A method for extracting extracellular vesicles of turmeric and its applications

Abstract

The present invention relates to the application of turmeric extracellular vesicles in the field of cosmetics. The present invention discovers for the first time that turmeric extracellular vesicles have anti-aging, repair, and antioxidant effects, and have good water solubility, excellent permeability, and are easily absorbed by the skin; they can (a) promote the proliferation of fibroblasts, (b) promote the collagen secretion of fibroblasts, (c) inhibit the level of MMP1 in fibroblasts, (d) promote the level of TMP1 in fibroblasts, (e) inhibit the level of ROS in fibroblasts, (f) inhibit the level of SA-β-Gal in fibroblasts, (g) promote the proliferation level of keratinocytes, (h) inhibit the level of ROS in keratinocytes, (i) promote the expression of keratinization-related proteins in keratinocytes, and (j) promote the migration level of keratinocytes.

Description

Technical Field

[0001] The present invention relates to the technical field of biological extraction, and particularly relates to a method for extracting turmeric extracellular vesicles and its application. Background Art

[0002] Extracellular vesicles are membrane-bound vesicles with a diameter of about 20 nm - 5 μm. They differ in size and composition and contain 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 facing various biotic and abiotic environmental stresses (such as pathogen infection and attack), and have similar size distribution, surface charge, surface morphology, content composition, etc. to animal-derived exosomes. Extracellular vesicles contain various molecules such as RNA, proteins, and lipids, thus playing a role in regulating physiological processes. They can serve as both transport carriers and possess biological functions themselves. Plant exosomes have a wide source, are safe, non-toxic, have low immunogenicity, can be produced on a large scale, and have low cost, and have good biocompatibility and environmental friendliness; and plant exosomes can be directly eaten and absorbed through the gastrointestinal tract, and play a role in interspecies communication between plants and mammalian cells.

[0003] Turmeric (Curcuma longa L.), as a tropical perennial herb, its rhizome is rich in various active ingredients, especially curcumin, showing excellent application value. Curcumin not only has strong antioxidant and anti-inflammatory abilities, can effectively scavenge free radicals in the body and inhibit inflammatory reactions, 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 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 values.

[0004] In the currently reported studies, it has been proven that curcumin has functions such as anti-inflammatory, antioxidant, and regulation of apoptosis, and has potential application values in tumor treatment, inflammation regulation, skin repair, etc., but no study on the anti-aging effect of turmeric extracellular vesicles has been found. At the same time, due to the poor water solubility and absorbability of curcumin itself, it may limit its bioavailability on the skin, resulting in less-than-satisfactory effects in delaying skin aging, repairing the skin, etc. Therefore, there is an urgent need in this field for a turmeric extract with good water solubility, good permeability, and easy skin absorption. Summary of the Invention

[0005] Problems to be Solved by the Invention

[0006] In view of the above problems existing in the prior art, the object of the present invention is to provide curcumin extracellular vesicles with good water solubility, excellent permeability, easy skin absorption, and anti-aging, repair, and antioxidant effects.

[0007] Solutions for Solving the Problems

[0008] The present invention provides an application of curcumin extracellular vesicles in the field of cosmetics.

[0009] Preferably, the application includes:

[0010] (1) Application in the preparation of skin anti-wrinkle and anti-aging products;

[0011] (2) Application in the preparation of skin repair and antioxidant products;

[0012] (3) Application in the preparation of skin whitening and freckle-removing products.

[0013] Preferably, the preparation method of the curcumin extracellular vesicles includes:

[0014] (1) Soaking and breaking the wall of the washed curcumin to obtain curcumin juice;

[0015] (2) Using the curcumin juice obtained in step (1) to extract curcumin extracellular vesicles.

[0016] Preferably, the solution for soaking in step (1) is PBS;

[0017] and / or, the soaking time in step (1) is 6 to 24 hours.

[0018] Preferably, the soaking time in step (1) is 6 to 12 hours.

[0019] and / or, the time for breaking the wall in step (1) is 5 to 10 min.

[0020] 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.

[0021] Preferably, the specific steps of the ultracentrifugation include:

[0022] (I) Centrifuging the curcumin juice obtained in step (1) at 400 - 800×g for 5 - 20 min, and collecting the supernatant;

[0023] (II) Then centrifuging at 1000 - 3000×g for 10 - 30 min, and collecting the supernatant;

[0024] (III) Centrifuge at 3000 - 5000×g for 20 - 40 min, and collect the supernatant;

[0025] (IV) Centrifuge at 8000 - 12000×g for 40 - 70 min, and collect the supernatant;

[0026] (V) Centrifuge at 100000 - 120000×g for 60 - 100 min, collect the precipitate, and resuspend it with PBS;

[0027] (VI) Centrifuge at 100000 - 120000×g for 60 - 100 min, collect the precipitate, and resuspend it with PBS to obtain the turmeric extracellular vesicles.

[0028] Preferably, the specific steps of the ultracentrifugation are as follows:

[0029] (I) Centrifuge the turmeric juice obtained in step (1) at 500×g for 10 min, and collect the supernatant;

[0030] (II) Then centrifuge at 2000×g for 20 min, and collect the supernatant;

[0031] (III) Then centrifuge at 4000×g for 30 min, and collect the supernatant;

[0032] (IV) Then centrifuge at 10000×g for 60 min, and collect the supernatant;

[0033] (V) Then centrifuge at 110200×g for 70 min, collect the precipitate, and resuspend it with PBS;

[0034] (VI) Then centrifuge at 110200×g for 70 min, collect the precipitate, and resuspend it with PBS to obtain the turmeric extracellular vesicles.

