Extraction method and application of turmeric extracellular vesicles

Abstract

The invention relates to application of turmeric extracellular vesicles in the field of cosmetics. It is found for the first time that the turmeric extracellular vesicles have the anti-aging, repairing and anti-oxidation effects, are good in water solubility and permeability and are easily absorbed by skin; the composition can (a) promote fibroblast proliferation, (b) promote fibroblast collagen secretion, (c) inhibit the fibroblast MMP1 level, (d) promote the fibroblast TMP1 level, (e) inhibit the fibroblast ROS level, (f) inhibit the fibroblast SA-beta-Gal level, (g) promote the keratinocyte proliferation level, (h) inhibit the keratinocyte ROS level, (i) promote the expression of keratinocyte keratinization-related proteins, and (ii) promote the keratinocyte keratinization-related proteins. And (j) promoting the keratinocyte migration level.

Description

Technical Field

[0001] The invention relates to the technical field of biological extraction, and in particular to a method for extracting turmeric extracellular vesicles and an application thereof. Background Art

[0002] Extracellular vesicles are membrane-structured vesicles with a diameter of about 20 nm-5 μm. They vary in size and composition and include various types, such as exosomes (about 30-100 nm), ectosomes, microvesicles (about 100-1,000 nm), microparticles, apoptotic bodies (about 1-5 μm), etc. Exosomes can be derived not only from animal cells, but also from plant cells. Plant cells secrete exosomes when responding to various biological and non-biological environmental stresses (such as pathogen infection and attack). They have similar size distribution, surface charge, surface morphology, content composition, etc. to animal exosomes. Extracellular vesicles contain a variety of molecules such as RNA, proteins and lipids, which play a role in regulating physiological processes. They can serve as transport carriers and have biological functions. Plant exosomes are widely available, safe and non-toxic, have low immunogenicity, can be mass-produced at low cost, and have good biocompatibility and environmental friendliness. Plant exosomes can be eaten directly, absorbed through the gastrointestinal tract, and play a role in interspecies communication between plant and mammalian cells.

[0003] Turmeric (Curcuma longa L.), as a tropical perennial herb, is rich in a variety of active ingredients, especially curcumin, which shows excellent application value. Curcumin not only has strong antioxidant and anti-inflammatory capabilities, can effectively scavenge free radicals in the body and inhibit inflammatory responses, but also shows significant immunomodulatory and anti-tumor potential, providing a new perspective for cancer prevention and treatment. In addition, turmeric also performs well in regulating lipid metabolism, promoting bile secretion and enhancing liver detoxification function, further broadening its application prospects in the fields of medicine and health care. In summary, turmeric and its extracts have shown extremely high scientific research and application potential in the fields of pharmaceutical research and development, functional food development, and health management due to their multiple biological activities and wide application value.

[0004] In the studies that have been reported so far, it has been proven that curcumin has anti-inflammatory, anti-oxidative, and apoptosis regulating functions, and has potential application value in tumor treatment, inflammation regulation, skin repair, etc., but no research has been found on the efficacy of turmeric extracellular vesicles in anti-aging. At the same time, due to the poor water solubility and absorption of curcumin itself, its bioavailability on the skin may be limited, resulting in its effects in delaying skin aging, repairing skin, etc. are often unsatisfactory. Therefore, the art urgently needs a turmeric extract with good water solubility, good permeability, and easy absorption by the skin. Summary of the invention

[0005] Problem that the invention aims to solve In view of the above problems existing in the prior art, the purpose of the present invention is to provide a turmeric extracellular vesicle which has good water solubility, good permeability, is easily absorbed by the skin, and has anti-aging, repairing and antioxidant effects.

[0006] Solutions for solving problems The invention provides application of turmeric extracellular vesicles in the field of cosmetics.

[0007] Preferably, the application includes: (1) Application in the preparation of skin anti-wrinkle and anti-aging products; (2) Application in the preparation of skin repair and antioxidant products; (3) Application in the preparation of whitening and anti-freckle products.

[0008] Preferably, the method for preparing the turmeric extracellular vesicles comprises: (1) Soak the washed turmeric and break the wall to obtain turmeric juice; (2) Using the turmeric juice obtained in step (1), extracting turmeric extracellular vesicles.

[0009] Preferably, the immersion solution in step (1) is PBS; And / or, the soaking time in step (1) is 6 to 24 hours.

[0010] Preferably, the soaking time in step (1) is 6 to 12 hours.

[0011] And / or, the cell wall breaking time in step (1) is 5 to 10 minutes.

[0012] Preferably, the extraction method in step (2) is selected from one or more of sucrose density gradient centrifugation, ultracentrifugation, fractional filtration, and tangential flow filtration.

[0013] Preferably, the specific steps of the ultracentrifugation include: (I) centrifuging the turmeric juice obtained in step (1) at 400-800×g for 5-20 min and collecting the supernatant; (II) Centrifuge again at 1000-3000 × g for 10-30 min and collect the supernatant; (III) Centrifuge at 3000-5000 × g for 20-40 min and collect the supernatant; (IV) Centrifuge at 8000-12000 × g for 40-70 min and collect the supernatant; (V) Centrifuge again at 100,000-120,000 × g for 60-100 min, collect the precipitate, and resuspend in PBS; (VI) Centrifuging at 100,000-120,000×g for 60-100 min, collecting the precipitate, and resuspending it in PBS to obtain the turmeric extracellular vesicles.

[0014] Preferably, the specific steps of the ultracentrifugation are: (I) centrifuging the turmeric juice obtained in step (1) at 500×g for 10 min and collecting the supernatant; (II) Centrifuge at 2000 × g for 20 min and collect the supernatant; (III) Centrifuge at 4000 × g for 30 min and collect the supernatant; (IV) Centrifuge at 10,000 × g for 60 min and collect the supernatant; (V) Centrifuge at 110200 × g for 70 min, collect the precipitate, and resuspend in PBS; (VI) Centrifuging at 110200×g for 70 min, collecting the precipitate, and resuspending it in PBS to obtain the turmeric extracellular vesicles.

[0015] Preferably, the specific steps of the tangential flow filtration include: (a) subjecting the turmeric juice obtained in step (1) to gradient centrifugation and collecting the supernatant; (b) diluting the supernatant obtained in step (a) with PB buffer until the turbidity is less than 900 NTU; (c) filtering the diluted solution in step (b) through a tangential flow depth filtration membrane package, and collecting the filtrate; (d) The filtrate obtained in step (c) is concentrated and purified using a hollow fiber column to obtain the turmeric extracellular vesicles.

[0016] Preferably, the specific steps of the gradient centrifugation in step (a) include: (a1) centrifuging the turmeric juice obtained in step (1) at 400-800×g for 5-20 min and collecting the supernatant; (a2) Centrifuge at 1000-3000 × g for 10-30 min and collect the supernatant; (a3) Centrifuge at 3000-5000 × g for 20-40 min and collect the supernatant; (a4) Centrifuge at 8000-12000×g for 40-70 min and collect the supernatant.

