A milk thistle-derived plant extracellular vesicle, a preparation method thereof, and application thereof in soothing and oil-controlling products

Abstract

The present invention relates to the field of cosmetic technology, and specifically to a milk thistle-derived plant extracellular vesicle, a preparation method thereof, and an application thereof in soothing and oil-controlling products. The preparation method of the milk thistle-derived plant extracellular vesicle comprises the following steps: (1) crushing and juicing milk thistle fruit and filtering to obtain filtrate A; (2) subjecting filtrate A to differential centrifugation to obtain supernatant B; (3) filtering supernatant B to obtain filtrate C; (4) adding 5%-20% polyethylene glycol solution to filtrate C and centrifuging for the first time, collecting the precipitate, resuspending the precipitate and filtering it, further purifying it by molecular exclusion chromatography, centrifuging it for the second time, and collecting the liquid. The present invention has the advantages of significant soothing, redness-removing and oil-controlling effects, good anti-inflammatory, repairing and antioxidant effects, simple preparation process, easy large-scale production, and stable properties.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and in particular to a silymarin-derived plant extracellular vesicle, a preparation method thereof, and application thereof in soothing and oil-controlling products. Background Art

[0002] Skin is the largest organ in the human body, with protective, perceptual and regulatory functions. It is composed of the epidermis, dermis and subcutaneous tissue, and undertakes important functions such as protecting the body from external environmental damage, maintaining water balance, regulating body temperature and perceiving external stimuli. As people pay more and more attention to beauty and health, skin care has become an indispensable part of people's daily lives. However, more and more consumers' skin barriers are damaged due to problems such as severe oil secretion caused by the environment and stress, resulting in a series of problems such as skin sensitivity, redness and itching, and enlarged pores.

[0003] In order to solve the problems of damaged skin, such as sunburn, allergies, etc., soothing and repairing ingredients such as Centella asiatica and Purslane are widely used in skin care products to help relieve skin discomfort caused by sunburn, allergies, etc. and promote the repair of damaged skin. However, these plant extracts cannot solve a series of skin problems caused by severe oily skin. Therefore, the development of a cosmetic raw material that can control oil, repair and soothe has gradually become a research hotspot.

[0004] Plant extracellular vesicles are vesicle-like nanoparticles secreted by plant cells, with diameters generally ranging from 30 to 140 nanometers. These vesicles are mainly wrapped by biological membranes and contain rich biologically active components such as nucleic acids, proteins, lipids, enzymes and hormones. Plant extracellular vesicles play an important role in plant growth and development. They not only participate in signal transmission and material exchange between plant cells, but also play an important role in plant physiological processes such as disease resistance and resistance to adversity.

[0005] Plant extracellular vesicles have certain therapeutic value in improving skin, including accelerating wound healing, reducing skin pigmentation, reducing wrinkles and preventing scar formation. Existing studies have shown that extracellular vesicles have a wide range of applications in facial aesthetics and have many potentials in scar removal, facial rejuvenation, anti-aging and anti-pigmentation.

[0006] Existing methods for extracting plant extracellular vesicles include ultracentrifugation, density gradient ultracentrifugation, antibody affinity magnetic beads, microfluidic separation, etc. However, all of the above methods have problems such as high equipment requirements and difficulty in large-scale production. Therefore, the development of an extraction and purification method that is easy to mass produce and has stable extraction quality is of great significance for the implementation of plant extracellular vesicles in the cosmetics field.

[0007] Milk thistle (Silybum marianum.), a herbaceous plant belonging to the genus Silybum of the Asteraceae family, has the effects of clearing away heat, detoxifying, protecting the liver and promoting bile secretion. The extract of milk thistle can capture oxygen free radicals and has strong antioxidant properties; it has a good inhibitory effect on elastase and can also tighten skin pores, so it is also used in soothing and oil-controlling cosmetics.

[0008] For example, Chinese patent publication number CN101966130A discloses a silybin nanocrystalline cosmetic, which is composed of surfactant, silybin, oil, essence, preservative, and the balance is distilled water. According to the HLB emulsification value, the oil phase and emulsifier of appropriate proportion and type are selected to prepare water / oil, oil / water type silybin anti-wrinkle and skin-rejuvenating cosmetics for different purposes. The cosmetic not only promotes absorption, has a fine and smooth texture, has strong wrinkle removal and skin rejuvenation, and has good appearance, but also has the effects of long-lasting moisturizing, resisting UV damage, and increasing skin elasticity.

[0009] In addition to the antioxidant and other regulatory effects of milk thistle extract on the human body, milk thistle extracellular vesicles have a variety of similar regulatory effects on the human body. Currently, there are no related reports on milk thistle extracellular vesicles in the cosmetics field, showing great potential in the cosmetics field.

