Exosome-like vesicle as well as extraction method and application thereof

Abstract

The invention relates to the field of biological medicine, and discloses an exosome-like vesicle as well as an extraction method and application thereof. The exosome-like vesicles are extracted and separated from spinach, and the exosome-like vesicles have self-adaptive surface characteristics, can play a role in relieving neuroinflammation injury, and have a remarkable application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly relates to an exosome-like vesicle and its extraction method and application. Background Art

[0002] Neuroinflammation is closely related to a variety of neurological diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, depression, anxiety, stroke, traumatic brain injury, etc. Although there are currently anti-neuroinflammatory drugs in clinical practice, these drugs generally have side effects, and some drugs are difficult to cross the blood-brain barrier to reach nerve cells and exert their effects. In the context of "using food as medicine" in the field of big health, it is particularly important to extract natural products from fruits and vegetables and find safer and more effective solutions to relieve neuroinflammation.

[0003] Plant-derived exosome-like vesicles have similar structures, components, and physiological functions to animal-derived exosomes. Exosome-like vesicles are composed of specific proteins, lipids, nucleic acids, and secondary metabolites, and play an important role in maintaining cell homeostasis and intercellular information communication. Exosome-like vesicles have advantages such as good biocompatibility, low immunogenicity, oral safety, and the ability to cross physiological barriers. In recent years, their applications in the fields of food and medicine have attracted much attention. The latest research has confirmed that edible plant-derived exosome-like vesicles utilize the advantages of their vesicle structure in oral and gastrointestinal digestive juices to avoid the influence of harsh environments such as gastric acid, successfully reach the intestine, are distributed in distal tissues through the body fluid circulation, especially in brain nerves, release active ingredients, and exert regulatory functions. There is a close connection between the above-mentioned vesicle transport mechanism and the surface characteristics of exosome-like vesicles. Therefore, exploring the surface characteristics of edible plant-derived exosome-like vesicles, their ability to be taken up by nerve cells, and their anti-neuroinflammatory effects is expected to become an effective strategy for relieving neuroinflammatory damage.

[0004] Therefore, there is an urgent need to provide an exosome-like vesicle with adaptive surface characteristics to play a role in relieving neuroinflammatory damage. Summary of the Invention

[0005] The purpose of the present invention is to overcome the problems of side effects and insignificant efficacy of existing drugs for relieving neuroinflammatory damage, and to provide an exosome-like vesicle and its extraction method and application. The exosome-like vesicle has adaptive surface characteristics, can play a role in relieving neuroinflammatory damage, and has significant application prospects.

[0006] To achieve the above purpose, in the first aspect of the present invention, an exosome-like vesicle is provided, which is extracted and separated from spinach.

[0007] Preferably, the particle size of the exosome-like vesicle is 50 - 200 nm.

[0008] Preferably, the exosome-like vesicles have a saucer-like structure.

[0009] Preferably, the exosome-like vesicles contain proteins, lipids and nucleic acids.

[0010] Preferably, based on 100 mg of the exosome-like vesicles, the content of the protein is 3 - 5 mg, the content of the lipid is 0.5 - 1 mg, and the content of the nucleic acid is 15 - 25 μg.

[0011] The second aspect of the present invention provides a method for extracting exosome-like vesicles, comprising the following steps: extracting and separating spinach.

[0012] Preferably, the method for extracting exosome-like vesicles comprises the following steps:

[0013] (1) After crushing the spinach to obtain spinach juice, performing solid-liquid separation on the spinach juice to obtain a supernatant;

[0014] (2) Passing the supernatant through a filter membrane to obtain a filtrate, and performing ultrafiltration on the filtrate.

[0015] Preferably, in step (1), the solid-liquid separation includes a first centrifugal separation and a second centrifugal separation; the conditions of the first centrifugal separation include at least: a temperature of 2 - 5 °C, a centrifugal force of 2000 - 3000 g, and a time of 20 - 30 min; the conditions of the second centrifugal separation include at least: a temperature of 2 - 5 °C, a centrifugal force of 8000 - 10000 g, and a time of 50 - 70 min.