[0035] Preferably, the specific steps of the tangential flow filtration include:

[0036] (a) Centrifuge the turmeric juice obtained in step (1) by gradient centrifugation, and collect the supernatant;

[0037] (b) Dilute the supernatant obtained in step (a) with PB buffer until the turbidity is less than 900 NTU;

[0038] (c) Deep filter the solution diluted in step (b) using a tangential flow deep filtration membrane module, and collect the filtrate;

[0039] (d) Concentrate and purify the filtrate obtained in step (c) using a hollow fiber column to obtain the turmeric extracellular vesicles.

[0040] Preferably, the specific steps of the gradient centrifugation in step (a) include:

[0041] (a1) Centrifuge the turmeric juice obtained in step (1) at 400 - 800×g for 5 - 20 min, and collect the supernatant;

[0042] (a2) Centrifuge at 1000 - 3000×g for 10 - 30 min, and collect the supernatant;

[0043] (a3) Centrifuge at 3000 - 5000×g for 20 - 40 min, and collect the supernatant;

[0044] (a4) Centrifuge at 8000 - 12000×g for 40 - 70 min, and collect the supernatant.

[0045] Effects of the Invention

[0046] The method for extracting turmeric extracellular vesicles of the present invention uses dehydrated turmeric as the raw material, which is more convenient to store compared to fresh turmeric, does not require refrigeration, and the active ingredients are stable. Dehydrated turmeric usually undergoes standardized treatment, and the quality is more consistent, which is beneficial 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 lie in the diversity of their natural components, bioavailability, and simplicity of processing.

[0047] The turmeric extracellular vesicles prepared by the extraction method according to the present invention have anti - aging, repair, and antioxidant effects, and have good water solubility, good permeability, and are easily absorbed by the skin; they can (a) promote the proliferation of fibroblasts, (b) promote the collagen secretion of fibroblasts, (c) inhibit the level of MMP1 in fibroblasts, (d) promote the level of TMP1 in fibroblasts, (e) inhibit the level of ROS in fibroblasts, (f) inhibit the level of SA - β - Gal in fibroblasts, (g) promote the proliferation level of keratinocytes, (h) inhibit the level of ROS in keratinocytes, (i) promote the expression of keratinization - related proteins in keratinocytes, (j) promote the migration level of keratinocytes. Description of the Drawings

[0048] Figure 1 It is the detection result of the particle size of turmeric extracellular vesicles.

[0049] Figure 2 It is the characterization result of turmeric extracellular vesicles by transmission electron microscopy (TEM).

[0050] Figure 3 It is the detection result of the total flavonoid concentration in turmeric extracellular vesicles.

[0051] Figure 4 It is the detection result of the total sugar concentration in turmeric extracellular vesicles.

[0052] Figure 5 It is the fluorescence microscope observation and photographing result of turmeric extracellular vesicles infiltrating into ex vivo porcine skin.

[0053] Figure 6 Effect of turmeric extracellular vesicles on keratinocyte proliferation.

[0054] Figure 7 Effect of turmeric extracellular vesicles on keratinocyte migration.

[0055] Figure 8 Effect of turmeric extracellular vesicles on the expression level of keratin-related proteins.

[0056] Figure 9 Effect of turmeric extracellular vesicles on the level of reactive oxygen species (ROS) in keratinocytes.

[0057] Figure 10 Effect of turmeric extracellular vesicles on the secretion of type I collagen (MMP1) by dermal fibroblasts.

[0058] Figure 11 Effect of turmeric extracellular vesicles on the secretion of extracellular matrix proteases by dermal fibroblasts.

[0059] Figure 12 Effect of turmeric extracellular vesicles on the senescence of dermal fibroblasts.

[0060] Figure 13 Overall test results of the human effects of a facial cream containing turmeric extracellular vesicles.

[0061] Figure 14 Main excellent cases of the human effects of a facial cream containing turmeric extracellular vesicles. Detailed implementation manners

[0062] To make the technical solutions and beneficial effects of the present invention more obvious and understandable, the following provides a detailed description by way of listing specific embodiments. Among them, the drawings are not necessarily drawn to scale, and local features can be enlarged or reduced to more clearly show the details of local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical and scientific fields to which this application belongs.

[0063] As used herein, the term "extracellular vesicles" or "EVs" refers to nanoscale particles secreted by organisms, with diameters of about 20 nm - 5 μm, which mediate the interaction between the environment and organisms, and between organisms by carrying and transmitting functional molecules such as RNA, proteins, and metabolites, and have become signal communication carriers that have received wide attention.

[0064] As used herein, the term "exosomes" refers to small membrane vesicles containing complex RNA and proteins, which are one type of extracellular vesicles and have diameters of about 30 - 100 nm.

[0065] The present invention provides an application of turmeric extracellular vesicles in the field of cosmetics.

[0066] In some embodiments, the application includes:

[0067] (1) Application in the preparation of skin anti-wrinkle and anti-aging products;

[0068] (2) Application in the preparation of skin repair and antioxidant products;

[0069] (3) Application in the preparation of skin whitening and freckle-removing products.

[0070] In some embodiments, the extracellular vesicles are exosomes.

[0071] In some embodiments, the method for preparing turmeric extracellular vesicles includes:

[0072] (1) Soaking and breaking the wall of washed turmeric to obtain turmeric juice;

[0073] (2) Extracting turmeric extracellular vesicles using the turmeric juice obtained in step (1).

