powder as well as preparation method and application thereof

Abstract

The invention relates to chlorella exosome freeze-dried powder as well as a preparation method and application thereof. The chlorella exosome freeze-dried powder is prepared from the following raw materials: chlorella exosome and a freeze-drying protection solution, the freeze-drying protection liquid comprises a freeze-drying protection agent and a buffer solution; the freeze-drying protective agent comprises erythritol. The erythritol in the chlorella exosome freeze-dried powder interacts with certain polar groups on the surface of the chlorella exosome, in the cold drying process, aggregation of the exosome can be reduced, the integrity of active ingredients of the exosome can be kept, and then the stability of the exosome in the storage process is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant exosomes, and specifically relates to a chlorella exosome freeze-dried powder and a preparation method and application thereof. Background Art

[0002] Exosomes are the most essential part of stem cells. Exosomes refer to small membrane vesicles (30-150nm) containing complex RNA and proteins. Nowadays, they specifically refer to disc-shaped vesicles with a diameter of 40-100nm. Exosomes are a type of extracellular vesicles secreted by cells. Their main components are phospholipid bilayers and related contents. Algae exosomes contain a large number of active ingredients and have excellent anti-aging, moisturizing, anti-inflammatory and soothing effects. Therefore, they are increasingly used in the beauty industry.

[0003] CN115992052A discloses a microalgae exosome, a preparation method and application thereof. The present invention uses ultracentrifugation to extract microalgae exosomes from Chlorella vulgaris. After testing, the microalgae exosomes can carry pigment proteins and miRNAs, and do not contain any CD proteins homologous to human or animal exosomes. The use of microalgae exosomes is safer and has broad prospects in the preparation and application of cosmetics, drugs and vaccines.

[0004] KR20240016207A discloses exosomes from a chlorella culture medium and uses thereof. According to the present invention, exosomes derived from a chlorella culture medium are safe for skin application because they are non-cytotoxic and are used for wound healing, immunomodulation, skin whitening, wrinkle improvement, etc. It is highly effective and can be provided as a functional cosmetic derived from natural products, drugs, quasi-drugs, health functional foods, etc.

[0005] However, although algae exosomes are increasingly used in beauty and skin care in the existing technology, the shelf life of exosomes is very short, which greatly limits the scope of their application. Therefore, the preservation of exosomes is an urgent problem that needs to be solved. Summary of the invention

[0006] In view of the deficiencies in the prior art, the object of the present invention is to provide a Chlorella exosome freeze-dried powder and a preparation method and application thereof.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a lyophilized powder of Chlorella exosomes, wherein the raw materials for preparing the lyophilized powder of Chlorella exosomes include Chlorella exosomes and lyophilization protection solution;

[0009] The freeze-drying protection solution includes a freeze-drying protection agent and a buffer solution;

[0010] The lyoprotectant includes erythritol.

[0011] The erythritol in the Chlorella exosome freeze-dried powder of the present invention interacts with certain polar groups on the surface of Chlorella exosomes, and can reduce the aggregation of exosomes during the freeze-drying process, maintain the integrity of the active ingredients of the exosomes, and thus improve the stability of the exosomes during storage.

[0012] Preferably, the volume ratio of the Chlorella exosomes to the freeze-dried protective solution is 1:(0.5-1.5), for example, it can be 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3 or 1:1.4, etc.

[0013] Preferably, the buffer is a phosphate buffer of 0.2-0.5 mol / L (for example, 0.25 mol / L, 0.3 mol / L, 0.35 mol / L, 0.4 mol / L or 0.45 mol / L, etc.).

[0014] Preferably, the concentration of erythritol in the freeze-dried protective solution is 3-15%, for example, it can be 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13% or 14%.

[0015] Preferably, the lyoprotectant further comprises β-cyclodextrin.

[0016] Preferably, the mass ratio of erythritol to β-cyclodextrin is 1:(0.1-0.5), for example, it can be 1:0.15, 1:0.2, 1:0.25, 1:0.3, 1:0.35, 1:0.4 or 1:0.45, etc.