[0017] Effects of the Invention The turmeric extracellular vesicle extraction method of the present invention uses dehydrated turmeric as a raw material, which is convenient to store compared to fresh turmeric, does not need to be refrigerated, and has stable active ingredients. Dehydrated turmeric is usually standardized and has more consistent quality, which is conducive to ensuring the consistency and predictability of the product. Compared with artificially prepared extracellular vesicles loaded with curcumin, the advantages of natural turmeric extracellular vesicles are the diversity of their natural ingredients, bioavailability and ease of processing.

[0018] The turmeric extracellular vesicles prepared according to the extraction method of the present invention have anti-aging, repairing and antioxidant effects, and have good water solubility, good permeability and are easily absorbed by the skin; they can (a) promote fibroblast proliferation, (b) promote fibroblast collagen secretion, (c) inhibit fibroblast MMP1 level, (d) promote fibroblast TMP1 level, (e) inhibit fibroblast ROS level, (f) inhibit fibroblast SA-β-Gal level, (g) promote keratinocyte proliferation level, (h) inhibit keratinocyte ROS level, (i) promote keratinocyte keratinization-related protein expression, and (j) promote keratinocyte migration level. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 These are the results of turmeric extracellular vesicle size detection.

[0020] Figure 2 These are the transmission electron microscopy (TEM) characterization results of turmeric extracellular vesicles.

[0021] Figure 3 This is the detection result of total flavonoids concentration in turmeric extracellular vesicles.

[0022] Figure 4 The results of total sugar concentration detection in turmeric extracellular vesicles.

[0023] Figure 5 Fluorescence microscopy observation of turmeric extracellular vesicles penetrating into isolated pig skin.

[0024] Figure 6 Effects of turmeric extracellular vesicles on keratinocyte proliferation.

[0025] Figure 7 Effects of turmeric extracellular vesicles on keratinocyte migration.

[0026] Figure 8 The effect of turmeric extracellular vesicles on the expression level of keratin-related proteins.

[0027] Fig. 9 To investigate the effects of turmeric extracellular vesicles on reactive oxygen species (ROS) levels in keratinocytes.

[0028] Fig.10To investigate the effect of turmeric extracellular vesicles on the secretion of type Ⅰ collagen (MMP1) in dermal fibroblasts.

[0029] Fig.11 Effects of turmeric extracellular vesicles on the secretion of extracellular matrix proteinases in dermal fibroblasts.

[0030] Fig.12 Effects of turmeric extracellular vesicles on senescence of dermal fibroblasts.

[0031] Fig.13 Overall results of human testing of a face cream containing turmeric extracellular vesicles.

[0032] Fig.14 This is an excellent example of the human effect of a face cream containing turmeric extracellular vesicles. DETAILED DESCRIPTION

[0033] In order to make the technical solutions and beneficial effects of the present invention more clearly understandable, the following is a detailed description by listing specific embodiments. The drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly show the details of the local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application belongs.

[0034] As used in this article, the term "extracellular vesicles" or "EVs" are nanoscale particles secreted by organisms with a diameter of approximately 20 nm-5 μm. They mediate the interaction between the environment and organisms, and between organisms by carrying and delivering functional molecules such as RNA, proteins, and metabolites, becoming a widely-watched signal communication carrier.

[0035] As used herein, the term "exosome" refers to a small membrane vesicle containing complex RNA and proteins, a type of extracellular vesicle, with a diameter of approximately 30-100 nm.

[0036] The invention provides application of turmeric extracellular vesicles in the field of cosmetics.

[0037] In certain embodiments, the application comprises: (1) Application in the preparation of skin anti-wrinkle and anti-aging products; (2) Application in the preparation of skin repair and antioxidant products; (3) Application in the preparation of whitening and anti-freckle products.

[0038] In certain embodiments, the extracellular vesicles are exosomes.

[0039] In certain embodiments, the method for preparing the turmeric extracellular vesicles comprises: (1) Soak the washed turmeric and break the wall to obtain turmeric juice; (2) Using the turmeric juice obtained in step (1), extracting turmeric extracellular vesicles.

[0040] In certain embodiments, the turmeric is fresh turmeric.

[0041] In certain embodiments, the method for preparing turmeric extracellular vesicles further comprises: washing and drying fresh turmeric.

[0042] In certain embodiments, the method for preparing the turmeric extracellular vesicles comprises: washing and drying fresh turmeric, soaking and breaking the wall to obtain turmeric juice, and using the turmeric juice to extract turmeric extracellular vesicles.

[0043] In certain embodiments, the method for preparing the turmeric extracellular vesicles comprises: washing the processed turmeric, soaking and breaking the wall to obtain turmeric juice, and using the turmeric juice to extract the turmeric extracellular vesicles.

[0044] In some embodiments, the drying temperature is 40-60°C.

[0045] In certain embodiments, the drying temperature is 40°C, or 41°C, or 42°C, or 43°C, or 44°C, or 45°C, or 46°C, or 47°C, or 48°C, or 49°C, or 50°C, or 51°C, or 52°C, or 53°C, or 54°C, or 55°C, or 56°C, or 57°C, or 58°C, or 59°C, or 60°C.

[0046] In certain embodiments, the drying temperature is 50°C.

[0047] In some embodiments, the drying time is 12 to 48 hours.

[0048] In some embodiments, the drying time is 12 hours, or 13 hours, or 14 hours, or 15 hours, or 16 hours, or 17 hours, or 18 hours, or 19 hours, or 20 hours, or 21 hours, or 22 hours, or 23 hours, or 24 hours, or 25 hours, or 26 hours, or 27 hours, or 28 hours, or 29 hours, or 30 hours, or 31 hours, or 32 hours, or 33 hours, or 34 hours, or 35 hours, or 36 hours, or 37 hours, or 38 hours, or 39 hours, or 40 hours, or 41 hours, or 42 hours, or 43 hours, or 44 hours, or 45 hours, or 46 hours, or 47 hours, or 48 hours.

[0049] In some embodiments, the drying time is 24 hours.

[0050] In certain embodiments, the immersion solution in step (1) is PBS.

[0051] In certain embodiments, the soaking solution in step (1) is sterile PBS.

[0052] In certain embodiments, the soaking time in step (1) is 6 to 24 hours.

[0053] In certain embodiments, the soaking time in step (1) is 6 hours, or 7 hours, or 8 hours, or 9 hours, or 10 hours, or 11 hours, or 12 hours, or 13 hours, or 14 hours, or 15 hours, or 16 hours, or 17 hours, or 18 hours, or 19 hours, or 20 hours, or 21 hours, or 22 hours, or 23 hours, or 24 hours.

[0054] In certain embodiments, the soaking time in step (1) is 6 to 12 hours.

[0055] In certain embodiments, the soaking time in step (1) is 12 hours.

[0056] In certain embodiments, the cell wall breaking time in step (1) is 5 to 10 minutes.