[0010] Therefore, it is very necessary to develop a milk thistle-derived plant extracellular vesicle that can solve the above-mentioned technical problems, a preparation method thereof, and an application thereof in soothing and oil-controlling products. Summary of the invention

[0011] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a milk thistle-derived plant extracellular vesicle with significant soothing, redness-removing and oil-control effects, good anti-inflammatory, repairing and antioxidant effects, simple preparation process, easy large-scale production, stable properties, and a preparation method thereof, as well as an application thereof in soothing and oil-control products.

[0012] The present invention is achieved through the following technical solutions:

[0013] In one aspect, a method for preparing milk thistle-derived plant extracellular vesicles comprises the following steps:

[0014] (1) crushing milk thistle fruit, squeezing juice and filtering to obtain filtrate A;

[0015] (2) Filtrate A is subjected to differential centrifugation to obtain supernatant B;

[0016] (3) Filter the supernatant B to obtain filtrate C;

[0017] (4) After adding polyethylene glycol (PEG) solution to filtrate C, centrifuge for the first time to collect the precipitate, resuspend the precipitate and filter it, and then use size exclusion chromatography (SEC) to further purify it. Then centrifuge for the second time and collect the liquid.

[0018] In some embodiments of the present invention, the milk thistle fruit is pre-treated before being crushed in step (1), and the specific steps of the pre-treatment are as follows: cooling in a low temperature environment, then separating the tissues, and washing with water.

[0019] In some embodiments of the present invention, the pretreatment steps are as follows: cooling the milk thistle fruit in an environment of -4°C to 8°C for 30-60 minutes, then separating the tissues at 0-4°C, and washing with water. The washing times are two or more.

[0020] The tissue separation includes cutting and separating the complete tissue by mechanical methods, including but not limited to separating the fruit shell from the fruit pulp.

[0021] The water includes but is not limited to at least one of double distilled water and ultrapure water.

[0022] In some embodiments of the present invention, the pulverization in step (1) includes grinding at low temperature to obtain a ground product, and if necessary, adding an appropriate amount of pre-cooled 1× PBS. The grinding temperature is 0-4°C.

[0023] In some embodiments of the present invention, the filtration in step (1) is preliminary filtration using a filter.

[0024] Preferably, step (1) comprises the following steps:

[0025] The milk thistle fruit was cooled in an environment of -4℃~8℃ for 30-60min, then the tissue was separated at 0-4℃, and washed with water. Then the fruit was crushed and juiced, and then filtered with a filter to obtain filtrate A.

[0026] In some embodiments of the present invention, the specific steps of the differential centrifugation in step (2) are as follows: centrifugation at -4°C~25°C at a centrifugal force of 500-1000g, 1000-3000g, 5000-8000g, and 10000-12000g for 30-60min each.

[0027] Preferably, step (2) comprises the following steps:

[0028] The filtrate A was subjected to differential centrifugation at -4°C to 25°C at a centrifugal force of 500-1000g, 1000-3000g, 5000-8000g, and 10000-12000g for 30-60 min each to obtain supernatant B.

[0029] In some embodiments of the present invention, the molecular weight of the polyethylene glycol in step (4) is 2000-10000 Da.

[0030] In some embodiments of the present invention, the mass concentration of the polyethylene glycol solution in step (4) is 5%-20%.

[0031] In some embodiments of the present invention, the volume ratio of polyethylene glycol to filtrate C in step (4) is 1:1-1:5.

[0032] In some embodiments of the present invention, in step (4), pre-cooled 1×PBS is used to resuspend the precipitate. The pre-cooling temperature is 4°C.

[0033] In some embodiments of the present invention, the resuspended precipitate in step (4) is filtered using a 0.45 μm or 0.22 μm filter membrane;

[0034] In step (4), the mixture is incubated for 30-60 min by size exclusion chromatography and then centrifuged a second time;

[0035] First centrifugation parameters: centrifugal force 3000-8000g, 4℃ centrifugation for 30-120min;

[0036] Second centrifugation parameters: centrifugal force 500-1500g, centrifugation 10-30min.

[0037] Preferably, step (4) comprises the following steps:

[0038] An equal volume of PEG solution (PEG molecular weight 2000-10000Da, PEG solution mass concentration 5%-20%) is added to the filtrate C, and then the mixture is centrifuged at 3000-8000g and 4°C for 30-120min. The precipitate is collected and resuspended in 1×PBS precooled at 4°C. The precipitate is filtered through a 0.45μm or 0.22μm filter membrane, and then incubated by size exclusion chromatography (SEC) for 30-60min. The mixture is then centrifuged at 500-1500g for 10-30min, and the liquid is collected.

[0039] In a second aspect, the present invention also relates to the milk thistle-derived plant extracellular vesicles prepared by the above-mentioned preparation method.

[0040] In a third aspect, the present invention also relates to the use of the above-mentioned milk thistle-derived plant extracellular vesicles in the preparation of soothing and / or oil-control products.

[0041] In some embodiments of the present invention, the product is a cosmetic or a medicine.

[0042] In some embodiments of the present invention, the product is a cosmetic, and the dosage forms include emulsions, creams, lotions, and gels.