[0017] Preferably, the method for preparing Chlorella exosomes comprises:

[0018] (1) treating the cell wall of Chlorella vulgaris, wherein the pH value of the cell wall treatment is 8-10, the temperature is 70-80° C., and the time is 20-60 min;

[0019] (2) subjecting the cell fluid after cell wall disruption to gradient centrifugation, wherein the specific steps of the gradient centrifugation are: centrifugation at 300-400 g for 5-15 min, centrifugation of the supernatant at 1000-2000 g for 10-30 min, centrifugation of the supernatant at 4000-5000 g for 10-30 min, centrifugation of the supernatant at 6000-7000 g for 10-30 min, and then taking the supernatant to obtain a crude exosome extract;

[0020] (3) resuspending the crude exosome extract with Tween 80 aqueous solution at a volume ratio of 1:(0.3-1), and then ultracentrifuging 2-4 times, each time at a speed of 15000-20000g for 30-60 min to obtain purified exosomes;

[0021] (4) The purified exosomes, polyglycerol-10 laurate and phosphate nanoparticle solution are mixed, and then homogenized by ultra-speed microfluidization to obtain the Chlorella exosomes.

[0022] Preferably, the concentration of the Tween 80 aqueous solution in step (3) is 20-30 mg / mL, for example, 21 mg / mL, 23 mg / mL, 25 mg / mL, 27 mg / mL or 29 mg / mL.

[0023] Preferably, the volume ratio of the purified exosomes, polyglycerol-10 laurate and phosphate nanoparticle solution in step (4) is (2-5):(0.1-1):(0.1-1).

[0024] Wherein "2-5" can be 2.2, 2.5, 2.8, 3, 3.2, 3.5, 3.8, 4, 4.2, 4.5 or 4.8, etc.;

[0025] The first "0.1-1" can be 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or 0.9, etc.;

[0026] The second "0.1-1" can be 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or 0.9, etc.

[0027] Preferably, the concentration of the phosphate nanoparticle solution in step (4) is 0.2-0.5%, for example, it can be 0.25%, 0.3%, 0.35%, 0.4% or 0.45%, etc.

[0028] Preferably, the jet velocity of the ultrasonic microjet in step (4) is 5000-7000 m / s, for example, it can be 5200 m / s, 5400 m / s, 5600 m / s, 5800 m / s, 6000 m / s, 6200 m / s, 6400 m / s, 6600 m / s or 6800 m / s, etc.

[0029] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0030] In a second aspect, the present invention provides a method for preparing the Chlorella exosome freeze-dried powder as described in the first aspect, the preparation method comprising: mixing the Chlorella exosomes with a freeze-drying protection liquid to obtain a freeze-dried mixed liquid, and then pre-freezing, refrigerating, vacuuming and analytical drying to obtain the Chlorella exosome freeze-dried powder.

[0031] Preferably, the pre-freezing step comprises: cooling the freeze-dried mixture to -20 to -30°C (for example, -21°C, -23°C, -25°C, -27°C or -29°C, etc.), and maintaining it for 1-3h (for example, 1.2h, 1.5h, 1.8h, 2h, 2.2h, 2.5h or 2.8h, etc.).

[0032] Preferably, the refrigeration step comprises: cooling the pre-frozen freeze-dried mixed solution from -20 to -30°C to -35 to -50°C (for example, -38°C, -40°C, -42°C, -44°C, -46°C or -48°C) for 20-60s (for example, 25s, 30s, 35s, 40s, 45s, 50s or 55s, etc.), and maintaining it for 1-2h (for example, 1.1h, 1.3h, 1.5h, 1.7h or 1.9h, etc.).

[0033] Preferably, the vacuum degree after evacuation is 0.01-0.1 mbar, for example, it can be 0.02 mbar, 0.03 mbar, 0.04 mbar, 0.05 mbar, 0.06 mbar, 0.07 mbar, 0.08 mbar or 0.09 mbar, etc.