[0016] Preferably, before the centrifugal separation, mixing the spinach juice and a phosphate buffer solution.

[0017] Preferably, the mass ratio of the spinach juice to the phosphate buffer solution is 1:1.5 - 2.

[0018] Preferably, in step (2), the filter diameter of the filter membrane is 1 - 2.2 μm.

[0019] Preferably, the process of ultrafiltration includes: performing a first ultrafiltration on the filtrate through an ultrafiltration cup with a filter membrane having a molecular weight cut-off of 90 - 100 kDa and collecting the ultrafiltrate, and performing a second ultrafiltration on the ultrafiltrate through an ultrafiltration cup with a filter membrane having a molecular weight cut-off of 2 - 4 kDa.

[0020] The third aspect of the present invention provides the application of the exosome-like vesicles described in the first aspect and / or the exosome-like vesicles obtained by the extraction method described in the second aspect in the preparation of products for relieving nerve inflammatory injury.

[0021] Preferably, the product for relieving nerve inflammatory injury is a product that down-regulates the secretion amount of pro-inflammatory factors and / or up-regulates the release amount of anti-inflammatory factors.

[0022] Preferably, the pro-inflammatory factor is selected from at least one of TNF-α, IFN-γ and IL-6, and the anti-inflammatory factor is IL-10.

[0023] The fourth aspect of the present invention provides a composition for alleviating neuroinflammatory damage, wherein the composition contains the exosome-like vesicles described in the first aspect and / or the exosome-like vesicles obtained by the extraction method described in the second aspect.

[0024] Through the above technical solution, the beneficial effects of the present invention are:

[0025] The exosome-like vesicles provided by the present invention are extracted and separated from spinach, have adaptive surface characteristics, are easy to cross physiological barriers, are quickly taken up by glial cells to play a regulatory function, play a role in alleviating neuroinflammatory damage, and have both biocompatibility and neuroprotective effects, and have significant application prospects. Moreover, the extraction method is simple, and no chemical reagents are added during the preparation process, which is green, pollution-free, and highly safe.

[0026] The exosome-like vesicles provided by the present invention have a particle size of 50-200nm, a saucer-shaped structure, are rich in proteins, lipids and nucleic acids, can significantly downregulate the secretion of pro-inflammatory factors TNF-α, IFN-γ and IL-6, and simultaneously upregulate the release of anti-inflammatory factor IL-10, and show good anti-inflammatory effects on lipopolysaccharide-induced neuroinflammation, thereby being able to alleviate neuroinflammatory damage.

[0027] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a flow chart of extracting spinach exosome-like vesicles in Example 1 of the present invention;

[0029] Figure 2 This is a transmission electron micrograph of the spinach exosome-like vesicles extracted in Example 1 of the present invention;

[0030] Figure 3 This is a particle size distribution diagram of the spinach exosome-like vesicles extracted in Example 1 of the present invention;

[0031] Figure 4Results of the adaptive surface properties of spinach exosome-like vesicles obtained in Example 1 of the present invention. Among them, (A) shows the results of the hydrated particle size of spinach exosome-like vesicles under different pH conditions, (B) shows the results of the ζ-potential changes of spinach exosome-like vesicles under different pH conditions, (C) shows the results of the complexation constants of spinach exosome-like vesicles under different incubation times, and (D) shows the results of the surface hydrophilicity of spinach exosome-like vesicles evaluated by the mucin affinity experiment under different pH conditions;

[0032] Figure 5 Shows the uptake of spinach exosome-like vesicles by BV-2 cells over time after treatment;

[0033] Figure 6 Is a graph of the survival rate of BV-2 cells before and after treatment with spinach exosome-like vesicles obtained in Example 1 of the present invention;