[0043] The aqueous solution includes an essence.

[0044] In some embodiments of the present invention, the product further comprises excipients, which include any one or a combination of at least two of a diluent, a dispersant, a binder, a filler, a thickener, a lubricant, a pH adjuster, a masking agent, a colorant, an antioxidant or an antibacterial agent.

[0045] Further preferably, the product contains, by mass percentage, 1%-5% glycerol, 1%-3% butylene glycol, 0.3%-0.5% p-hydroxyacetophenone, 0.4%-0.6% hexylene glycol, 0.01%-0.5% sodium acrylate copolymer, 2.5%-3.5% squalane, 0.5%-1% xanthan gum, 1%-2% diethoxyethyl succinate, 0.3%-0.8% jojoba seed oil, 0.1%-0.3% glyceryl stearate, 0.05%-0.15% PEG-100 stearate, 0.04%-0.06% EDTA-2Na, 0.01%-0.5% milk thistle-derived extracellular vesicles, and the balance water.

[0046] The beneficial effects of the present invention are:

[0047] The silybum-derived plant extracellular vesicles of the present invention are prepared by a differential centrifugation-PEG-SEC combination method, the preparation process is simple, and large-scale production is easy. The prepared silybum-derived plant extracellular vesicles have high purity, significant soothing, redness-removing and oil-controlling effects, good anti-inflammatory, repairing and antioxidant effects, and stable properties.

[0048] The present invention optimizes the concentration of the PEG solution and the parameters of the differential centrifugation, so that the prepared silybum-derived plant extracellular vesicles have the advantages of high purity, high yield, small particle size, and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is a particle size distribution diagram of the milk thistle-derived extracellular vesicles prepared in Example 1 of the present invention;

[0050] Figure 2 This is a potential characterization diagram of the silybum-derived extracellular vesicles prepared in Example 1 of the present invention;

[0051] Figure 3A This is the morphology of macrophage RAW 264.7 cells;

[0052] Figure 3B This is a cell morphology diagram of human epidermal cells HaCat;

[0053] Figure 4AThis is a statistical chart showing the results of detecting the toxic effects of extracellular vesicles derived from silymarin on HaCat cells using the MTT assay;

[0054] Figure 4B This is a statistical diagram of the results of detecting the toxic effects of extracellular vesicles derived from milk thistle on RAW 264.7 cells using the MTT assay;

[0055] Figure 5A The figure shows the results of the migration scratch experiment of HaCat cells using silymarin-derived extracellular vesicles;

[0056] Figure 5B This is a statistical diagram of the migration scratch experiment of HaCat cells using silymarin-derived extracellular vesicles;

[0057] Figure 6 This is an experimental diagram showing the effect of extracellular vesicles derived from silymarin on the IL-1α content after cell stimulation;

[0058] Figure 7 This is an experimental diagram showing the effect of extracellular vesicles derived from silymarin on the TNF-α content after cell stimulation;

[0059] Figure 8 This is an experimental diagram showing the effect of extracellular vesicles derived from silymarin on the IL-6 content after cell stimulation;

[0060] Fig. 9 The silymarin-derived extracellular vesicles are 2 O 2 Figure 2. Effects of induced cell damage on the experimental results.

[0061] Fig.10 This is an experimental diagram showing the effect of silymarin-derived extracellular vesicles on linoleic acid-induced lipid synthesis in SZ95 cells. DETAILED DESCRIPTION

[0062] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0063] The SEC columns used in the embodiments and comparative examples of the present invention were purchased from Beijing Enze Kangtai Biotechnology Co., Ltd.

[0064] Example 1

[0065] Extraction and purification of extracellular vesicles from milk thistle:

[0066] (1) The milk thistle fruit was cooled at -4°C for 30 min, then the tissue was separated and cut into segments at 0°C, washed with ultrapure water, and soaked for 1 h.

[0067] (2) Drain the water, crush and squeeze the juice at 0℃, and add pre-cooled PBS if necessary. Use 100 mesh, 200 mesh, and 400 mesh filters to perform preliminary filtration to obtain filtrate A;

[0068] (3) Filtrate A was subjected to differential centrifugation at -4°C at 500g, 1000g, 5000g, and 10000g for 30 min each to obtain supernatant B;

[0069] (4) Filter the supernatant B using 0.8 μm, 0.45 μm, and 0.22 μm filter membranes in sequence to obtain filtrate C;

[0070] (5) Add 5 times the volume of PEG solution (molecular weight 2000Da, mass concentration 5%) to filtrate C, mix thoroughly, and let stand overnight at low temperature. Centrifuge at 3000g and 4℃ for 90min, collect the precipitate, resuspend the precipitate in 1×PBS precooled at 4℃, filter with 0.45μm and 0.22μm filter membranes in turn, load into the pre-washed SEC column, incubate at room temperature for 30min, centrifuge at 1500g for 20min, and collect the liquid.