[0034] Preferably, the analytical drying parameters include: 40-50 min (for example, 41 min, 43 min, 45 min, 47 min or 49 min, etc.) heating from -35 to -50 ° C to -5 to -15 ° C (for example, -6 ° C, -8 ° C, -10 ° C, -12 ° C or -14 ° C, etc.), maintaining for 2-4 h (for example, 2.2 h, 2.5 h, 2.8 h, 3 h, 3.2 h, 3.5 h or 3.8 h, etc.); 20-40 min (for example, 22 min, 25 min, 28 min, 30 min, 32 min, 35 min or 38 min, etc.) heating from -5 to -15 ° C to -5 -0℃ (for example, -4℃, -3℃, -2℃ or -1℃, etc.), keep for 2-4h (for example, it can be 2.2h, 2.5h, 2.8h, 3h, 3.2h, 3.5h or 3.8h, etc.); 20-40min (for example, it can be 22min, 25min, 28min, 30min, 32min, 35min or 38min, etc.) increase the temperature from -5-0℃ to 20-35℃ (for example, it can be 21℃, 23℃, 25℃, 27℃, 30℃, 32℃ or 34℃, etc.), keep for 2-4h (for example, it can be 2.2h, 2.5h, 2.8h, 3h, 3.2h, 3.5h or 3.8h, etc.).

[0035] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0036] In a third aspect, the present invention provides a use of the Chlorella exosome freeze-dried powder as described in the first aspect in the preparation of skin care products.

[0037] Preferably, the skin care product comprises any one of a gel, an essence, a lotion, a cream, an ointment or a mask.

[0038] Preferably, the addition amount of the Chlorella exosome freeze-dried powder in the skin care product is 0.5-5%, for example, it can be 0.8%, 1%, 1.2%, 1.5%, 1.8%, 2%, 2.2%, 2.5%, 2.8%, 3%, 3.2%, 3.5%, 3.8%, 4%, 4.2%, 4.5% or 4.8%, etc.

[0039] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] (1) The erythritol in the Chlorella exosome freeze-dried powder of the present invention interacts with certain polar groups on the surface of Chlorella exosomes, which can reduce the aggregation of exosomes during the freeze-drying process, maintain the integrity of the active ingredients of the exosomes, and thus improve the stability of the exosomes during storage.

[0042] (2) The method provided by the present invention can control the process parameters so that the final exosome freeze-dried powder can be fluffy in appearance, compact in powder quality, not easy to fall off, and can be quickly dissolved when re-dissolved. The solution is clear and transparent, and the particle size, potential and morphology of the exosomes after re-dissolution do not change significantly. The membrane of the freeze-dried and re-dissolved exosomes is intact and the surface is smooth, which is of great value to the long-term stability of the exosomes. DETAILED DESCRIPTION

[0043] In order to further explain the technical means and effects adopted by the present invention, the technical solution of the present invention is further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0044] The polyglycerol-10 laurate mentioned in the raw materials involved in the following examples, comparative examples and preparation examples was purchased from: Guangzhou Jahwa Chemical Co., Ltd.;

[0045] The phosphate nanoparticles were purchased from Suzhou Element Chemical Industry Co., Ltd.

[0046] The rest of the raw materials can be used as long as they are purchased from regular dealers.

[0047] Preparation Example 1

[0048] This preparation example provides a Chlorella exosome, and the preparation method of the Chlorella exosome is:

[0049] (1) treating the cell wall of Chlorella vulgaris at a pH of 8.5, a temperature of 78° C., and a time of 45 minutes;

[0050] (2) subjecting the cell fluid after cell wall disruption to gradient centrifugation, wherein the specific steps of the gradient centrifugation are: centrifugation at 400 g for 15 min, centrifugation at 1500 g for 10 min, centrifugation at 4500 g for 30 min, and centrifugation at 6500 g for 10 min, and then taking the supernatant to obtain a crude exosome extract;

[0051] (3) The crude exosome extract was resuspended in a 28 mg / mL Tween 80 aqueous solution at a volume ratio of 1:0.7, and then ultracentrifuged three times at a speed of 16000 g for 35 min each time to obtain purified exosomes;

[0052] (4) The purified exosomes, polyglycerol-10 laurate and phosphate nanoparticle solution (concentration 0.4%) were mixed in a volume ratio of 4:0.2:0.7, and then homogenized by ultrafast microfluidization (jet velocity 6500 m / s) to obtain the Chlorella exosomes.