[0057] In certain embodiments, the cell wall breaking time in step (1) is 5 min, or 6 min, or 7 min, or 8 min, or 9 min, or 10 min.

[0058] In certain embodiments, the extraction method in step (2) is selected from one or more of sucrose density gradient centrifugation, ultracentrifugation, fractional filtration, and tangential flow filtration.

[0059] In certain embodiments, the extraction method in step (2) is sucrose density gradient centrifugation.

[0060] In certain embodiments, the extraction method in step (2) is graded filtration.

[0061] In certain embodiments, the extraction method in step (2) is ultracentrifugation and / or tangential flow filtration.

[0062] In certain embodiments, the extraction method in step (2) is ultracentrifugation.

[0063] In certain embodiments, the ultracentrifugation step in step (2) comprises: (I) centrifuging the turmeric juice obtained in step (1) at 400-800×g for 5-20 min and collecting the supernatant; (II) Centrifuge again at 1000-3000 × g for 10-30 min and collect the supernatant; (III) Centrifuge at 3000-5000 × g for 20-40 min and collect the supernatant; (IV) Centrifuge at 8000-12000 × g for 40-70 min and collect the supernatant; (V) Centrifuge again at 100,000-120,000 × g for 60-100 min, collect the precipitate, and resuspend in PBS; (VI) Centrifuging at 100,000-120,000×g for 60-100 min, collecting the precipitate, and resuspending it in PBS to obtain the turmeric extracellular vesicles.

[0064] In certain embodiments, the centrifugal force of the centrifugation in step (I) is 400×g, or 450×g, or 500×g, or 550×g, or 600×g, or 650×g, or 700×g, or 750×g, or 800×g.

[0065] In certain embodiments, the centrifugal force of the centrifugation in step (I) is 500×g.

[0066] In certain embodiments, the centrifugation time in step (I) is 5 min, or 6 min, or 7 min, or 8 min, or 9 min, or 10 min, or 11 min, or 12 min, or 13 min, or 14 min, or 15 min, or 16 min, or 17 min, or 18 min, or 19 min, or 20 min.

[0067] In certain embodiments, the centrifugation time in step (I) is 10 min.

[0068] In certain embodiments, the centrifugal force of the centrifugation in step (II) is 1000×g, or 1100×g, or 1200×g, or 1300×g, or 1400×g, or 1500×g, or 1600×g, or 1700×g, or 1800×g, or 1900×g, or 2000×g, or 2100×g, or 2200×g, or 2300×g, or 2400×g, or 2500×g, or 2600×g, or 2700×g, or 2800×g, or 2900×g, or 3000×g.

[0069] In certain embodiments, the centrifugal force of the centrifugation in step (II) is 2000×g.

[0070] In certain embodiments, the centrifugation time in step (II) is 10 min, or 11 min, or 12 min, or 13 min, or 14 min, or 15 min, or 16 min, or 17 min, or 18 min, or 19 min, or 20 min, or 21 min, or 22 min, or 23 min, or 24 min, or 25 min, or 26 min, or 27 min, or 28 min, or 29 min, or 30 min.

[0071] In certain embodiments, the centrifugation time in step (II) is 20 min.

[0072] In certain embodiments, the centrifugal force of the centrifugation in step (III) is 3000×g, or 3100×g, or 3200×g, or 3300×g, or 3400×g, or 3500×g, or 3600×g, or 3700×g, or 3800×g, or 3900×g, or 4000×g, or 4100×g, or 4200×g, or 4300×g, or 4400×g, or 4500×g, or 4600×g, or 4700×g, or 4800×g, or 4900×g, or 5000×g.

[0073] In certain embodiments, the centrifugal force of the centrifugation in step (III) is 4000×g.

[0074] In certain embodiments, the centrifugation time in step (III) is 20 min, or 21 min, or 22 min, or 23 min, or 24 min, or 25 min, or 26 min, or 27 min, or 28 min, or 29 min, or 30 min, or 31 min, or 32 min, or 33 min, or 34 min, or 35 min, or 36 min, or 37 min, or 38 min, or 39 min, or 40 min.

[0075] In certain embodiments, the centrifugation time in step (III) is 30 min.

[0076] In certain embodiments, the centrifugal force of the centrifugation in step (IV) is 8000×g, or 8100×g, or 8200×g, or 8300×g, or 8400×g, or 8500×g, or 8600×g, or 8700×g, or 8800×g, or 8900×g, or 9000×g, or 9100×g, or 9200×g, or 9300×g, or 9400×g, or 9500×g, or 9600×g, or 9700×g, or 9800×g, or 9900×g, or 10 000×g, or 10100×g, or 10200×g, or 10300×g, or 10400×g, or 10500×g, or 10600×g, or 10700×g, or 10800×g, or 10900×g, or 11000×g, or 11100×g, or 11200×g, or 11300×g, or 11400×g, or 11500×g, or 11600×g, or 11700×g, or 11800×g, or 11900×g, or 12000×g.

[0077] In certain embodiments, the centrifugal force of the centrifugation in step (IV) is 10,000×g.

[0078] In certain embodiments, the centrifugation time in step (IV) is 40 min, or 41 min, or 42 min, or 43 min, or 44 min, or 45 min, or 46 min, or 47 min, or 48 min, or 49 min, or 50 min, or 51 min, or 52 min, or 53 min, or 54 min, or 55 min, or 56 min, or 57 min, or 58 min, or 59 min, or 60 min, or 61 min, or 62 min, or 63 min, or 64 min, or 65 min, or 66 min, or 67 min, or 68 min, or 69 min, or 70 min.

[0079] In certain embodiments, the centrifugation time in step (IV) is 60 min.

[0080] In certain embodiments, the centrifugal force of the centrifugation in step (V) is 100,000×g, or 101,000×g, or 102,000×g, or 103,000×g, or 104,000×g, or 105,000×g, or 106,000×g, or 107,000×g, or 108,000×g, or 109,000×g, or 110,000×g, or 110,200×g, or 111,000×g, or 112,000×g, or 113,000×g, or 114,000×g, or 115,000×g, or 116,000×g, or 117,000×g, or 118,000×g, or 119,000×g, or 120,000×g.

[0081] In certain embodiments, the centrifugal force of the centrifugation in step (V) is 110200×g.

[0082] In certain embodiments, the centrifugation time in step (V) is 60 min, or 61 min, or 62 min, or 63 min, or 64 min, or 65 min, or 66 min, or 67 min, or 68 min, or 69 min, or 70 min, or 71 min, or 72 min, or 73 min, or 74 min, or 75 min, or 76 min, or 77 min, or 78 min, or 79 min, or 80 min, or 81 min, or 82 min, or 83 min, or 84 min, or 85 min, or 86 min, or 87 min, or 88 min, or 89 min, or 90 min, or 91 min, or 92 min, or 93 min, or 94 min, or 95 min, or 96 min, or 97 min, or 98 min, or 99 min, or 100 min.