[0071] The maximum processing capacity of the above operation can reach 3kg.

[0072] Example 2

[0073] Extraction and purification of extracellular vesicles from milk thistle:

[0074] (1) The milk thistle fruit was cooled at 4°C for 45 min, then the tissue was separated and cut into sections at 4°C, washed with ultrapure water, and soaked for 1 h.

[0075] (2) Drain the water and crush and squeeze the juice at 4°C. If necessary, add an appropriate amount of pre-cooled PBS. Use 100 mesh, 200 mesh, and 400 mesh filters for preliminary filtration to obtain filtrate A;

[0076] (3) Filtrate A was subjected to differential centrifugation at 800g, 2000g, 6000g, and 11000g at 4°C for 40 min each to obtain supernatant B;

[0077] (4) Filter the supernatant B using 0.8 μm, 0.45 μm, and 0.22 μm filter membranes in sequence to obtain filtrate C;

[0078] (5) Add an equal volume of PEG solution (molecular weight 6000Da, mass concentration 10%) to filtrate C and mix thoroughly, then let stand overnight at low temperature. Centrifuge at 6000g and 4℃ for 30min, collect the precipitate, resuspend the precipitate in 1×PBS precooled at 4℃, filter with 0.45μm and 0.22μm filter membranes in turn, load into a pre-washed SEC column, incubate at room temperature for 60min, centrifuge at 1000g for 30min, and collect the liquid.

[0079] The maximum processing capacity of the above operation can reach 2.8kg.

[0080] Example 3

[0081] Extraction and purification of extracellular vesicles from milk thistle:

[0082] (1) The milk thistle fruit was cooled at 8°C for 60 min, then the tissue was separated and cut into sections at 4°C, washed with ultrapure water, and soaked for 1 h.

[0083] (2) Drain the water and crush and squeeze the juice at 4°C. If necessary, add an appropriate amount of pre-cooled PBS. Use 100 mesh, 200 mesh, and 400 mesh filters for preliminary filtration to obtain filtrate A;

[0084] (3) Filtrate A was subjected to differential centrifugation at 1000g, 3000g, 8000g, and 12000g at 25°C for 60 min each to obtain supernatant B;

[0085] (4) Filter the supernatant B using 0.8 μm, 0.45 μm, and 0.22 μm filter membranes in sequence to obtain filtrate C;

[0086] (5) Add an equal volume of PEG solution (molecular weight 10000Da, mass concentration 20%) to filtrate C and mix thoroughly, then let stand overnight at low temperature. Centrifuge at 8000g and 4℃ for 120min, collect the precipitate, resuspend the precipitate in 1×PBS precooled at 4℃, filter with 0.45μm and 0.22μm filter membranes in turn, load into a pre-washed SEC column, incubate at room temperature for 60min, centrifuge at 500g for 10min, and collect the liquid.

[0087] The maximum processing capacity of the above operation can reach 2.5kg.

[0088] Comparative Example 1

[0089] The only difference from Example 1 is that the mass concentration of the PEG solution in step (5) is different, which is 30%, and the other conditions are the same, as follows:

[0090] Extraction and purification of extracellular vesicles from milk thistle:

[0091] (1) The milk thistle fruit was cooled at -4°C for 30 min, then the tissue was separated and cut into segments at 0°C, washed with ultrapure water, and soaked for 1 h.

[0092] (2) Drain the water, crush and squeeze the juice at 0℃, and add pre-cooled PBS if necessary. Use 100 mesh, 200 mesh, and 400 mesh filters to perform preliminary filtration to obtain filtrate A;

[0093] (3) Filtrate A was subjected to differential centrifugation at -4°C at 500g, 1000g, 5000g, and 10000g for 30 min each to obtain supernatant B;

[0094] (4) Filter the supernatant B using 0.8 μm, 0.45 μm, and 0.22 μm filter membranes in sequence to obtain filtrate C;

[0095] (5) Add an equal volume of PEG solution (molecular weight 2000Da, mass concentration 30%) to filtrate C and mix thoroughly, then let stand overnight at low temperature. Centrifuge at 8000g and 4℃ for 120min, collect the precipitate, resuspend the precipitate in 1×PBS precooled at 4℃, filter with 0.45μm and 0.22μm filter membranes in turn, load into a pre-washed SEC column, incubate at room temperature for 60min, centrifuge at 500g for 10min, and collect the liquid.

[0096] Comparative Example 2

[0097] The only difference from Example 1 is that the mass concentration of the PEG solution in step (5) is different, which is 3%, and the other conditions are the same, as follows:

[0098] Extraction and purification of extracellular vesicles from milk thistle:

[0099] (1) The milk thistle fruit was cooled at -4°C for 30 min, then the tissue was separated and cut into segments at 0°C, washed with ultrapure water, and soaked for 1 h.