[0053] Example 1

[0054] This embodiment provides a lyophilized powder of Chlorella exosomes, and the preparation method of the lyophilized powder of Chlorella exosomes is as follows:

[0055] (1) mixing a lyophilization protective agent (erythritol and β-cyclodextrin in a mass ratio of 1:0.3) and 0.3 mol / L phosphate buffer to obtain a lyophilization protective solution, wherein the concentration of erythritol in the lyophilization protective solution is 10%;

[0056] (2) mixing the Chlorella exosomes of Preparation Example 1 with the freeze-dried protective solution in a volume ratio of 1:1 to obtain a freeze-dried mixed solution;

[0057] (3) Cool the lyophilized mixture to -25°C and keep it for 2 h, then cool the lyophilized protective solution to -40°C within 40 s and keep it for 1.5 h;

[0058] (4) After the freeze-dried mixed solution treated in step (3) is evacuated, the vacuum degree is controlled to be 0.05 mbar, and then analytical drying is performed. The analytical drying parameters are as follows: heating from -40°C to -10°C for 45 minutes and maintaining for 3 hours; heating from -10°C to 0°C for 30 minutes and maintaining for 3 hours; heating from 0°C to 25°C for 30 minutes and maintaining for 3 hours, to obtain the Chlorella exosome freeze-dried powder.

[0059] Example 2

[0060] This embodiment provides a lyophilized powder of Chlorella exosomes, and the preparation method of the lyophilized powder of Chlorella exosomes is as follows:

[0061] (1) mixing a lyophilization protective agent (erythritol and β-cyclodextrin in a mass ratio of 1:0.5) and 0.25 mol / L phosphate buffer to obtain a lyophilization protective solution, wherein the concentration of erythritol in the lyophilization protective solution is 8%;

[0062] (2) mixing the Chlorella exosomes of Preparation Example 1 with the freeze-dried protective solution at a volume ratio of 1:0.8 to obtain a freeze-dried mixed solution;

[0063] (3) Cool the lyophilized mixture to -20°C and keep it for 2.5 h, then cool the lyophilized protective solution to -45°C within 60 s and keep it for 1 h;

[0064] (4) After the freeze-dried mixed solution treated in step (3) is evacuated, the vacuum degree is controlled to be 0.03 mbar, and then analytical drying is performed. The analytical drying parameters are as follows: heating from -45°C to -15°C for 50 minutes and maintaining for 2 hours; heating from -15°C to -5°C for 40 minutes and maintaining for 4 hours; heating from -5°C to 20°C for 40 minutes and maintaining for 2 hours, to obtain the Chlorella exosome freeze-dried powder.

[0065] Example 3

[0066] This embodiment provides a lyophilized powder of Chlorella exosomes, and the preparation method of the lyophilized powder of Chlorella exosomes is as follows:

[0067] (1) mixing a lyophilization protective agent (erythritol and β-cyclodextrin in a mass ratio of 1:0.2) and 0.45 mol / L phosphate buffer to obtain a lyophilization protective solution, wherein the concentration of erythritol in the lyophilization protective solution is 12%;

[0068] (2) mixing the Chlorella exosomes of Preparation Example 1 with the freeze-dried protective solution at a volume ratio of 1:1.3 to obtain a freeze-dried mixed solution;

[0069] (3) Cool the lyophilized mixture to -30°C and keep it for 1.5 h, then cool the lyophilized protective solution to -50°C within 40 s and keep it for 2 h;

[0070] (4) After the freeze-dried mixed solution treated in step (3) is evacuated, the vacuum degree is controlled to be 0.08 mbar, and then analytical drying is performed. The analytical drying parameters are as follows: heating from -50°C to -12°C in 40 minutes and maintaining for 4 hours; heating from -10°C to 0°C in 40 minutes and maintaining for 2 hours; heating from 0°C to 30°C in 40 minutes and maintaining for 4 hours, to obtain the Chlorella exosome freeze-dried powder.

[0071] Example 4

[0072] This embodiment provides a lyophilized powder of Chlorella exosomes, and the preparation method of the lyophilized powder of Chlorella exosomes is different from that of Example 1 only in that step (1) is: mixing erythritol and 0.3 mol / L phosphate buffer to obtain a lyophilization protection solution, wherein the concentration of erythritol in the lyophilization protection solution is 10%, and the other steps and parameters remain unchanged.

[0073] Example 5

[0074] This embodiment provides a lyophilized powder of Chlorella exosomes. The preparation method of the lyophilized powder of Chlorella exosomes is different from that of Example 1 only in that the concentration of erythritol in the lyophilization protection solution in step (1) is 20%, and the other steps and parameters remain unchanged.