[0074] In some embodiments, the turmeric is fresh turmeric.

[0075] In some embodiments, the method for preparing turmeric extracellular vesicles further includes: washing and drying fresh turmeric.

[0076] In some embodiments, the method for preparing turmeric extracellular vesicles is: washing and drying fresh turmeric, soaking and breaking the wall to obtain turmeric juice, and extracting turmeric extracellular vesicles using the turmeric juice.

[0077] In some embodiments, the method for preparing turmeric extracellular vesicles is: washing processed turmeric, soaking and breaking the wall to obtain turmeric juice, and extracting turmeric extracellular vesicles using the turmeric juice.

[0078] In some embodiments, the drying temperature is 40 - 60 °C.

[0079] In some 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.

[0080] In some embodiments, the drying temperature is 50 °C.

[0081] In some embodiments, the drying time is 12 - 48 hours.

[0082] 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.

[0083] In some embodiments, the drying time is 24 hours.

[0084] In some embodiments, the solution for soaking in step (1) is PBS.

[0085] In some embodiments, the solution for soaking in step (1) is sterile PBS.

[0086] In some embodiments, the soaking time in step (1) is 6 - 24 hours.

[0087] In some 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.

[0088] In some embodiments, the soaking time in step (1) is 6 - 12 hours.

[0089] In some embodiments, the soaking time in step (1) is 12 hours.

[0090] In some embodiments, the cell wall breaking time in step (1) is 5 - 10 min.

[0091] In some 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.

[0092] In some 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.

[0093] In some embodiments, the extraction method in step (2) is sucrose density gradient centrifugation.

[0094] In some embodiments, the extraction method described in step (2) is hierarchical filtration.

[0095] In some embodiments, the extraction method described in step (2) is ultracentrifugation and / or tangential flow filtration.

[0096] In some embodiments, the centrifugation in the extraction method described in step (2) is ultracentrifugation.

[0097] In some embodiments, the specific steps of the ultracentrifugation described in step (2) include:

[0098] (I) Centrifuge the turmeric juice obtained in step (1) at 400 - 800×g for 5 - 20 min, and collect the supernatant;

[0099] (II) Then centrifuge at 1000 - 3000×g for 10 - 30 min, and collect the supernatant;

[0100] (III) Then centrifuge at 3000 - 5000×g for 20 - 40 min, and collect the supernatant;

[0101] (IV) Then centrifuge at 8000 - 12000×g for 40 - 70 min, and collect the supernatant;

[0102] (V) Then centrifuge at 100000 - 120000×g for 60 - 100 min, collect the precipitate, and resuspend it with PBS;

[0103] (VI) Then centrifuge at 100000 - 120000×g for 60 - 100 min, collect the precipitate, and resuspend it with PBS to obtain the turmeric extracellular vesicles.

[0104] In some embodiments, the centrifugal force of the centrifuge 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.

[0105] In some embodiments, the centrifugal force of the centrifuge in step (I) is 500×g.

[0106] In some 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.

[0107] In some embodiments, the centrifugation time in step (I) is 10 min.

[0108] In some embodiments, the centrifugal force 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.

[0109] In some embodiments, the centrifugal force in step (II) is 2000×g.

[0110] In some 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 21min, 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.

[0111] In some embodiments, the centrifugation time in step (II) is 20 min.

[0112] In some embodiments, the centrifugal force 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.

[0113] In some embodiments, the centrifugal force in step (III) is 4000×g.

[0114] 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.

[0115] In certain embodiments, the centrifugation time in step (III) is 30 min.

[0116] In certain embodiments, the centrifugal force 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 10000×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.

[0117] In certain embodiments, the centrifugal force in step (IV) is 10000×g.

[0118] 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.

[0119] In some embodiments, the centrifugation time in step (IV) is 60 min.

[0120] In some embodiments, the centrifugal force 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.

[0121] In some embodiments, the centrifugal force in step (V) is 110,200×g.

[0122] In some 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.

[0123] In some embodiments, the centrifugation time in step (V) is 70 min.

[0124] In some embodiments, the centrifugal force for the centrifugation in step (VI) is 100000×g, or 101000×g, or 102000×g, or 103000×g, or 104000×g, or 105000×g, or 106000×g, or 107000×g, or 108000×g, or 109000×g, or 110000×g, or 110200×g, or 111000×g, or 112000×g, or 113000×g, or 114000×g, or 115000×g, or 116000×g, or 117000×g, or 118000×g, or 119000×g, or 120000×g.

[0125] In some embodiments, the centrifugal force for the centrifugation in step (VI) is 110200×g.

[0126] In some embodiments, the centrifugation time for 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 71min, 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 88min, 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.

[0127] In some embodiments, the centrifugation time for step (VI) is 70 min.

[0128] In some embodiments, the specific steps of the ultracentrifugation are as follows:

[0129] (I) Centrifuge the turmeric juice obtained in step (1) at 500×g for 10 min, and collect the supernatant;

[0130] (II) Then centrifuge at 2000×g for 20 min, and collect the supernatant;

[0131] (III) Then centrifuge at 4000×g for 30 min, and collect the supernatant;

[0132] (IV) Then centrifuge at 10000×g for 60 min, and collect the supernatant;

[0133] (V) Centrifuge at 110200×g for 70 min, collect the precipitate, and resuspend it with PBS.

[0134] (VI) Centrifuge at 110200×g for 70 min, collect the precipitate, and resuspend it with PBS to obtain the turmeric extracellular vesicles.