[0083] In certain embodiments, the centrifugation time in step (V) is 70 min.

[0084] In certain embodiments, the centrifugal force of the centrifugation in step (VI) is 100,000×g, or 101,000×g, or 102,000×g, or 103,000×g, or 104,000×g, or 105,000×g, or 106,000×g, or 107,000×g, or 108,000×g, or 109,000×g, or 110,000×g, or 110,200×g, or 111,000×g, or 112,000×g, or 113,000×g, or 114,000×g, or 115,000×g, or 116,000×g, or 117,000×g, or 118,000×g, or 119,000×g, or 120,000×g.

[0085] In certain embodiments, the centrifugal force of the centrifugation in step (VI) is 110200×g.

[0086] In certain embodiments, the centrifugation time in step (VI) is 60 min, or 61 min, or 62 min, or 63 min, or 64 min, or 65 min, or 66 min, or 67 min, or 68 min, or 69 min, or 70 min, or 71 min, or 72 min, or 73 min, or 74 min, or 75 min, or 76 min, or 77 min, or 78 min, or 79 min, or 80 min, or 81 min, or 82 min, or 83 min, or 84 min, or 85 min, or 86 min, or 87 min, or 88 min, or 89 min, or 90 min, or 91 min, or 92 min, or 93 min, or 94 min, or 95 min, or 96 min, or 97 min, or 98 min, or 99 min, or 100 min.

[0087] In certain embodiments, the centrifugation time in step (VI) is 70 min.

[0088] In certain embodiments, the specific steps of the ultracentrifugation are: (I) centrifuging the turmeric juice obtained in step (1) at 500×g for 10 min and collecting the supernatant; (II) Centrifuge at 2000 × g for 20 min and collect the supernatant; (III) Centrifuge at 4000 × g for 30 min and collect the supernatant; (IV) Centrifuge at 10,000 × g for 60 min and collect the supernatant; (V) Centrifuge at 110200 × g for 70 min, collect the precipitate, and resuspend in PBS; (VI) Centrifuging at 110200×g for 70 min, collecting the precipitate, and resuspending it in PBS to obtain the turmeric extracellular vesicles.

[0089] In certain embodiments, the extraction method in step (2) is graded filtration.

[0090] In certain embodiments, the extraction method in step (2) is tangential flow filtration.

[0091] In certain embodiments, the specific steps of the tangential flow filtration include: (a) subjecting the turmeric juice obtained in step (1) to gradient centrifugation and collecting the supernatant; (b) diluting the supernatant obtained in step (a) with PB buffer until the turbidity is less than 900 NTU; (c) filtering the diluted solution in step (b) through a tangential flow depth filtration membrane package, and collecting the filtrate; (d) The filtrate obtained in step (c) is concentrated and purified using a hollow fiber column to obtain the turmeric extracellular vesicles.

[0092] In certain embodiments, the specific steps of the gradient centrifugation in step (a) include: (a1) centrifuging the turmeric juice obtained in step (1) at 400-800×g for 5-20 min and collecting the supernatant; (a2) Centrifuge again at 1000-3000 × g for 10-30 min and collect the supernatant; (a3) Centrifuge at 3000-5000 × g for 20-40 min and collect the supernatant; (a4) Centrifuge again at 8000-12000×g for 40-70 min and collect the supernatant.

[0093] In certain embodiments, the centrifugal force of the centrifugation in step (a1) is 400×g, or 450×g, or 500×g, or 550×g, or 600×g, or 650×g, or 700×g, or 750×g, or 800×g.

[0094] In certain embodiments, the centrifugal force of the centrifugation in step (a1) is 500×g.

[0095] In certain embodiments, the centrifugation time in step (a1) is 5 min, or 6 min, or 7 min, or 8 min, or 9 min, or 10 min, or 11 min, or 12 min, or 13 min, or 14 min, or 15 min, or 16 min, or 17 min, or 18 min, or 19 min, or 20 min.

[0096] In certain embodiments, the centrifugation time in step (a1) is 10 min.

[0097] In certain embodiments, the centrifugal force of the centrifugation in step (a2) is 1000×g, or 1100×g, or 1200×g, or 1300×g, or 1400×g, or 1500×g, or 1600×g, or 1700×g, or 1800×g, or 1900×g, or 2000×g, or 2100×g, or 2200×g, or 2300×g, or 2400×g, or 2500×g, or 2600×g, or 2700×g, or 2800×g, or 2900×g, or 3000×g.

[0098] In certain embodiments, the centrifugal force of the centrifugation in step (a2) is 2000×g.

[0099] In certain embodiments, the centrifugation time in step (a2) is 10 min, or 11 min, or 12 min, or 13 min, or 14 min, or 15 min, or 16 min, or 17 min, or 18 min, or 19 min, or 20 min, or 21 min, or 22 min, or 23 min, or 24 min, or 25 min, or 26 min, or 27 min, or 28 min, or 29 min, or 30 min.

[0100] In certain embodiments, the centrifugation time in step (a2) is 20 min.

[0101] In certain embodiments, the centrifugal force of the centrifugation in step (a3) ​​is 3000×g, or 3100×g, or 3200×g, or 3300×g, or 3400×g, or 3500×g, or 3600×g, or 3700×g, or 3800×g, or 3900×g, or 4000×g, or 4100×g, or 4200×g, or 4300×g, or 4400×g, or 4500×g, or 4600×g, or 4700×g, or 4800×g, or 4900×g, or 5000×g.

[0102] In certain embodiments, the centrifugal force of the centrifugation in step (a3) ​​is 4000×g.

[0103] In certain embodiments, the centrifugation time in step (a3) ​​is 20 min, or 21 min, or 22 min, or 23 min, or 24 min, or 25 min, or 26 min, or 27 min, or 28 min, or 29 min, or 30 min, or 31 min, or 32 min, or 33 min, or 34 min, or 35 min, or 36 min, or 37 min, or 38 min, or 39 min, or 40 min.

[0104] In certain embodiments, the centrifugation time in step (a3) ​​is 30 min.

[0105] In certain embodiments, the centrifugal force of the centrifugation in step (a4) is 8000×g, or 8100×g, or 8200×g, or 8300×g, or 8400×g, or 8500×g, or 8600×g, or 8700×g, or 8800×g, or 8900×g, or 9000×g, or 9100×g, or 9200×g, or 9300×g, or 9400×g, or 9500×g, or 9600×g, or 9700×g, or 9800×g, or 9900×g, or 10 000×g, or 10100×g, or 10200×g, or 10300×g, or 10400×g, or 10500×g, or 10600×g, or 10700×g, or 10800×g, or 10900×g, or 11000×g, or 11100×g, or 11200×g, or 11300×g, or 11400×g, or 11500×g, or 11600×g, or 11700×g, or 11800×g, or 11900×g, or 12000×g.

[0106] In certain embodiments, the centrifugal force of the centrifugation in step (a4) is 10,000×g.