[0034] Figure 7 Results of the secretion levels of TNF-α, IFN-γ, IL-6, and IL-10 in lipopolysaccharide-induced nerve cells before and after treatment with spinach exosome-like vesicles obtained in Example 1 of the present invention. Among them, (A) is a bar graph of the TNF-α secretion level in lipopolysaccharide-induced nerve cells before and after treatment with spinach exosome-like vesicles, (B) is a bar graph of the IFN-γ secretion level in lipopolysaccharide-induced nerve cells before and after treatment with spinach exosome-like vesicles, (C) is a bar graph of the IL-6 secretion level in lipopolysaccharide-induced nerve cells before and after treatment with spinach exosome-like vesicles, and (D) is a bar graph of the IL-10 secretion level in lipopolysaccharide-induced nerve cells before and after treatment with spinach exosome-like vesicles;

[0035] Figure 8 Results of the secretion levels of IL-6 and IL-10 in lipopolysaccharide-induced nerve cells before and after treatment with green amaranth exosome-like vesicles obtained in the comparative example of the present invention. Among them, (A) is a bar graph of the IL-6 secretion level in lipopolysaccharide-induced nerve cells before and after treatment with green amaranth exosome-like vesicles, and (B) is a bar graph of the IL-10 secretion level in lipopolysaccharide-induced nerve cells before and after treatment with green amaranth exosome-like vesicles. Detailed implementation methods

[0036] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoints of each range, between the endpoints of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be considered as specifically disclosed herein.

[0037] To achieve the above object, in a first aspect, the present invention provides an exosome-like vesicle, which is extracted and isolated from spinach.

[0038] During the research process, the inventors of the present invention unexpectedly found that the exosome-like vesicles extracted and isolated from spinach have adaptive surface characteristics, are easy to cross physiological barriers, can be quickly taken up by glial cells to play a regulatory function, play a role in alleviating nerve inflammation damage, and have both biocompatibility and neuroprotective effects, showing significant application prospects.

[0039] In the present invention, spinach is a common vegetable with relatively rich nutritional components, and it has the effects of supplementing vitamins, promoting digestion, preventing anemia, protecting eyesight, enhancing immunity, and strengthening the physique, being suitable for both the young and the old. The exosome-like vesicles can be extracted and isolated from the leaves of spinach or from the roots of spinach.

[0040] According to the present invention, preferably, the particle size of the exosome-like vesicles is 50 - 200 nm, specifically, it can be 50 nm, 100 nm, 150 nm, 200 nm, or any value between the aforementioned two values. The exosome-like vesicles with the above nano-level particle size can be more easily taken up by glial cells, thereby further enhancing the effect of alleviating nerve inflammation damage.

[0041] In the present invention, the particle size of the exosome-like vesicles can be measured by a particle size tracking analyzer to determine its size distribution.

[0042] According to the present invention, preferably, the exosome-like vesicles have a saucer-like structure. The exosome-like vesicles with the above structure can be more easily taken up by glial cells, thereby further enhancing the effect of alleviating nerve inflammation damage.

[0043] In the present invention, a transmission electron microscope can be used to observe the structure of the exosome-like vesicles.

[0044] According to the present invention, preferably, the exosome-like vesicles contain proteins, lipids, and nucleic acids (RNA). The exosome-like vesicles containing proteins, lipids, and nucleic acids can effectively ensure that the exosome-like vesicles play a regulatory function in cells, thereby further enhancing its effect of alleviating nerve inflammation damage.

[0045] According to the present invention, in order to further enhance the effect of exosome-like vesicles in alleviating neuroinflammatory injury, preferably, based on 100 mg of the exosome-like vesicles, the content of the protein is 3 - 5 mg, specifically it can be 3 mg, 4 mg, 5 mg, or any value between the aforementioned two values; the content of the lipid is 0.5 - 1 mg, specifically it can be 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, or any value between the aforementioned two values; the content of the nucleic acid is 15 - 25 μg, specifically it can be 15 μg, 20 μg, 25 μg, or any value between the aforementioned two values.

[0046] In the present invention, for the determination of the contents of protein, lipid, and nucleic acid in exosome-like vesicles, conventional testing methods in the art can be used for determination. Exemplarily, the Coomassie Brilliant Blue reagent method can be used to determine the protein content in the extracted spinach exosome-like vesicles; the Soxhlet extraction method can be used to extract the lipid in the spinach exosome-like vesicles and determine the total lipid content; the Trizol reagent can be used to extract the nucleic acid in the spinach exosome-like vesicles and determine the RNA content.