[0075] Example 6

[0076] This embodiment provides a lyophilized powder of Chlorella exosomes. The preparation method of the lyophilized powder of Chlorella exosomes is different from that of Example 1 only in that the concentration of erythritol in the lyophilization protection solution in step (1) is 1%, and the other steps and parameters remain unchanged.

[0077] Example 7

[0078] This embodiment provides a lyophilized powder of Chlorella exosomes. The preparation method of the lyophilized powder of Chlorella exosomes is different from that of Example 1 only in that the volume ratio of Chlorella exosomes to lyophilization protection solution in step (2) is 1:2, and the other steps and parameters remain unchanged.

[0079] Example 8

[0080] This embodiment provides a lyophilized powder of Chlorella exosomes. The preparation method of the lyophilized powder of Chlorella exosomes is different from that of Example 1 only in that the volume ratio of Chlorella exosomes to lyophilization protection solution in step (2) is 1:0.3, and the other steps and parameters remain unchanged.

[0081] Example 9

[0082] The present embodiment provides a lyophilized powder of Chlorella exosomes, and the preparation method of the lyophilized powder of Chlorella exosomes is different from that of Example 1 only in that: step (4) is: evacuating the lyophilized mixed solution treated in step (3) and controlling the vacuum degree to -0.05 mbar, and then performing analytical drying, and the analytical drying parameters are: heating from -40°C to 0°C for 45 minutes and maintaining for 3 hours; heating from 0°C to 25°C for 30 minutes and maintaining for 3 hours, to obtain the lyophilized powder of Chlorella exosomes, and the other steps and parameters remain unchanged.

[0083] Example 10

[0084] The present embodiment provides a lyophilized powder of Chlorella exosomes, and the preparation method of the lyophilized powder of Chlorella exosomes is different from that of Example 1 only in that: step (4) is: evacuating the lyophilized mixed solution treated in step (3) and controlling the vacuum degree to -0.05 mbar, and then performing analytical drying, and the analytical drying parameters are: heating from -40°C to -20°C for 45 minutes and maintaining for 3 hours; heating from -20°C to -10°C for 30 minutes and maintaining for 3 hours; heating from -10°C to 0°C for 30 minutes and maintaining for 3 hours; heating from 0°C to 25°C for 30 minutes and maintaining for 3 hours, to obtain the lyophilized powder of Chlorella exosomes, and the remaining steps and parameters remain unchanged.

[0085] Comparative Example 1

[0086] This comparative example provides a lyophilized powder of Chlorella exosomes, and the preparation method of the lyophilized powder of Chlorella exosomes is different from that of Example 1 only in that step (1) is: mixing β-cyclodextrin and 0.3 mol / L phosphate buffer to obtain a lyophilization protection solution, wherein the concentration of β-cyclodextrin in the lyophilization protection solution is 10%, and the other steps and parameters remain unchanged.

[0087] Comparative Example 2

[0088] This comparative example provides a lyophilized powder of Chlorella exosomes, and the preparation method of the lyophilized powder of Chlorella exosomes is different from that of Example 1 only in that step (1) is: mixing mannitol and 0.3 mol / L phosphate buffer to obtain a lyophilization protection solution, wherein the concentration of mannitol in the lyophilization protection solution is 10%, and the other steps and parameters remain unchanged.

[0089] Comparative Example 3

[0090] This comparative example provides a lyophilized powder of Chlorella exosomes, and the preparation method of the lyophilized powder of Chlorella exosomes is different from that of Example 1 only in that step (1) is: mixing trehalose and 0.3 mol / L phosphate buffer to obtain a lyophilization protection solution, wherein the concentration of trehalose in the lyophilization protection solution is 10%, and the other steps and parameters remain unchanged.

[0091] Comparative Example 4

[0092] This comparative example provides a lyophilized powder of Chlorella exosomes, and the preparation method of the lyophilized powder of Chlorella exosomes is as follows:

[0093] (1) mixing a lyophilization protective agent (erythritol and β-cyclodextrin in a mass ratio of 1:0.3) and 0.3 mol / L phosphate buffer to obtain a lyophilization protective solution, wherein the concentration of erythritol in the lyophilization protective solution is 10%;

[0094] (2) mixing the Chlorella exosomes and the freeze-dried protective solution in a volume ratio of 1:1 to obtain a freeze-dried mixed solution;

[0095] (3) Cool the freeze-dried mixture to -25°C and keep it for 3.5 hours;

[0096] (4) After the freeze-dried mixed solution treated in step (3) is evacuated, the vacuum degree is controlled to be -0.05 mbar, and then analytical drying is performed. The analytical drying parameters are as follows: heating from -25°C to -10°C for 45 minutes and maintaining for 3 hours; heating from -10°C to 0°C for 30 minutes and maintaining for 3 hours; heating from 0°C to 25°C for 30 minutes and maintaining for 3 hours, to obtain the Chlorella exosome freeze-dried powder.