[0107] In certain embodiments, the centrifugation time in step (a4) is 40 min, or 41 min, or 42 min, or 43 min, or 44 min, or 45 min, or 46 min, or 47 min, or 48 min, or 49 min, or 50 min, or 51 min, or 52 min, or 53 min, or 54 min, or 55 min, or 56 min, or 57 min, or 58 min, or 59 min, or 60 min, or 61 min, or 62 min, or 63 min, or 64 min, or 65 min, or 66 min, or 67 min, or 68 min, or 69 min, or 70 min.

[0108] In certain embodiments, the centrifugation time in step (a4) is 60 min.

[0109] In certain embodiments, the specific steps of the gradient centrifugation in step (a) are: (a1) centrifuging the turmeric juice obtained in step (1) at 500×g for 10 min and collecting the supernatant; (a2) Centrifuge at 2000 × g for 20 min and collect the supernatant; (a3) Centrifuge at 4000 × g for 30 min and collect the supernatant; (a4) Centrifuge at 10,000 × g for 60 min and collect the supernatant.

[0110] The invention also provides a turmeric extracellular vesicle prepared according to the method.

[0111] In certain embodiments, the extracellular vesicles are exosomes.

[0112] The present invention also provides a composition, which comprises the turmeric extracellular vesicles.

[0113] Example 1: Extraction of turmeric extracellular vesicles by ultracentrifugation 1. Take fresh turmeric, wash it, and dry it at 50℃ for 24 hours.

[0114] 2. Accurately weigh 50 g of dried turmeric, add 500 mL of sterile (filtered using a 0.22 μm filter membrane) PBS, and soak overnight.

[0115] 3. Use a wall-breaking machine to homogenize the turmeric into tissue fluid (5-10 min) without obvious lumps and fragments.

[0116] 4. Ultracentrifugation extraction: (1) Transfer the dried turmeric homogenate to a 50 mL sterile centrifuge tube and centrifuge at 500 × g for 10 min at 4°C. Discard the precipitate and take the supernatant.

[0117] (2) Centrifuge at 2000 × g for 20 min at 4°C, discard the precipitate and take the supernatant.

[0118] (3) Centrifuge at 4000 × g for 30 min at 4°C, discard the precipitate and take the supernatant.

[0119] (4) Centrifuge at 10,000 × g for 1 h at 4°C, discard the precipitate and take the supernatant.

[0120] (5) Centrifuge at 110,200 × g for 70 min at 4°C, discard the supernatant, and resuspend the precipitate in PBS.

[0121] (6) Centrifuge at 110200 × g for 70 min in a 4°C ultracentrifuge, discard the supernatant, and resuspend the precipitate in PBS to obtain turmeric extracellular vesicles (CL-Exo).

[0122] Example 2: Extraction of extracellular vesicles of turmeric by tangential flow filtration Steps 1-3 are the same as in Example 1 4. Tangential flow filtration extraction: (1) Pretreatment centrifugation: Transfer the dried turmeric homogenate to a centrifuge tube and use a desktop centrifuge set to 4°C for gradient centrifugation: 500g for 10 min, 2000g for 20 min, 4000g for 30 min, and 10000g for 60 min; after centrifugation, collect the supernatant and discard the precipitate.

[0123] (2) Supernatant dilution: Use a standard with a turbidity of 800 NTU to calibrate the turbidity meter. Take the supernatant obtained in step 3 for turbidity measurement. If the turbidity is greater than 900 NTU, dilute it with PB buffer until the turbidity is less than 900 NTU.

[0124] (3) Deep filtration: The supernatant diluted in step (2) is deep filtered through a tangential flow deep filtration membrane package, and the deep filtered liquid is collected.

[0125] (4) Concentration and purification: The deep filtrate obtained in step (3) is concentrated and purified by passing it through a hollow fiber column. The suspension obtained after the concentration is completed is the turmeric extracellular vesicles.

[0126] Example 3: Characterization of Curcuma longa extracellular vesicles (1) Particle size determination Test instrument: microfluidic resistive pulse induction instrument.

[0127] The results are as follows Figure 1 As shown, the particle size of CL-Exo is 162.2.

[0128] (2) Transmission electron microscopy (TEM) characterization The CL-Exo prepared in Example 1 was resuspended in pure water to obtain an EVs solution. The EVs solution (resuspended in pure water) was diluted with an appropriate amount of pure water to a protein concentration of 6 mg / mL, and then dripped onto a sample copper grid, evaporated naturally, stained with phosphotungstic acid, and the morphology was observed under a transmission electron microscope.

[0129] The results are as follows Figure 2 As shown, the particle density in the EVs stock solution is relatively high, the particle size distribution of most particles is about 160 nm, and the nanoparticles have a typical teacup shape.

[0130] Example 4: Analysis of components of turmeric extracellular vesicles (1) Determination of total flavonoids (using the total flavonoids (Flavonoid) kit from Suzhou Grace Biotechnology Co., Ltd.) 1. Sample preparation: Weigh about 0.1g of fresh sample (CL-Exo prepared in Example 1, if the water content is sufficient, the sample sampling mass can be increased); or weigh about 0.03g of dried sample (the sample is sterilized at 105℃ for 3min, then dried at 60℃ to constant weight, crushed, and passed through a 40-60 mesh sieve to obtain a dried sample), add 1.5mL of 60% ethanol, and extract at 60℃ for 2h. Centrifuge at 25℃×12000rpm for 10min, take the supernatant, and dilute to 1.5mL with 60% ethanol for testing. [Note]: If the sample size is small, the sample size can be reduced in the same proportion, such as taking 0.02g of dry sample, adding 1mL of 60% ethanol, and extracting at 60℃ for 2h. Centrifuge at 25℃×12000rpm for 10min, take the supernatant, and dilute to 1mL with 60% ethanol for testing.

[0131] 2. On-machine detection: ① Preheat the microplate reader for 30 minutes and adjust the wavelength to 510nm. ② Select two samples for pre-determination. If the A measurement value exceeds 1.5, dilute the supernatant with 60% ethanol to determine the dilution multiple D suitable for this batch of samples. The corresponding dilution multiple D needs to be substituted into the formula for calculation. ③ Add samples, distilled water, reagent 1 (5% sodium nitrite), reagent 2 (10% aluminum nitrate) and reagent 3 (4% sodium hydroxide) to the 96-well plate in sequence according to the instructions, mix well, let stand at 25℃ for 15 minutes, and measure the absorbance at 510nm.

[0132] 3. Calculation of results: 1. Standard curve equation: y = 1.6277x - 0.0049, where x is the concentration of the standard (mg / mL) and y is ΔA.

[0133] Total flavonoid content (mg / g dry weight) = [(ΔA+0.0049)÷1.6277×V1]÷(V1÷V×W) ×D =0.6×(ΔA+0.0049)÷W×V×D Wherein, V is the volume of the extract, 1.5 mL; V1 is the volume of the sample in the reaction, 50 µL = 0.05 ml; W is the mass of the sample, g; D is the dilution factor, undiluted is 1; The results are as follows Figure 3 As shown, the concentration of total flavonoids in turmeric extracellular vesicles was 0.43 mg / mL.