[0100] (2) Drain the water, crush and squeeze the juice at 0℃, and add pre-cooled PBS if necessary. Use 100 mesh, 200 mesh, and 400 mesh filters to perform preliminary filtration to obtain filtrate A;

[0101] (3) Filtrate A was subjected to differential centrifugation at -4°C at 500g, 1000g, 5000g, and 10000g for 30 min each to obtain supernatant B;

[0102] (4) Filter the supernatant B using 0.8 μm, 0.45 μm, and 0.22 μm filter membranes in sequence to obtain filtrate C;

[0103] (5) Add an equal volume of PEG solution (molecular weight 2000Da, mass concentration 3%) to filtrate C and mix thoroughly, then let stand overnight at low temperature. Centrifuge at 8000g and 4℃ for 120min, collect the precipitate, resuspend the precipitate in 1×PBS precooled at 4℃, filter with 0.45μm and 0.22μm filter membranes in turn, load into the pre-washed SEC column, incubate at room temperature for 60min, centrifuge at 500g for 10min, and collect the liquid.

[0104] Comparative Example 3

[0105] The only difference from Example 1 is that the parameters of differential centrifugation in step (3) are different, and the other conditions are the same, as follows:

[0106] Extraction and purification of extracellular vesicles from milk thistle:

[0107] (1) The milk thistle fruit was cooled at -4°C for 30 min, then the tissue was separated and cut into segments at 0°C, washed with ultrapure water, and soaked for 1 h.

[0108] (2) Drain the water, crush and squeeze the juice at 0℃, and add pre-cooled PBS if necessary. Use 100 mesh, 200 mesh, and 400 mesh filters to perform preliminary filtration to obtain filtrate A;

[0109] (3) Filtrate A was subjected to differential centrifugation at -4°C at 400g, 4000g, and 14000g for 40 min each to obtain supernatant B;

[0110] (4) Filter the supernatant B using 0.8 μm, 0.45 μm, and 0.22 μm filter membranes in sequence to obtain filtrate C;

[0111] (5) Add an equal volume of PEG solution (molecular weight 2000Da, mass concentration 5%) to filtrate C and mix thoroughly, then let stand overnight at low temperature. Centrifuge at 8000g and 4℃ for 120min, collect the precipitate, resuspend the precipitate in 1×PBS precooled at 4℃, filter with 0.45μm and 0.22μm filter membranes in turn, load into the pre-washed SEC column, incubate at room temperature for 60min, centrifuge at 500g for 10min, and collect the liquid.

[0112] Comparative Example 4

[0113] 4-1: Compared with Example 3, differential centrifugation is not used

[0114] Extraction and purification of extracellular vesicles from milk thistle:

[0115] (1) The milk thistle fruit was cooled at 8°C for 60 min, then the tissue was separated and cut into sections at 4°C, washed with ultrapure water, and soaked for 1 h.

[0116] (2) Drain the water and crush and squeeze the juice at 4°C. If necessary, add an appropriate amount of pre-cooled PBS. Use 100 mesh, 200 mesh, and 400 mesh filters for preliminary filtration to obtain filtrate A;

[0117] (3) Filtrate A is filtered using 0.8 μm, 0.45 μm, and 0.22 μm filter membranes in sequence to obtain filtrate B;

[0118] (4) Add an equal volume of PEG solution (molecular weight 10000Da, mass concentration 20%) to filtrate B and mix thoroughly, then let stand overnight at low temperature. Centrifuge at 8000g and 4℃ for 120min, collect the precipitate, resuspend the precipitate in 1×PBS precooled at 4℃, filter with 0.45μm and 0.22μm filter membranes in turn, load into a pre-washed SEC column, incubate at room temperature for 60min, centrifuge at 500g for 10min, and collect the liquid.

[0119] This method does not use differential centrifugation for preliminary purification, resulting in a large amount of impurities such as plant fibers and cell walls in the crude extract after direct use of the PEG precipitation method. When further purification is performed using a SEC column, the SEC column becomes clogged.

[0120] 4-2: Compared with Example 3, PEG method is not used

[0121] Extraction and purification of extracellular vesicles from milk thistle:

[0122] (1) The milk thistle fruit was cooled at 8°C for 60 min, then the tissue was separated and cut into sections at 4°C, washed with ultrapure water, and soaked for 1 h.

[0123] (2) Drain the water and crush and squeeze the juice at 4°C. If necessary, add an appropriate amount of pre-cooled PBS. Use 100 mesh, 200 mesh, and 400 mesh filters for preliminary filtration to obtain filtrate A;

[0124] (3) Filtrate A was subjected to differential centrifugation at 1000g, 3000g, 8000g, and 12000g at 25°C for 60 min each to obtain supernatant B;

[0125] (4) Filter the supernatant B using 0.8 μm, 0.45 μm, and 0.22 μm filter membranes in sequence to obtain filtrate C;

[0126] (5) Filtrate C was filtered through 0.45 μm and 0.22 μm filter membranes and directly loaded into a pre-washed SEC column. The column was incubated at room temperature for 60 min and centrifuged at 500 g for 10 min. The liquid was collected.