[0135] In certain embodiments, the extraction method described in step (2) is fractional filtration.

[0136] In certain embodiments, the extraction method described in step (2) is tangential flow filtration.

[0137] In certain embodiments, the specific steps of the tangential flow filtration include:

[0138] (a) Centrifuge the turmeric juice obtained in step (1) at a gradient, and collect the supernatant.

[0139] (b) Dilute the supernatant obtained in step (a) with PB buffer until the turbidity is less than 900 NTU.

[0140] (c) Deep filter the solution diluted in step (b) using a tangential flow depth filtration membrane package, and collect the filtrate.

[0141] (d) Concentrate and purify the filtrate obtained in step (c) using a hollow fiber column to obtain the turmeric extracellular vesicles.

[0142] In certain embodiments, the specific steps of the gradient centrifugation described in step (a) include:

[0143] (a1) Centrifuge the turmeric juice obtained in step (1) at 400 - 800×g for 5 - 20 min, and collect the supernatant.

[0144] (a2) Then centrifuge at 1000 - 3000×g for 10 - 30 min, and collect the supernatant.

[0145] (a3) Then centrifuge at 3000 - 5000×g for 20 - 40 min, and collect the supernatant.

[0146] (a4) Then centrifuge at 8000 - 12000×g for 40 - 70 min, and collect the supernatant.

[0147] In certain embodiments, the centrifugal force 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.

[0148] In certain embodiments, the centrifugal force in step (a1) is 500×g.

[0149] In some 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.

[0150] In some embodiments, the centrifugation time in step (a1) is 10 min.

[0151] In some embodiments, the centrifugal force 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.

[0152] In some embodiments, the centrifugal force in step (a2) is 2000×g.

[0153] In some 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.

[0154] In some embodiments, the centrifugation time in step (a2) is 20 min.

[0155] In some embodiments, the centrifugal force 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.

[0156] In certain embodiments, the centrifugal force for the centrifugation in step (a3) is 4000×g.

[0157] 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.

[0158] In certain embodiments, the centrifugation time in step (a3) is 30 min.

[0159] In certain embodiments, the centrifugal force for 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 10000×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.

[0160] In certain embodiments, the centrifugal force for the centrifugation in step (a4) is 10000×g.

[0161] 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.

[0162] In certain embodiments, the centrifugation time in step (a4) is 60 min.

[0163] In certain embodiments, the specific steps of the gradient centrifugation in step (a) are as follows:

[0164] (a1) Centrifuge the turmeric juice obtained in step (1) at 500×g for 10 min, and collect the supernatant;

[0165] (a2) Then centrifuge at 2000×g for 20 min, and collect the supernatant;

[0166] (a3) Then centrifuge at 4000×g for 30 min, and collect the supernatant;

[0167] (a4) Then centrifuge at 10000×g for 60 min, and collect the supernatant.

[0168] The present invention also provides a turmeric extracellular vesicle prepared according to the above method.

[0169] In certain embodiments, the extracellular vesicle is an exosome.

[0170] The present invention also provides a composition, which comprises the turmeric extracellular vesicle.

[0171] Example 1: Extraction of turmeric extracellular vesicles by ultracentrifugation method

[0172] 1. Take fresh turmeric, wash it, and dry it at 50°C for 24 hours.

[0173] 2. Weigh 50 g of dry turmeric precisely, add 500 mL of sterile (filtered through a 0.22 μm filter membrane) PBS, and soak overnight.

[0174] 3. Use a blender to homogenize the turmeric into tissue fluid (5 - 10 min) without obvious lumpy fragments.

[0175] 4. Extraction by ultracentrifugation method:

[0176] (1) Transfer the dry turmeric homogenate to a 50 mL sterile centrifuge tube, centrifuge at 500×g for 10 min using a low-speed centrifuge at 4°C, discard the precipitate, and take the supernatant.

[0177] (2) Centrifuge at 2000×g for 20 min using a low-speed centrifuge at 4°C, discard the precipitate, and take the supernatant.

[0178] (3) Centrifuge at 4000×g for 30 min using a low-speed centrifuge at 4°C, discard the precipitate, and take the supernatant.

[0179] (4) Centrifuge at 10000×g for 1 h using a high-speed centrifuge at 4°C, discard the precipitate, and take the supernatant.

[0180] (5) Centrifuge at 110200×g for 70 min using an ultracentrifuge at 4°C, discard the supernatant, and resuspend the precipitate in PBS.

[0181] (6) Centrifuge at 110200×g for 70 min using an ultracentrifuge at 4°C, discard the supernatant, and resuspend the precipitate in PBS to obtain curcuma longa extracellular vesicles (CL-Exo).

[0182] Example 2: Extraction of curcuma longa extracellular vesicles by tangential flow filtration method

[0183] Steps 1-3 are the same as in Example 1

[0184] 4. Extraction by tangential flow filtration method:

[0185] (1) Pretreatment centrifugation: Transfer the dry turmeric homogenate to a centrifuge tube, and perform gradient centrifugation using a tabletop centrifuge at 4°C: 500g for 10 min, 2000g for 20 min, 4000g for 30 min, 10000g for 60 min; after centrifugation, collect the supernatant and discard the precipitate.

[0186] (2) Dilution of supernatant: Calibrate the turbidimeter using a standard with a turbidity of 800 NTU, take the supernatant obtained in step 3 for turbidity measurement, and if the turbidity is greater than 900 NTU, dilute it with PB buffer until the turbidity is less than 900 NTU.