[0097] Comparative Example 5

[0098] This comparative example provides a lyophilized powder of Chlorella exosomes, and the preparation method of the lyophilized powder of Chlorella exosomes is as follows:

[0099] (1) mixing a lyophilization protective agent (erythritol and β-cyclodextrin in a mass ratio of 1:0.3) and 0.3 mol / L phosphate buffer to obtain a lyophilization protective solution, wherein the concentration of erythritol in the lyophilization protective solution is 10%;

[0100] (2) mixing the Chlorella exosomes and the freeze-dried protective solution in a volume ratio of 1:1 to obtain a freeze-dried mixed solution;

[0101] (3) Cool the freeze-dried mixture to -40°C and keep it for 3.5 hours;

[0102] (4) After the freeze-dried mixed solution treated in step (3) is evacuated, the vacuum degree is controlled to be -0.05 mbar, and then analytical drying is performed. The analytical drying parameters are as follows: heating from -40°C to -10°C for 45 minutes and maintaining for 3 hours; heating from -10°C to 0°C for 30 minutes and maintaining for 3 hours; heating from 0°C to 25°C for 30 minutes and maintaining for 3 hours, to obtain the Chlorella exosome freeze-dried powder.

[0103] Test example:

[0104] The Chlorella exosomes (D0) obtained in Preparation Example 1 were subjected to moisturizing, anti-aging and anti-inflammatory tests, and then the exosomes obtained in Preparation Example 1 (control example), Examples 1-10, and the samples provided in Comparative Examples 1-5 were placed at 25° C. for 6 months and then subjected to moisturizing, anti-aging and anti-inflammatory tests, wherein the samples obtained in Examples 1-10 and Comparative Examples 1-5 were tested using 0.3 mol / L phosphate buffer for reconstitution (dosage: 0.3 g / mL).

[0105] (1) Moisturizing performance test

[0106] The moisturizing performance of the test samples was tested, and the changes in skin moisture content of the test subjects were compared before and after use (the usage operation was kept consistent). A total of 170 volunteers were included, including 85 males and 85 females, aged 20-40 years old with an average age of 28 years old. They were randomly divided into 17 groups, with 5 females and 5 males in each group.

[0107] The subjects washed their forearms and sat quietly for 30 minutes in a set humidity environment. A 3×3cm2 square experimental area was selected on each of the subjects' left and right forearms. The left arm was used as the test area for the test sample, and the symmetrical area of ​​the right arm was used as a blank control. The moisture content of each experimental part was tested with Corneometer CM825. This was repeated 3 times and the average value was recorded.

[0108] The formula for calculating skin hydration rate is:

[0109] The average skin hydration rate of the subjects after using the samples for 2 hours is shown in Table 1:

[0110] Table 1

[0111]

[0112]

[0113] (2) Evaluation of anti-aging effects

[0114] Test method:

[0115] (1) Inhibition rate of metalloproteinase (MMP-1) expression:

[0116] Test principle: One of the main causes of skin aging is the change in the structure of the dermis. Due to the action of some external factors, the matrix metalloproteinases (MMPs) in the body are activated, which can lead to excessive degradation of collagen and elastin in the dermis that support the skin structure, resulting in aging symptoms such as wrinkles and inelasticity. Metalloproteinase MMP-1, also known as collagenase-1, can degrade collagen gelatin and proteoglycans of types I, II, III, VII and X. The anti-aging effect of these samples is evaluated by testing the inhibition rate of the samples on MMP-1;

[0117] Test method: Fibroblasts were seeded into 12-well cell culture plates, each well containing 0.75×10 5 Cells were starved and cultured in serum-free medium for 24 h. The starved cells were rinsed with PBS and treated with blue light (40 mJ). Then, the test sample was added to the cells twice within 48 h. The MMP-1 isolated in the culture medium was measured using a kit (BIOTRAK, RPN2610). The strength of the anti-aging effect was evaluated by calculating the expression inhibition rate of MMP-1;