[0127] This method does not use PEG precipitation for extraction, resulting in a large volume of supernatant B after differential centrifugation, which cannot be used for batch incubation in the SEC column. Therefore, the processing volume of this comparative example is only 200 g.

[0128] 4-3: Compared with Example 3, the SEC method is not used.

[0129] Extraction and purification of extracellular vesicles from milk thistle:

[0130] (1) The milk thistle fruit was cooled at 8°C for 60 min, then the tissue was separated and cut into sections at 4°C, washed with ultrapure water, and soaked for 1 h.

[0131] (2) Drain the water and crush and squeeze the juice at 4°C. If necessary, add an appropriate amount of pre-cooled PBS. Use 100 mesh, 200 mesh, and 400 mesh filters for preliminary filtration to obtain filtrate A;

[0132] (3) Filtrate A was subjected to differential centrifugation at 1000g, 3000g, 8000g, and 12000g at 25°C for 60 min each to obtain supernatant B;

[0133] (4) Filter the supernatant B using 0.8 μm, 0.45 μm, and 0.22 μm filter membranes in sequence to obtain filtrate C;

[0134] (5) Add an equal volume of PEG solution (molecular weight 10,000 Da, mass concentration 20%) to filtrate C, mix thoroughly, and let stand overnight at low temperature. Centrifuge at 8,000 g and 4°C for 120 min, collect the precipitate, and resuspend the precipitate in 1× PBS precooled at 4°C.

[0135] This method did not use a SEC column for further purification and separation. After testing by protein immunoblotting, the comparative example contained plant impurities in addition to extracellular vesicles, which reduced the purity of the extraction.

[0136] The silybum-derived extracellular vesicles prepared in the above comparative example and Examples 1-3 were freeze-dried, and the specific operation was as follows:

[0137] 1) Pre-freeze at -40℃ for 24h;

[0138] 2) Place in freeze dryer, set vacuum degree to 20Pa, and freeze dry for 24 hours.

[0139] The yield, total protein concentration, and particle size of freeze-dried silybum-derived extracellular vesicles were evaluated. Yield = freeze-dried silybum-derived extracellular vesicle mass (referred to as freeze-dried mass) / silybum fruit mass*100%. The total protein concentration was determined using the BCA protein concentration assay kit. The results are shown in Table 1. The higher the total protein concentration, the higher the purity.

[0140] Table 1 Yield, total protein concentration and particle size test results of each embodiment and comparative example

[0141]

[0142] Compared with Example 1, the mass concentration of the PEG solution in Comparative Example 1-2 is different. As a result, the total protein concentration of Comparative Example 1 is significantly lower than that of Example 1, and the particle size is significantly higher than that of Example 1; the yield and total protein concentration of Comparative Example 2 are significantly lower than those of Example 1. This indicates that the PEG solution of a specific concentration of the present application must be used so that the silymarin-derived extracellular vesicles can simultaneously achieve excellent results in terms of yield, purity and particle size.

[0143] Compared with Example 1, the parameters of differential centrifugation were changed in Comparative Example 3. As a result, the total protein concentration of Comparative Example 3 was significantly lower than that of Example 1, and the particle size was significantly higher than that of Example 1, indicating that the parameters of differential centrifugation have a significant effect on the purity and particle size of silymarin-derived extracellular vesicles.

[0144] Compared with Example 3, Comparative Example 4-1 did not use differential centrifugation for purification, resulting in SEC column clogging and failure to prepare silymarin-derived extracellular vesicles.

[0145] Compared with Example 3, Comparative Example 4-2 did not use PEG solution for purification. As a result, the yield and total protein concentration were lower than those in Example 3, and the processing volume was only 200 g, far lower than 2.5 kg in Example 3.

[0146] Compared with Example 3, Comparative Example 4-3 did not use SEC column for purification. As a result, the total protein concentration was significantly lower than that of Example 3, and the particle size was significantly higher than that of Example 3.

[0147] Therefore, differential centrifugation, PEG and SEC purification must be used simultaneously to achieve excellent results in terms of yield, purity, particle size and processing volume.

[0148] Application Example 1

[0149] Preparation of a soothing and anti-redness serum containing extracellular vesicles derived from milk thistle

[0150] (1) Add 1% glycerol, 3% butylene glycol, 0.5% p-hydroxyacetophenone, 0.6% hexylene glycol, 0.05% sodium acrylate copolymer, 0.5% xanthan gum, 2% diethoxyethyl succinate, and the balance water into a 200 g beaker and heat at 80°C for 20 min.

[0151] (2) Add 2.5% squalane, 2% diethoxyethyl succinate, 0.5% jojoba seed oil, 0.3% glyceryl stearate, 0.1% PEG-100 stearate, and 0.05% EDTA-2Na, homogenize at 7000 rpm for 3 min, and stir to cool.