[0187] (3) Depth filtration: Pass the supernatant diluted in step (2) through a tangential flow depth filtration membrane module for depth filtration, and collect the liquid after depth filtration.

[0188] (4) Concentration and purification: Concentrate and purify the depth filtrate obtained in step (3) through a hollow fiber column, and the suspension obtained after concentration is the curcuma longa extracellular vesicles.

[0189] Example 3: Characterization of curcuma longa extracellular vesicles

[0190] (1)Particle size measurement

[0191] Testing instrument: microfluidic resistive pulse sensing instrument.

[0192] The results are as Figure 1 shown, the particle size of CL-Exo is 162.2.

[0193] (2)Characterization by transmission electron microscopy (TEM)

[0194] The CL-Exo prepared in Example 1 was resuspended with pure water to obtain an EVs solution. Take the EVs solution (resuspended with pure water), dilute it with an appropriate amount of pure water to a protein concentration of 6 mg / mL, then drop it on a sample copper grid, allow it to dry naturally, stain with phosphotungstic acid, and observe the morphology by transmission electron microscopy.

[0195] The results are as Figure 2 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 are typically cup-shaped.

[0196] Example 4: Composition analysis of Curcuma longa extracellular vesicles

[0197] (1)Total flavonoid determination (using the Total Flavonoid kit from Suzhou Grees Biotechnology Co., Ltd.)

[0198] 1. Sample preparation: Weigh approximately 0.1 g of fresh sample (CL-Exo prepared in Example 1, if the moisture content is sufficient, the sample sampling mass can be increased); or weigh approximately 0.03 g of dried sample (the sample is blanched at 105 °C for 3 min, then dried at 60 °C to constant weight, pulverized, and sieved through a 40 - 60 mesh sieve to obtain the dried sample), add 1.5 mL of 60% ethanol, and extract by shaking at 60 °C for 2 h. Centrifuge at 25 °C × 12000 rpm for 10 min, take the supernatant, and make up the volume to 1.5 mL with 60% ethanol for testing. [Note]: If the sample amount is small, the sample amount can be reduced proportionally, such as taking 0.02 g of dry sample, adding 1 mL of 60% ethanol, extracting by shaking at 60 °C for 2 h. Centrifuge at 25 °C × 12000 rpm for 10 min, take the supernatant, and make up the volume to 1 mL with 60% ethanol for testing.

[0199] 2. Detection on the machine: ① Preheat the microplate reader for 30 min and adjust the wavelength to 510 nm. ② Two samples can be selected for preliminary determination first. If the measured value of A exceeds 1.5, the supernatant can be diluted with 60% ethanol to determine the dilution factor D suitable for this batch of samples, and the corresponding dilution factor 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) into the 96-well plate in sequence according to the requirements of the instruction manual, mix well, let stand at 25 °C for 15 min, and measure the absorbance at 510 nm.

[0200] 3. Result calculation: 1. Standard curve equation: y = 1.6277x - 0.0049, where x is the standard concentration (mg / mL) and y is ΔA.

[0201] Total flavonoid content (mg / g dry weight) = [(ΔA + 0.0049) ÷ 1.6277 × V1] ÷ (V1 ÷ V × W) × D

[0202] = 0.6 × (ΔA + 0.0049) ÷ W × V × D

[0203] Among them, V is the volume of the extraction solution, 1.5 mL; V1 is the volume of the sample in the reaction, 50 μL = 0.05 mL; W is the sample mass, g; D is the dilution factor, which is 1 if not diluted;

[0204] The result is as Figure 3 shown, and the concentration of total flavonoids in turmeric extracellular vesicles is 0.43 mg / mL.

[0205] (2)Determination of total sugar (Shanghai Yuanye Bio-Technology - Plant Total Sugar and Reducing Sugar Detection Kit (DNS Microplate Method))

[0206] 1. Hydrolysis and extraction of total sugar: 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 onto a glass slide, then add 1 drop of the chromogenic solution (about 50 μL) to check whether the hydrolysis is complete. If it is completely hydrolyzed, it will not show blue. ④ After hydrolysis, cool to room temperature, add 6M sodium hydroxide solution to adjust the solution pH to 7.4, make up the volume to 100 mL with distilled water, mix well, centrifuge at 4000 g for 5 min or filter. ⑤ Take 10 mL of the supernatant or filtrate, make up the volume to 100 mL with distilled water to form a 10-fold diluted total sugar hydrolysis solution (extraction solution), and take 50 μL of the total sugar hydrolysis solution to measure its reducing sugar content.

[0207] 2. Dilute glucose standard: Take a clean centrifuge tube or test tube and operate according to the kit instruction manual to obtain a series of Glu standards with different concentrations in sequence.

[0208] 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.

[0209] 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.

[0210] 5. Calculation: Percentage of total sugar: Total sugar content in 100g sample

[0211] (g) = (c × N × V T ) / (m×1000)×100×0.9=(c×N×VT) / (m×10)×0.9

[0212] 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.

[0213] The results are as follows Figure 4 As shown, the concentration of total sugars in turmeric extracellular vesicles was 12.07 mg / mL.

[0214] Example 5: Penetration of Curcuma Extracellular Vesicles into Ex vivo Pig Skin

[0215] 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.