[0134] (2) Determination of total sugar (Shanghai Yuanye Biotechnology-Plant Total Sugar and Reducing Sugar Detection Kit (DNS Microplate Method)) 1. Hydrolysis and extraction of total sugars: ① Weigh the plant sample (CL-Exo prepared in Example 1) and transfer it to a container. ② Add 10 ml of 6M hydrochloric acid solution to the container, stir evenly, boil for 30 min, and stir from time to time. ③ Take 2 drops and add them to a glass slide, add 1 drop of color developing solution (about 50 μL), check whether the hydrolysis is complete. If the hydrolysis is complete, no blue color will be displayed. ④ After the hydrolysis is completed, cool to room temperature, add 6M sodium hydroxide solution to adjust the solution pH to 7.4, dilute to 100 ml with distilled water, mix well, centrifuge at 4000g for 5 min or filter. ⑤ Take 10 ml of the supernatant or filtrate, dilute to 100 ml with distilled water to form a 10-fold diluted total sugar hydrolyzate (extract), take 50 ul of the total sugar hydrolyzate, and determine its reducing sugar content.

[0135] 2. Dilute the glucose standard: Take a clean centrifuge tube or test tube and follow the instructions of the kit to obtain a series of Glu standards.

[0136] 3. Sample addition: Take a 1ml centrifuge tube and follow the instructions of the kit to set up blank wells, standard wells, and measurement wells. The solutions should be added in order, and be careful to avoid bubbles and mix carefully. If the sugar concentration in the sample is too high, you can reduce the sample amount or dilute it appropriately before measuring. It is best to set up 2~3 parallel wells for sample testing and calculate the average value.

[0137] 4. Reducing sugar determination: Mix well, transfer 300ul to the corresponding 96-well plate in turn, adjust to zero with the blank well, and measure the absorbance of the standard well and the measurement well at 540nm.

[0138] 5. Calculation: Percentage of total sugar: Total sugar content in 100g sample (g) = (c × N × V T ) / (m×1000)×100×0.9=(c×N×VT) / (m×10)×0.9 Where: c = sugar content (mg / ml) from the standard curve, V T =Total volume of extract (ml) = 100, m = mass of plant sample (g), N = dilution factor of total sugar hydrolyzate = 10.

[0139] The results are as follows Figure 4 As shown, the concentration of total sugars in turmeric extracellular vesicles was 12.07 mg / mL.

[0140] Example 5: Penetration of Curcuma Extracellular Vesicles into Ex vivo Pig Skin Cut the back or abdominal skin of 8-week-old Bama miniature pigs into 1 cm × 1 cm pieces for later use. Take 1 mg of CL-Exo prepared in Example 1, and dilute the sample to 1 mL with diluent C in the kit (Sigma, PKH26 Red Fluorescent Cell Labeling Kit MINI26-1KT). Take 6 μL of PKH26 dye in the kit and add it to a test tube containing 1 mL of diluent C. Use a pipette to blow gently and mix continuously for 30 seconds. Let stand at room temperature for 5 minutes. Add 2 mL of 10% BSA in PBS (Sigma-Aldrich, D8537) for quenching. Use serum-free medium to make the volume 30 mL. Centrifuge at 110,000 g for 2 hours at 2-8°C. Use a pipette to blow gently and resuspend the extracellular vesicle pellet in 1 ml of serum-free medium for later use.

[0141] Place 1cm×1cm 8-week-old Bama miniature pig skin in a 32℃ environment, take 50μL 1mg / ml PKH26-labeled CL-Exo and drip it onto 1cm×1cm 8-week-old Bama miniature pig skin, evenly cover the entire epidermis, and incubate for 2 hours. After incubation, cryosections were performed with a thickness of 10 μm. After DAPI staining the cell nucleus, the cells were observed and photographed under a fluorescence microscope. The results are shown in Figure 5 As shown, turmeric-derived extracellular vesicles can penetrate into the skin of 8-week-old Bama miniature pigs.

[0142] Example 6: Effect of turmeric extracellular vesicles on keratinocyte proliferation The CCK-8 method was used to determine the proliferation effect of turmeric extracellular vesicles (CL-Exo prepared in Example 1) on keratinocytes. A control group (NC: DMEM basal medium), a positive control group (PC: DMEM basal medium + 100 ng / mL EGF) and an experimental group (DMEM basal medium containing different concentrations of turmeric EVs) were set up. Take the turmeric EVs solution and dilute it with serum-free DMEM medium to a mother solution with a protein concentration of 1 mg / mL. Prepare 5 μg / mL, 10 μg / mL, and 20 μg / mL sample solutions, which are recorded as experimental groups 1 to 3, respectively, for later use.

[0143] In addition to the blank group, cells with good growth status were taken and 1×10 4 The cells were seeded at a density of 100 cells / well in a 96-well plate and cultured for 24 h before use.

[0144] The experimental groups were given 100 μL / well of the sample solutions with the above concentration gradients, and the blank and normal groups were given the same dose of incomplete culture medium (i.e., culture medium without serum). The culture medium was discarded after incubation at 37°C and 5% CO2 for 24 h.

[0145] The cell proliferation activity was detected according to the instructions of the CCK-8 kit. Each group was tested in triplicate, and the cell survival rate and cell proliferation rate were calculated according to the following formula.

[0146] Cell proliferation rate%=(OD 实验组 -OD 空白 ) / (OD 对照组 -OD 空白 )×100%; The results are as follows Figure 6 As shown, the addition of 10 μg / mL of turmeric extracellular vesicles can significantly promote keratinocyte proliferation, and the proliferation ability of keratinocytes was upregulated by 20.7%.

[0147] Example 7: Effect of Curcuma longa extracellular vesicles on keratinocyte migration A control group (NC: basal medium), a positive control group (PC: basal medium + 100 ng / mL EGF) and an experimental group (basal medium containing turmeric Evs) were set up. Take the turmeric extracellular vesicle (CL-Exo prepared in Example 1) solution and dilute it with serum-free DMEM medium to a mother solution with a protein concentration of 1 mg / mL. Prepare a 20 μg / mL sample solution, record it as the experimental group, and set aside.

[0148] Keratinocytes in good growth condition were taken and 2×10 5 The cells were seeded at a density of 100 cells / well in a 24-well plate and cultured for 24 h before use.

[0149] Use a 300 μL pipette tip to scrape each well once (the tip should be vertical, not tilted) to form an artificial wound, wash the cells three times with PBS, and remove the scratched cells. Use a microscope to take pictures of each scratch position and record the scratch area at 0h.

[0150] The positive control group and the experimental group were given 500 μL / well of the sample solution with the above concentration gradient, and the control group was given the same dose of incomplete culture medium (i.e., culture medium without serum) and incubated at 37°C and 5% CO2 for 24 h.