[0152] (3) When the temperature of the material drops to 45°C, add 0.5% of extracellular vesicles derived from milk thistle and the rest of water. The material is cooled to obtain the product.

[0153] Test Example 1

[0154] Characterization of Milk Thistle-Derived Extracellular Vesicles

[0155] (1) The particle size distribution of the silymarin-derived extracellular vesicles prepared in Example 1 was analyzed using a nanoparticle laser particle size analyzer. The average particle size D50 was 126 nm. The concentration was 2.4×10 9 psc / cm 3 The polymer dispersibility index (PDI) is 0.400, and the average potential is -38.9mv. Its particle size is consistent with the particle size range of 30 to 150nm of general plant extracellular vesicles. The particles are negatively charged, which is consistent with the charge property of plant extracellular vesicles. The particle size distribution and potential characterization are as follows: Figure 1 , Figure 2 shown.

[0156] Test Example 2

[0157] Cytotoxic effects of extracellular vesicles derived from milk thistle

[0158] MTT assay was used to detect the cytotoxic effects of silymarin-derived extracellular vesicles on HaCaT cells and RAW 264.7 cells. The cell density of HaCaT or RAW 264.7 was 8000 cells / cm 2 The silymarin-derived extracellular vesicles (1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9) were co-cultured with HaCaT cells or RAW 264.7 cells for 48 h, and then MTT reagent was added. After incubation for 4 h, DMSO was added, and the absorbance of each group of cells was measured at 490 nm to calculate the cell survival rate (%). Figure 4A , 4B As shown, the results showed that the cell survival rate was no less than 95%, indicating that silymarin-derived extracellular vesicles had no cytotoxicity to HaCaT cells and RAW 264.7 cells and had good biosafety.

[0159] The cell viability calculation formula is as follows:

[0160] ;

[0161] Wherein, A represents the absorbance value, the negative control group is the absorbance of cells not treated with the drug, the blank control group is the absorbance of the 96-well plate itself, and the sample group is the absorbance of cells treated with the drug according to Example 1.

[0162] Test Example 3

[0163] Effects of extracellular vesicles derived from silymarin on wound healing assay of HaCaT cells

[0164] Migration scratch assay to detect the effect of silymarin-derived extracellular vesicles on the wound healing of HaCaT cells: HaCaT cells were cultured at a cell density of 50,000 cells / cm 2 The cells were inoculated in a 6-well plate. When the natural logarithm of the cells grew to 90%, the cells were scratched with a pipette and 1×10 9 , 1×10 8 , 1×10 7 The silymarin-derived extracellular vesicles prepared in Example 1 with three particle concentrations were incubated with HaCaT cells for 48 h, and the changes in the scratch width were observed and the cell migration rate (%) was calculated. Figure 5A , 5B As shown, the results showed that the cell migration rate in the sample group was significantly improved compared with the blank group (control), and the blank group did not receive any medication, indicating that silymarin-derived extracellular vesicles have the effect of repairing and promoting healing of HaCaT cells.

[0165] Test Example 4

[0166] Effects of extracellular vesicles derived from silymarin on inflammatory factors in HaCaT cells

[0167] RAW264.7 cells in logarithmic growth phase with good morphology were selected and inoculated into 24-well plates with a cell density of 2×10 per well. 5 Pieces / cm 2 . Set up blank control group, stimulation group, sample group (10 7Group, 10 8 Group, 10 9 group), at 37°C, 5% CO 2 The cells were incubated in an incubator for 12 h. Except for the blank control group, lipopolysaccharide (LPS) was added to each well for stimulation and induction. The silybum-derived extracellular vesicles (1×10 7 , 1×10 8 , 1×10 9 ) and incubated for 24 h. The contents of IL-1α, TNF-α, and IL-6 in the collected cell supernatants were detected, and the inhibition rate of inflammatory factors in each group relative to the LPS-induced stimulation group was calculated. The results are shown in Figure 6 , 7 , as shown in Figure 8.

[0168] Test Example 5

[0169] Effect of silymarin-derived extracellular vesicles on H 2 O 2 Effects of induced HaCaT cell injury

[0170] RAW264.7 cells in logarithmic growth phase with good morphology were inoculated into 96-well plates with a cell density of 8000 cells / cm per well. 2 . Set up a blank control group (blank group), H 2 O 2 The stimulation group (model group) and sample group were kept at 37°C and 5% CO 2 Incubate in the incubator for 12 h. Except for the blank control group, each well was added with H 2 O 2 Stimulation induction was performed, and the extracellular vesicles derived from milk thistle prepared in Example 1 with different particle concentrations (1×10 7 , 1×10 8 , 1×10 9 ), incubate for 24 h. Add MTT reagent, incubate for 4 h, add DMSO, measure the absorbance of each group of cells at 490 nm, and calculate the cell survival rate (%). Fig. 9 As shown, the results show that the sample group (10 7 Group, 10 8 Group, 10 9 Group) Cell survival rate relative to H 2 O 2 The stimulation group showed a significant improvement, indicating that extracellular vesicles derived from silymarin have a significant protective effect against cell damage.