[0216] The skin of 8-week-old Bama minipigs measuring 1 cm × 1 cm was set aside in an environment of 32 °C. 50 μL of CL-Exo labeled with 1 mg / ml PKH26 was dropped onto the skin of 8-week-old Bama minipigs measuring 1 cm × 1 cm, evenly covering the entire epidermis, and incubated for 2 h. After incubation, frozen sections were made with a thickness of 10 μm. After staining the cell nuclei with DAPI, observation and photography were carried out using a fluorescence microscope. The results are as Figure 5 shown, the curcumin-derived extracellular vesicles can penetrate into the skin of 8-week-old Bama minipigs.

[0217] Example 6: Effect of curcumin extracellular vesicles on keratinocyte proliferation

[0218] The CCK-8 method was used to determine the proliferative effect of curcumin 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 curcumin EVs) were set up. The curcumin EVs solution was taken and diluted with serum-free DMEM medium into a stock solution with a protein concentration of 1 mg / mL. Sample solutions of 5 μg / mL, 10 μg / mL, and 20 μg / mL were prepared and recorded as experimental groups 1 to 3 respectively, and set aside.

[0219] Except for the blank group, cells in good growth state were seeded on a 96-well plate at a density of 1×10 4 cells / well, and the cells were cultured for 24 h and set aside.

[0220] The experimental groups were respectively given 100 μL / well of the above concentration gradient sample solutions, and the blank group and the normal group were given the same dose of incomplete medium (i.e., serum-free medium). After incubation at 37 °C and 5% CO2 for 24 h, the medium was discarded.

[0221] According to the instructions of the CCK-8 kit, the proliferative activity of the cells was detected. Each group was measured in parallel 3 times, and the cell survival rate and cell proliferation rate were calculated according to the following formula.

[0222] Cell proliferation rate % = (OD 实验组 - OD 空白 ) / (OD 对照组 - OD 空白 ) × 100%;

[0223] The results are as Figure 6 shown, adding 10 μg / mL of curcumin extracellular vesicles can significantly promote keratinocyte proliferation, and the keratinocyte proliferation ability is up-regulated by 20.7%.

[0224] Example 7: Effect of curcumin extracellular vesicles on keratinocyte migration

[0225] 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 into a stock solution with a protein concentration of 1 mg / mL. Prepare a sample solution of 20 μg / mL, denoted as the experimental group, and set aside.

[0226] Take keratinocytes in good growth state and inoculate them on a 24-well plate at a density of 2×10 5 cells / well, and culture the cells for 24 h for standby.

[0227] Scratch each well once with a 300 μL pipette tip (the pipette tip should be vertical and not tilted) to form an artificial wound, wash the cells 3 times with PBS to remove the scratched cells. Use a microscope to take pictures of each position of the scratch and record the scratch area at 0 h.

[0228] The positive control group and the experimental group were respectively given 500 μL / well of the above-mentioned concentration gradient sample solution, and the control group was given the same dose of incomplete medium (i.e., serum-free medium), and incubated at 37 °C and 5% CO2 for 24 h.

[0229] Use a microscope to take pictures of each position of the scratch and record the scratch area at 24 h, and use imageJ to statistically analyze the scratch area of keratinocytes at 0 h and 24 h respectively.

[0230] Cell migration rate % = (S 0h -S 24h ) / S 0h ×100%, where S 0h : Scratch area at 0 h; S 24h : Scratch area at 24 h.

[0231] The results are as Figure 7 shown. Adding 20 μg / mL of turmeric extracellular vesicles can significantly promote keratinocyte migration, and the cell migration level is up-regulated by 19.7%.

[0232] Example 8: Effect of turmeric extracellular vesicles on the expression level of keratin-related proteins

[0233] A blank group and a drug administration group were set up. Take the CL-Exo solution prepared in Example 1, and dilute it with serum-free DMEM medium into a stock solution with a protein concentration of 1 mg / mL. Dilute the stock solution to 20 μg / mL to obtain a sample solution, which is respectively denoted as the drug administration group and set aside.

[0234] Take keratinocytes in good growth state and inoculate them on a 12-well plate at a density of 2×10 5 cells / well, and incubate the cells for 24 h for standby.

[0235] The dosing groups were respectively 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 medium (i.e., DMEM medium without serum), and incubated at 37°C and 5% CO2 for 24 h.

[0236] Extract RNA using Novoprotein RNA extraction reagent R411 (VeZol Reagent R411) according to the instruction manual.

[0237] Reverse transcribe the RNA obtained in the previous step according to the instruction manual of Takara PrimeScript™ RT reagent Kit (PerfectReal Time).

[0238] Perform QPCR experiment on the cDNA obtained by reverse transcription in the previous step according to the instruction manual of Bimake 2x SYBRGreen qPCR Master Mix (High ROX).

[0239] Use the 2 -ΔΔCt -method to calculate the expression level of the corresponding gene:

[0240] ΔΔCt =(Ct(target gene)- Ct(reference gene))sample A-(Ct(target gene)- Ct(reference gene))sample B

[0241] Fold Change = 2 -ΔΔCt

[0242] Among them, Fold Change represents the multiple of the expression level of the target gene relative to the expression level of the reference gene.

[0243] The results are as Figure 8 shown. Adding 20 μg / mL of turmeric extracellular vesicles can significantly promote the expression of filaggrin (FLG) and involucrin (IVL) in keratinocytes, upregulating them by 48.3% and 92.2% respectively.

[0244] Example 9: Effect of turmeric extracellular vesicles on the level of reactive oxygen species (ROS) in keratinocytes

[0245] The DCFH-DA fluorescent probe was used to determine the effect of turmeric extracellular vesicles on the reactive oxygen species level 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 cell inoculation), 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. Take the turmeric extracellular vesicles (CL-Exo prepared in Example 1) solution, and dilute it with serum-free DMEM medium into a stock solution with a protein concentration of 1 mg / mL. Sample solutions with concentrations of 0.5 μg / mL, 5 μg / mL, and 20 μg / mL were prepared respectively, and were denoted as experimental groups 1 to 3 for standby.