[0151] Use a microscope to take pictures of each scratch position, record the scratch area for 24 h, and use imageJ to count the keratinocyte scratch area at 0 h and 24 h.

[0152] Cell migration rate % = (S 0h -S 24h ) / S 0h ×100%, where S 0h : Scratch area at 0 hours; S 24h :24 hours scratch area.

[0153] The results are as follows Figure 7 As shown, the addition of 20 μg / mL of turmeric extracellular vesicles can significantly promote keratinocyte migration, and the cell migration level was upregulated by 19.7%.

[0154] Example 8: Effect of turmeric extracellular vesicles on the expression level of keratin-related proteins A blank group and a drug administration group were set up. The CL-Exo solution prepared in Example 1 was taken and diluted with serum-free DMEM medium to a mother solution with a protein concentration of 1 mg / mL. The mother solution was diluted to 20 μg / mL to obtain sample solutions, which were recorded as drug administration groups for later use.

[0155] Keratinocytes in good growth condition were taken and 2×10 5 The cells were seeded at a density of 100 cells / well in a 12-well plate and cultured for 24 h before use.

[0156] The drug-treated groups were given 1 mL / well of the sample solution with the above concentration gradient, and the blank group and the normal group were given the same dose of incomplete culture medium (i.e., DMEM culture medium without serum), and incubated at 37°C and 5% CO2 for 24 h.

[0157] RNA was extracted using VeZol Reagent according to the instructions of VeZol Reagent R411.

[0158] The RNA extracted in the previous step was reverse transcribed according to the instructions of Takara PrimeScript™ RT reagent Kit (PerfectReal Time).

[0159] According to the instructions of Bimake 2x SYBRGreen qPCR Master Mix (High ROX), the cDNA obtained by reverse transcription in the previous step was used for QPCR experiment.

[0160] Use 2 -ΔΔCt Method to calculate the expression level of the corresponding gene: ΔΔCt = (Ct(target gene) - Ct(reference gene)) sample A - (Ct(target gene) - Ct(reference gene)) sample B Fold Change = 2 -ΔΔCt Among them, Fold Change represents the multiple of the expression level of the target gene relative to the expression level of the reference gene.

[0161] The results are as follows Figure 8 As shown in the results, the addition of 20 μg / mL of turmeric extracellular vesicles could significantly promote the expression of keratinocyte filaggrin (FLG) and involucrin (IVL), up-regulated by 48.3% and 92.2%, respectively.

[0162] Example 9: Effects of turmeric extracellular vesicles on reactive oxygen species (ROS) levels in keratinocytes The DCFH-DA fluorescent probe was used to determine the effect of turmeric extracellular vesicles on the level of reactive oxygen species in keratinocytes. A blank control group (BL: DMEM basal medium, without irradiation), a control group (NC: DMEM basal medium), a naked cell group (wells without cells inoculated), a positive control group (PC: DMEM basal medium + 5 μg / mL RA) and an experimental group (DMEM basal medium containing different concentrations of CL-Exo) were set up. A turmeric extracellular vesicle solution (CL-Exo prepared in Example 1) was taken and diluted with serum-free DMEM medium to a mother solution with a protein concentration of 1 mg / mL. Sample solutions of 0.5 μg / mL, 5 μg / mL, and 20 μg / mL were prepared respectively, and recorded as experimental groups 1 to 3, for later use.

[0163] Take cells in good growth state and add 1×10 4 The cells were seeded at a density of 100 cells / well in a 96-well plate and cultured for 24 h before use.

[0164] The cell culture supernatant was discarded, 50 μL DPBS was added to each well, and the well was placed 15 cm below the UV lamp and irradiated for 16 min.

[0165] The DPBS was discarded, and the positive control group and the drug-treated group were given 100 μL / well of the sample solution with the above concentration gradient, and the control group was given the same dose of incomplete culture medium (i.e., DMEM culture medium without serum). The culture medium was discarded after incubation at 37°C and 5% CO2 for 6 hours.

[0166] The DCFH-DA working solution was prepared at a dilution ratio of 1:1000, and DCFH-DA was diluted with DMEM basal medium to a final concentration of 10 μmol / L.

[0167] The culture medium in the remaining wells was discarded, and the wells were washed three times with PBS. Then, 100 μL of DCFH-DA working solution was added to each well and incubated in a CO2 incubator for 30 min.

[0168] After the incubation, the cells in each well were washed three times with basal medium, placed on the detection platform of the fluorescence microplate reader, and the incident light wavelength was set to 529 nm, the excitation light wavelength was set to 504 nm, and the reading was taken.

[0169] ROS level (%) = (T-C0) / (C-C0) × 100%, where T is the average of three fluorescence intensity of the test sample; C is the average of three fluorescence intensity of the control group; C0 is the average of three fluorescence intensity of the naked cell group.

[0170] The results are as follows Fig. 9 As shown, the addition of 20 μg / mL of turmeric extracellular vesicles can significantly inhibit the ROS level of keratinocytes, with the inhibition level reaching 20.7%.

[0171] Example 10: Effect of turmeric extracellular vesicle-like particles on type I collagen secretion in dermal fibroblasts Dermal fibroblasts were seeded into 96-well plates at a density of 5000 cells / 100 μL / well. The cells were cultured for 24 h. The cell supernatant was discarded, and 100 μL of negative control reagent (NC: T4 basal medium), positive control reagent (PC: 100 ng / mL TGF-β), and experimental group reagent (20 μg / mL, 50 μg / mL CL-Exo prepared in Example 1) were added and cultured for 24 h. The supernatant was taken and type I collagen secretion was detected using the Lianke type I collagen detection kit.

[0172] The results are as follows Fig.10 As shown, CL-Exo can significantly promote the secretion of type Ⅰ collagen in dermal fibroblasts.

[0173] Example 11: Effect of Curcuma longa extracellular vesicle-like particles on the secretion of extracellular matrix proteases by dermal fibroblasts Dermal fibroblasts were inoculated into 96-well plates at a density of 5000 cells / 100 μL / well. The cells were cultured for 24 h. The cells were irradiated with UVA to simulate the changes in matrix metalloenzymes caused by photoaging. The cell supernatant was discarded and 50 μL of pre-cooled PBS was added to each well. An ice bag was placed in the biosafety cabinet, and four layers of kraft paper were placed on the ice bag. The cell culture plate was then placed on the kraft paper. The position of the UVA lamp was adjusted so that the distance from the lamp to the cells was 5 cm. The cell culture plate was removed, the lamp was turned on for 5 min of equilibration, and then the cell culture plate was placed under the lamp. The lid was opened and irradiated for 1 h, for a total of 5 J / cm 2Energy. PBS was discarded, negative control reagent (NC: T4 basal medium), positive control reagent (PC: 100 ng / mL TGF-β), experimental group reagent (20 μg / mL, 50 μg / mL CL-Exo prepared in Example 1) were added, and T4 basal medium (BC) was added to the group not treated with UVA, and cultured for 24 hours. The supernatant was taken, and the secretion of matrix metalloproteinase 1 (MMP1) and matrix metalloproteinase tissue inhibitor 1 (TIMP1) proteins were detected by Lianke human MMP1 ELISA kit and human TIMP1 ELISA kit, respectively.