[0118] The calculation formula is as follows: inhibition rate = (M0-M1) / M0×100%;

[0119] M0 is the expression level of MMP-1 without adding the test sample but after irradiation with blue light, and M1 is the expression level of MMP-1 after adding the test sample and irradiation with blue light;

[0120] (2) Determination of the inhibitory effect of elastase activity

[0121] Test principle: One of the main causes of skin aging is the change in the structure of the dermis. Due to the action of some external factors, the elastase in the body is activated, which can lead to excessive degradation of collagen and elastin in the dermis that support the skin structure. The anti-aging effect of these samples is evaluated by testing the inhibition rate of elastase on the samples;

[0122] Test method: 200 μL of each test sample and 50 μL of 20 μg / mL elastase-III solution were mixed in a 96-well plate in a 10 mg / L Tris hydrochloride buffer. 250 μM EGCG was used as a positive control group, and distilled water was used as a negative control group. The reaction was carried out at 25°C for 15 minutes. After the reaction, the absorbance at a wavelength of 415 nm was measured.

[0123] The calculation formula is as follows: Elastase activity inhibition rate (%) = 1-(CD) / (AB) × 100%;

[0124] A is the absorbance at 415 nm when no test substance is added but enzyme is added, B is the absorbance at 415 nm when no test substance and enzyme are added, C is the absorbance at 415 nm when test substance and enzyme are added, and D is the absorbance at 415 nm when test substance is added but enzyme is not added;

[0125] The specific test results are shown in Table 2 below:

[0126] Table 2

[0127]

[0128]

[0129] (3) Anti-inflammatory efficacy test

[0130] The test method is as follows:

[0131] (1) Enzyme-linked immunosorbent assay (ELISA): The blood sample was mixed with an equal proportion of blood cell separation solution and centrifuged at 20°C for 40 min. The human peripheral blood mononuclear cell layer was obtained. After the cells were washed with a buffer solution, the cells were suspended in the culture medium RPMI-1640. Then, CD14+ monocytes from human peripheral blood mononuclear cells were purified using CD14+ microspheres (MiniMACS system). The cells were stimulated to differentiate into dendritic cells using cytokine IL-4 and growth hormone GM-CSF, and cultured at 37°C and 5% CO2 for 6 days. The differentiated dendritic cells were collected and seeded into a well plate. The above test samples were diluted to a concentration of 2 μg / mL and added to the well plate, and the wells with an equal volume of culture medium were set as blank controls. The wells were co-cultured at 37°C and 5% CO2 for 48 hours. The cell culture supernatant was collected. The levels of interleukin IL-10 and IL-12 in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA), and the content of anti-inflammatory cytokine IL-10 in the blank control group was defined as 0, and the content of pro-inflammatory cytokine IL-12 was defined as 100%;

[0132] (2) Inhibition of TNF-α expression: Fresh blood was treated with EDTA as anticoagulant, blood cells were separated by Ficoll, and the cells were resuspended in RIMP 1640 medium containing 10% fetal bovine serum. 100 mL of 1×10 5 cells / mL Freshly isolated cells, total number of cells per well: 10 4 3 wells for each sample; weigh the test sample, add sterile PBS solution to prepare the test sample stock solution (10 mg / mL), and then use sterile PBS to dilute the stock solution to the corresponding working concentration for use; add 10 mL of the test solution of the corresponding working concentration to the well of the culture plate. The culture plate is placed in an incubator at 37°C and 5% CO2 for 15 minutes; add 10 mL of LPS with a concentration of 100 mg / mL and incubate in an incubator at 37°C and 5% CO2 for 16 hours; the culture plate is centrifuged at 1000 rpm for 15 minutes, the supernatant is transferred to a new culture plate, and the TNF-α concentration in the culture supernatant is determined using an enzyme marker; or frozen at -20°C to avoid repeated freezing and thawing, and the inhibition rate is calculated.