[0246] Take cells in good growth state and inoculate them on a 96-well plate at a density of 1×10 4 cells / well, and culture the cells for 24 h for standby.

[0247] Aspirate and discard the cell culture supernatant, add 50 μL of DPBS to each well, place it at a distance of 15 cm below the ultraviolet lamp tube, and irradiate for 16 min.

[0248] Discard the DPBS, and the positive control group and the drug administration group were respectively given 100 μL / well of the above sample solutions with concentration gradients, and the control group was given the same dose of incomplete medium (i.e., serum-free DMEM medium). After incubating for 6 h at 37°C and 5% CO2, discard the medium.

[0249] The DCFH-DA working solution was prepared according to a dilution ratio of 1:1000, and DCFH-DA was diluted with DMEM basal medium to a final concentration of 10 μmol / L.

[0250] Discard the medium in the remaining wells, wash 3 times with PBS, then add 100 μL of DCFH-DA working solution to each well, and incubate in a CO2 incubator for 30 min.

[0251] After the incubation, wash the cells in each well 3 times with the basal medium, place them on the detection platform of a fluorescence microplate reader, set the incident light wavelength to 529 nm and the excitation light wavelength to 504 nm, and read the values.

[0252] ROS level (%) = (T - C0) / (C - C0)×100%, where T—the average value of the fluorescence intensity of the test sample in 3 times; C—the average value of the fluorescence intensity of the control group in 3 times; C0—the average value of the fluorescence intensity of the naked cell group in 3 times.

[0253] The results are as Figure 9As shown, adding 20 μg / mL of turmeric extracellular vesicles can significantly inhibit the ROS level in keratinocytes, and the inhibition level reaches 20.7%.

[0254] Example 10: Effect of turmeric extracellular vesicle-like particles on the secretion of type I collagen by dermal fibroblasts

[0255] 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 supernatants were discarded, and 100 μL of negative control reagent (NC: T4 basal medium), positive control group reagent (PC: 100 ng / mL of TGF-β), and experimental group reagent (20 μg / mL, 50 μg / mL of CL-Exo prepared in Example 1) were added respectively, and cultured for 24 h. The supernatants were taken, and the secretion of type I collagen was detected by the Lianke type I collagen detection kit.

[0256] The results are as Figure 10 shown, CL-Exo can significantly promote the secretion of type I collagen by dermal fibroblasts.

[0257] Example 11: Effect of turmeric extracellular vesicle-like particles on the secretion of extracellular matrix proteases by dermal fibroblasts

[0258] 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 matrix metalloenzyme changes caused by photoaging were simulated by irradiating the cells with UVA. The cell supernatants were discarded, and 50 μL of pre-cooled PBS was added to each well. An ice pack was placed in the biosafety cabinet, and four layers of kraft paper were laid on the ice pack. Then the cell culture plate was placed on the kraft paper, and the position of the UVA lamp tube was adjusted so that the distance from the lamp tube to the cells was 5 cm. The cell culture plate was removed, and after the lamp tube was turned on and balanced for 5 min, the cell culture plate was placed under the lamp tube again, the lid was opened, and irradiated for 1 h, with a total energy of 5 J / cm 2 2. The PBS was discarded, and negative control reagent (NC: T4 basal medium), positive control group reagent (PC: 100 ng / mL of TGF-β), and experimental group reagent (20 μg / mL, 50 μg / mL of CL-Exo prepared in Example 1) were added. The group without UVA treatment was added with T4 basal medium (BC), and cultured for 24 h. The supernatants were taken, and the protein secretion of matrix metalloproteinase 1 (MMP1) and tissue inhibitor of metalloproteinase 1 (TIMP1) was detected by the Lianke human MMP1 ELISA kit and human TIMP1 ELISA kit respectively.

[0259] The results are as Figure 11As shown, CL-Exo can not only promote the secretion of TIMP1 by dermal fibroblasts induced by UVA, but also inhibit the secretion of MMP1 by dermal fibroblasts induced by UVA.

[0260] Example 12: Effect of turmeric extracellular vesicles on the senescence of dermal fibroblasts

[0261] Dermal fibroblasts were seeded into 24-well plates at a density of 20,000 cells / 100 μL / well. The cells were cultured for 24 h. The cells were senesced by UVA irradiation. The cell supernatant was discarded, and 100 μL of pre-cooled PBS was added to each well. An ice pack was placed in the biosafety cabinet, and four layers of kraft paper were laid on the ice pack. Then the cell culture plate was placed on the kraft paper. The position of the UVA lamp tube was adjusted so that the distance from the lamp tube to the cells was 5 cm. The cell culture plate was removed. After the lamp tube was turned on and balanced for 5 min, the cell culture plate was placed under the lamp tube again, the lid was opened, and the cells were irradiated for 1 h, with a total energy of 5 J / cm². The PBS was discarded, and blank control reagent (BC: basal medium), negative control reagent (NC: basal medium), positive control group reagent (PC: 100 ng / mL TGF-β), and experimental group reagent (20 μg / mL CL-Exo prepared in Example 1) were added, and the cells were cultured for 24 h. The NC, PC, and experimental group cells were irradiated with UVA at an energy of 5 J / cm² again. After the drug incubation was performed again for 24 h, the senescent cell staining was performed using the β-galactosidase staining kit from Shanghai Shangbao Biotechnology Co., Ltd. 2 After the NC, PC, and experimental group cells were irradiated with UVA at an energy of 5 J / cm² again and the drug incubation was performed again for 24 h, the senescent cell staining was performed using the β-galactosidase staining kit from Shanghai Shangbao Biotechnology Co., Ltd.