[0174] The results are as follows Fig.11 As shown, CL-Exo can both promote the secretion of TIMP1 by UVA-induced dermal fibroblasts and inhibit the secretion of MMP1 by UVA-induced dermal fibroblasts.

[0175] Example 12: Effects of turmeric extracellular vesicles on senescence of dermal fibroblasts Dermal fibroblasts were inoculated into 24-well plates at a density of 20,000 cells / 100 μL / well. The cells were cultured for 24 hours. The cells were aged by UVA irradiation, the cell supernatant was discarded, and 100 μL of pre-cooled PBS was added to each well. An ice bag was placed in the biosafety cabinet, four layers of kraft paper were placed on the ice bag, and then the cell culture plate was placed on the kraft paper. The position of the UVA lamp was adjusted so that the distance from the lamp to the cells was 5 cm. The cell culture plate was removed, the lamp was turned on for 5 minutes of balance, and then the cell culture plate was placed under the lamp, the lid was opened, and irradiated for 1 hour, with a total energy of 5 J / cm2. PBS was discarded, and blank control reagent (BC: basal culture medium), negative control reagent (NC: basal culture medium), positive control reagent (PC: 100 ng / mL of TGF-β), and experimental group reagent (20 μg / mL of CL-Exo prepared in Example 1) were added and cultured for 24 hours. 5 J / cm 2 The NC, PC, and experimental group cells were irradiated with UVA of high energy, and then treated with drugs again and incubated for 24 h. Then, senescent cells were stained using the β-galactosidase staining kit of Shanghai Shangbao Biotechnology Co., Ltd.

[0176] The results are as follows Fig.12 As shown, CL-Exo can significantly reduce UVA-induced senescence of dermal fibroblasts.

[0177] Example 13: Human effect test of face cream containing turmeric extracellular vesicles The CL-Exo prepared in Example 1 was added to the matrix cream at an addition amount of 5%, 10%, and 20% (as shown in the formula Table 1), and a matrix cream without extracellular vesicle-like particles derived from turmeric was used as a control. It was applied once in the morning and evening, and no other skin care products were used during use. 30 testers were selected and divided into a matrix group and an experimental group. The matrix group used the matrix cream, and the experimental group used the matrix cream containing the extracellular vesicles of turmeric of the present invention. Visia photography was used to detect the skin status on day 0 and day 21 of use.

[0178] The results are as follows Fig.13 , 14 As shown, after using the cream containing turmeric-derived extracellular vesicles for 28 days, the subjects' skin SEw (skin wrinkles), wrinkle length, wrinkle depth, and wrinkle area ratio were reduced, and the wrinkles around the eyes were significantly reduced as detected by the skin rapid three-dimensional imaging system and Visia camera detection photos.

[0179] Table 1: Ingredients of base cream

[0180] It should be understood that the above embodiments are exemplary and are not intended to include all possible implementations included in the claims. Various modifications and changes may be made on the basis of the above embodiments without departing from the scope of the present disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only express several implementations of the present invention and do not limit the scope of protection of the patent of the present invention.

Claims

1. Application of turmeric extracellular vesicles in the field of cosmetics.

2. The use according to claim 1, characterized in that: The applications include: (1) Application in the preparation of skin anti-wrinkle and anti-aging products; (2) Application in the preparation of skin repair and antioxidant products; (3) Application in the preparation of whitening and anti-freckle products.

3. The use according to any one of claims 1-2, characterized in that: The preparation method of the turmeric extracellular vesicles comprises: (1) Soak the washed turmeric and break the wall to obtain turmeric juice; (2) Using the turmeric juice obtained in step (1), extracting turmeric extracellular vesicles.

4. The method according to claim 3, characterized in that The immersion solution in step (1) is PBS; And / or, the soaking time in step (1) is 6 to 24 hours.

5. The method according to claim 4, characterized in that The soaking time in step (1) is 6 to 12 hours; And / or, the cell wall breaking time in step (1) is 5 to 10 minutes.

6. The method according to claim 3, characterized in that: The extraction method in step (2) is selected from one or more of sucrose density gradient centrifugation, ultracentrifugation, graded filtration, and tangential flow filtration.

7. The method according to claim 6, characterized in that The specific steps of the ultracentrifugation include: (I) centrifuging the turmeric juice obtained in step (1) at 400-800×g for 5-20 min and collecting the supernatant; (II) Centrifuge again at 1000-3000 × g for 10-30 min and collect the supernatant; (III) Centrifuge at 3000-5000 × g for 20-40 min and collect the supernatant; (IV) Centrifuge at 8000-12000 × g for 40-70 min and collect the supernatant; (V) Centrifuge again at 100,000-120,000 × g for 60-100 min, collect the precipitate, and resuspend in PBS; (VI) Centrifuging at 100,000-120,000×g for 60-100 min, collecting the precipitate, and resuspending it in PBS to obtain the turmeric extracellular vesicles.

8. The method according to claim 7, characterized in that The specific steps of the ultracentrifugation are: (I) centrifuging the turmeric juice obtained in step (1) at 500×g for 10 min and collecting the supernatant; (II) Centrifuge at 2000 × g for 20 min and collect the supernatant; (III) Centrifuge at 4000 × g for 30 min and collect the supernatant; (IV) Centrifuge at 10,000 × g for 60 min and collect the supernatant; (V) Centrifuge at 110200 × g for 70 min, collect the precipitate, and resuspend in PBS; (VI) Centrifuging at 110200×g for 70 min, collecting the precipitate, and resuspending it in PBS to obtain the turmeric extracellular vesicles.

9. The method according to claim 6, characterized in that The specific steps of the tangential flow filtration include: (a) subjecting the turmeric juice obtained in step (1) to gradient centrifugation and collecting the supernatant; (b) diluting the supernatant obtained in step (a) with PB buffer until the turbidity is less than 900 NTU; (c) filtering the diluted solution in step (b) through a tangential flow depth filtration membrane package, and collecting the filtrate; (d) The filtrate obtained in step (c) is concentrated and purified using a hollow fiber column to obtain the turmeric extracellular vesicles.

10. The method according to claim 9, characterized in that The specific steps of the gradient centrifugation in step (a) include: (a1) centrifuging the turmeric juice obtained in step (1) at 400-800×g for 5-20 min and collecting the supernatant; (a2) Centrifuge at 1000-3000 × g for 10-30 min and collect the supernatant; (a3) Centrifuge at 3000-5000 × g for 20-40 min and collect the supernatant; (a4) Centrifuge at 8000-12000 × g for 40-70 min and collect the supernatant.

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