[0133] The specific test results are shown in Table 3 below:

[0134] Table 3

[0135]

[0136]

[0137] It can be seen from the test data in Tables 1-3 that compared with the control example, the lyophilized powder of Chlorella exosomes prepared by the method of the present invention in Examples 1-3 had a skin hydration rate of more than 41% after use by volunteers, an inhibition rate of metalloproteinase MMP-1 expression of more than 60%, an inhibition rate of elastase of more than 81%, an interleukin content of IL-10 of more than 156%, an interleukin IL-12 level of less than 6%, and an inhibition rate of TNF-a release of more than 86%, which is basically consistent with the effect of Chlorella exosomes (D0), indicating that the lyophilized powder obtained by the method can maintain the biological activity and stability of exosomes.

[0138] As shown in Example 4, the combination of β-cyclodextrin and erythritol can further improve the stability and biological activity of exosomes in freeze-dried powder;

[0139] As can be seen from Examples 5 and 6, the dosage of erythritol also has an effect on the protection of exosomes;

[0140] It can be seen from Examples 7-8 that only when the volume ratio of Chlorella exosomes to the freeze-dried protective solution is within the protection range of the present invention can the final freeze-dried powder have good stability and biological activity;

[0141] It can be seen from Examples 9-10 that when the number of analytical drying is too small, the biological activity of the lyophilized powder will be affected, and when the number of analytical drying is too large, the biological activity of the lyophilized powder will not be improved, and the process cost will be increased;

[0142] It can be seen from Comparative Examples 1-3 that the stability and biological activity of the lyophilized powder of Chlorella exosomes of the present invention can be improved only when the lyophilization protective agents are erythritol and β-cyclodextrin;

[0143] It can be seen from Comparative Examples 4 and 5 that pre-freezing and refrigeration will also have a certain impact on the activity of the freeze-dried powder.

[0144] The applicant declares that the present invention illustrates a lyophilized powder of Chlorella exosomes and its preparation method and application through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0145] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0146] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A lyophilized powder of Chlorella exosomes, characterized in that: The raw materials for preparing the Chlorella exosome freeze-dried powder include Chlorella exosomes and freeze-dried protective solution; The freeze-drying protection solution includes a freeze-drying protection agent and a buffer solution; The lyoprotectant includes erythritol.

2. The lyophilized Chlorella exosome powder according to claim 1, characterized in that The volume ratio of the Chlorella exosomes to the freeze-dried protective solution is 1:(0.5-1.5); Preferably, the buffer is 0.2-0.5 mol / L phosphate buffer; Preferably, the concentration of erythritol in the freeze-dried protective solution is 3-15%.

3. The lyophilized Chlorella exosome powder according to claim 1 or 2, characterized in that The freeze-drying protective agent also includes β-cyclodextrin; Preferably, the mass ratio of erythritol to β-cyclodextrin is 1:(0.1-0.5).

4. A method for preparing a lyophilized powder of Chlorella exosomes according to any one of claims 1 to 3, characterized in that: The preparation method comprises: mixing the Chlorella exosomes with a freeze-drying protection solution to obtain a freeze-dried mixed solution, and then pre-freezing, refrigerating, vacuumizing and analytical drying to obtain the Chlorella exosome freeze-dried powder.

5. The preparation method according to claim 4, characterized in that: The pre-freezing step includes: cooling the freeze-dried mixed solution to -20 to -30°C and maintaining it for 1 to 3 hours.

6. The preparation method according to claim 4 or 5, characterized in that: The refrigeration step comprises: cooling the pre-frozen freeze-dried mixed solution from -20 to -30°C to -35 to -50°C within 20 to 60 seconds, and maintaining the temperature for 1 to 2 hours.

7. The preparation method according to any one of claims 4 to 6, characterized in that: The vacuum degree after the vacuuming is 0.01-0.1 mbar.

8. The preparation method according to any one of claims 4 to 7, characterized in that: The analytical drying parameters include: heating from -35 to -50°C to -5 to -15°C in 40-50 minutes and maintaining for 2-4 hours; heating from -5 to -15°C to -5-0°C in 20-40 minutes and maintaining for 2-4 hours; heating from -5-0°C to 20-35°C in 20-40 minutes and maintaining for 2-4 hours.

9. Use of the Chlorella exosome freeze-dried powder according to any one of claims 1 to 3 in the preparation of skin care products.

10. The use according to claim 9, characterized in that The skin care product includes any one of a gel, an essence, a lotion, a cream, an ointment or a mask; Preferably, the amount of the Chlorella exosome freeze-dried powder added to the skin care product is 0.5-5%.

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