[0262] The results are as Figure 12 shown, CL-Exo can significantly reduce the senescence of dermal fibroblasts induced by UVA.

[0263] Example 13: Human efficacy test of the cream containing turmeric extracellular vesicles

[0264] CL-Exo prepared in Example 1 was added to the matrix cream at addition amounts of 5%, 10%, and 20% (as shown in Formulation Table 1). At the same time, a matrix cream without extracellular vesicle-like particles derived from turmeric was used as a control. It was applied once in the morning and once in the evening, and no other skin care products were used during the use period. Thirty experiencers were selected and divided into a matrix group and an experimental group. Among them, the matrix group used the matrix cream, and the experimental group used the matrix cream containing the turmeric extracellular vesicles of the present invention. The skin conditions were detected by Visia photography at 0 days and 21 days of use.

[0265] The results are as Figure 13 , 14 shown. After using the cream containing extracellular vesicles derived from turmeric for 28 days, the SEw (skin wrinkles), wrinkle length, wrinkle depth, and the proportion of wrinkle area of the experiencers' skin were all reduced, and the periorbital wrinkles were significantly reduced in the photos detected by the skin rapid three-dimensional imaging system and Visia photography.

[0266] Table 1: Ingredients List of Matrix Cream

[0267]

[0268] It should be understood that the above embodiments are all exemplary and are not used to cover all possible implementation manners included in the claims. Without departing from the scope of the present disclosure, various deformations and changes can also be made on the basis of the above embodiments. Similarly, various technical features of the above embodiments can also be arbitrarily combined to form additional embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only represent several implementation manners of the present invention and do not limit the protection scope of the present invention patent.

Claims

1. Use of turmeric extracellular vesicles in the preparation of cosmetics, characterized in that, The applications include: (1) Application in the preparation of skin anti-aging products; (2) Application in the preparation of skin repair and antioxidant products.

2. The application according to claim 1, wherein The preparation method of the turmeric extracellular vesicles includes: (1) Soaking and breaking the wall of the washed turmeric to obtain turmeric juice; (2) Extracting turmeric extracellular vesicles using the turmeric juice obtained in step (1).

3. The application according to claim 2, wherein The solution for soaking in step (1) is PBS; and / or, the soaking time in step (1) is 6 - 24 hours.

4. The application according to claim 3, characterized in that, The soaking time in step (1) is 6 - 12 hours; and / or, the time for breaking the wall in step (1) is 5 - 10 min.

5. The application according to claim 2, wherein The extraction method in step (2) is selected from one or more of sucrose density gradient centrifugation, ultracentrifugation, fractional filtration, and tangential flow filtration.

6. The application according to claim 5, 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) Then centrifuging at 1000 - 3000×g for 10 - 30 min, and collecting the supernatant; (III) Then centrifuging at 3000 - 5000×g for 20 - 40 min, and collecting the supernatant; (IV) Then centrifuging at 8000 - 12000×g for 40 - 70 min, and collecting the supernatant; (V) Then centrifuging at 100000 - 120000×g for 60 - 100 min, collecting the precipitate, and resuspending it with PBS; (VI) Then centrifuging at 100000 - 120000×g for 60 - 100 min, collecting the precipitate, and resuspending it with PBS to obtain the turmeric extracellular vesicles.

7. The application according to claim 6, characterized in that, (VI) Then centrifuging at 100000 - 120000×g for 60 - 100 min, collecting the precipitate, and resuspending it with PBS to obtain the turmeric extracellular vesicles. (I) Centrifuging the turmeric juice obtained in step (1) at 500×g for 10 min, and collecting the supernatant; (II) Then centrifuging at 2000×g for 20 min, and collecting the supernatant; (III) Then centrifuging at 4000×g for 30 min, and collecting the supernatant; (IV) Then centrifuging at 10000×g for 60 min, and collecting the supernatant; (V) Then centrifuging at 110200×g for 70 min, collecting the precipitate, and resuspending it with PBS; (VI) Then centrifuging at 110200×g for 70 min, collecting the precipitate, and resuspending it with PBS to obtain the turmeric extracellular vesicles.

8. The application according to claim 5, characterized in that, (VI) Then centrifuging at 110200×g for 70 min, collecting the precipitate, and resuspending it with PBS to obtain the turmeric extracellular vesicles. (a) Gradient centrifuging the turmeric juice obtained in step (1), and collecting the supernatant; (b) Diluting the supernatant obtained in step (a) with PB buffer until the turbidity is less than 900 NTU; (c) Deep filtering the solution diluted in step (b) using a tangential flow depth filtration membrane module, and collecting the filtrate; (d) Concentrating and purifying the filtrate obtained in step (c) using a hollow fiber column to obtain the turmeric extracellular vesicles.

9. The application according to claim 8, wherein (a) 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) Centrifuging at 1000 - 3000×g for 10 - 30 min, and collecting the supernatant; (a3) Centrifuging at 3000 - 5000×g for 20 - 40 min, and collecting the supernatant; (a4) Centrifuging at 8000 - 12000×g for 40 - 70 min, and collecting the supernatant.

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