[0171] Test Example 6

[0172] Application Example 1 Evaluation of the soothing effect of extracellular vesicles derived from milk thistle

[0173] The skin was stimulated with capsaicin for 30 minutes, and the skin redness reached the highest value. The soothing and anti-redness essence containing extracellular vesicles derived from silymarin was applied to the reddened area by patching, and the data were recorded every 10 minutes. The subjective visual evaluation of the subjects was combined with the skin color difference test probe Probe Mexameter MX18 (CK, Germany) for evaluation, and the results are shown in Table 2 below.

[0174] Table 2 Evaluation results of soothing effect of application example 1

[0175]

[0176] Test Example 7

[0177] Effects of extracellular vesicles derived from silymarin on lipid secretion of SZ95 cells induced by linoleic acid

[0178] SZ95 cells in logarithmic growth phase with good morphology were selected and inoculated into 96-well plates with a cell density of 8000 cells / cm per well. 2 A blank control group and a sample group were set up at 37°C and 5% CO 2 The cells were incubated in an incubator for 12 h. Linoleic acid was added to each well except for the blank control group for stimulation induction. The extracellular vesicles derived from silybum obtained in Example 1 with different particle concentrations (1×10 7 , 1×10 8 , 1×10 9 ), incubate for 24 hours. Add Nile red reagent and incubate, and use a multifunctional microplate reader to measure the fluorescence intensity. Fig.10 As shown, the results showed that the sample group (10 7 Group, 10 8 Group, 10 9 The fluorescence intensity of the silymarin vesicles in the SZ95 cells (group) was significantly decreased, proving that silymarin vesicles can inhibit linoleic acid-induced oil synthesis in SZ95 cells and have a significant oil-control effect.

[0179] Test Example 8

[0180] Stability test

[0181] Test samples: samples prepared in Example 1 and Application Example 1.

[0182] Experimental methods:

[0183] Each test sample was tested for 15d (days), 30d (days), and 40d (days) under five test conditions, namely -18℃, room temperature, light, 45℃, and -18~45℃ hot and cold cycle (-18℃ for 12h, 45℃ for 12h), to observe whether each test sample had obvious changes. If there was no change, it was qualified, and if there was a change, it was unqualified. The test results are shown in Table 3 below.

[0184] Table 3 Stability test results of Example 1 and Application Example 1

[0185]

[0186] The above detailed description is a specific description of one feasible embodiment of the present invention. The embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not deviate from the present invention should be included in the scope of the technical solution of the present invention.

Claims

1. A method for preparing milk thistle-derived plant extracellular vesicles, characterized in that: The steps include: (1) crushing milk thistle fruit, squeezing juice and filtering to obtain filtrate A; (2) Filtrate A is subjected to differential centrifugation to obtain supernatant B; (3) Filter the supernatant B to obtain filtrate C; (4) After adding polyethylene glycol solution to filtrate C, centrifugation is performed for the first time, and the precipitate is collected. The precipitate is resuspended and filtered, and then further purified by molecular exclusion chromatography, and then centrifugation is performed for the second time to collect the liquid; The specific steps of the differential centrifugation in step (2) are as follows: centrifuge at -4°C to 25°C at a centrifugal force of 500-1000g, 1000-3000g, 5000-8000g, and 10000-12000g for 30-60min each; The mass concentration of the polyethylene glycol solution in step (4) is 5%-20%; In step (4), the mixture is incubated for 30-60 min by size exclusion chromatography and then centrifuged a second time; First centrifugation parameters: centrifugal force 3000-8000g, 4℃ centrifugation for 30-120min; Second centrifugation parameters: centrifugal force 500g-1500g, centrifugation 10-30min.

2. The preparation method according to claim 1, characterized in that: In step (1), the milk thistle fruit is pre-treated before being crushed. The specific steps of the pre-treatment are as follows: the milk thistle fruit is cooled in an environment of -4°C to 8°C for 30-60 minutes, then the tissue is separated at 0-4°C and washed with water.

3. The preparation method according to claim 1, characterized in that: The molecular weight of the polyethylene glycol in step (4) is 2000-10000 Da.

4. The preparation method according to claim 1, characterized in that: In step (4), the resuspended precipitate is filtered using a 0.45 μm or 0.22 μm filter membrane.

5. Use of the milk thistle-derived plant extracellular vesicles prepared by the preparation method according to any one of claims 1 to 4 in the preparation of soothing and / or oil-controlling cosmetics.

6. The use according to claim 5, characterized in that: The dosage forms of the cosmetics include emulsions, creams, water solutions and gels.

7. The use according to claim 5, characterized in that: The cosmetics also include auxiliary materials, which include any one or a combination of at least two of a diluent, a dispersant, a binder, a filler, a thickener, a lubricant, a pH adjuster, a taste masking agent, a colorant, an antioxidant or an antibacterial agent.

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