[0047] In a second aspect, the present invention provides a method for extracting exosome-like vesicles, comprising the following steps: extracting and separating spinach.

[0048] The method for extracting exosome-like vesicles provided by the present invention is simple, and no chemical reagents are added during the preparation process, which is green, pollution-free, and has high safety.

[0049] In the present invention, the spinach can be the leaves and / or roots of spinach.

[0050] According to the present invention, in order to further improve the yield of exosome-like vesicles and the effect of alleviating neuroinflammatory injury, preferably, the method for extracting exosome-like vesicles comprises the following steps: (1) pulverizing the spinach to obtain spinach juice, and performing solid-liquid separation on the spinach juice to obtain a supernatant; (2) passing the supernatant through a filter membrane to obtain a filtrate, and performing ultrafiltration on the filtrate. The inventors found that in this preferred embodiment, when using the ultrafiltration method to extract exosome-like vesicles, no chemical reagents are added, which maximally ensures the natural extraction of spinach exosome-like vesicles, is green, pollution-free, and ensures oral safety; compared with methods such as ultra-high speed centrifugation, size exclusion chromatography, and microfluidic technology, this extraction method does not require expensive instrument equipment and separation aids, simplifies the separation steps, greatly reduces the operation time, and significantly improves the yield of exosome-like vesicles.

[0051] In the present invention, in step (1), the spinach can be pulverized by common pulverizing methods in the art, for example, juicing the spinach.

[0052] In the present invention, in step (1), before centrifugally separating the spinach juice, the spinach juice can also be manually squeezed and filtered through a gauze to remove residues.

[0053] According to the present invention, in order to further improve the extraction effect of exosome-like vesicles from spinach, preferably, in step (1), the solid-liquid separation includes a first centrifugal separation and a second centrifugal separation; the conditions of the first centrifugal separation include at least: the temperature is 2-5 °C, specifically it can be 2 °C, 3 °C, 4 °C, 5 °C, or any value between the aforementioned two values; the centrifugal force is 2000-3000 g, specifically it can be 2000 g, 2500 g, 3000 g, or any value between the aforementioned two values; the time is 20-30 min, specifically it can be 20 min, 25 min, 30 min, or any value between the aforementioned two values; the conditions of the second centrifugal separation include at least: the temperature is 2-5 °C, specifically it can be 2 °C, 3 °C, 4 °C, 5 °C, or any value between the aforementioned two values; the centrifugal force is 8000-10000 g, specifically it can be 8000 g, 9000 g, 10000 g, or any value between the aforementioned two values; the time is 50-70 min, specifically it can be 50 min, 60 min, 70 min, or any value between the aforementioned two values.

[0054] According to the present invention, in order to further improve the extraction effect of exosome-like vesicles from spinach, preferably, before the centrifugal separation, the spinach juice and a phosphate buffer are mixed.

[0055] According to the present invention, in order to further improve the extraction effect of exosome-like vesicles from spinach, preferably, the mass ratio of the spinach juice to the phosphate buffer is 1:1.5-2, specifically it can be 1:1.5, 1:1.8, 1:2, or any value between the aforementioned two values.

[0056] According to the present invention, in order to further improve the extraction effect of exosome-like vesicles from spinach, preferably, in step (2), the filtration diameter of the filter membrane is 1-2.2 μm, specifically it can be 1.2 μm, 1.6 μm, 2.2 μm, or any value between the aforementioned two values.

[0057] According to the present invention, the ultrafiltration of the filtrate can be carried out using an ultrafiltration cup. In order to further improve the extraction effect of exosome-like vesicles from spinach, preferably, the ultrafiltration process includes: subjecting the filtrate to a first ultrafiltration through an ultrafiltration cup with a filter membrane having a molecular weight cut-off of 90-100 kDa and collecting the ultrafiltrate, and subjecting the ultrafiltrate to a second ultrafiltration through an ultrafiltration cup with a filter membrane having a molecular weight cut-off of 2-4 kDa.

[0058] In the present invention, the extraction method further includes: drying the filtrate obtained in step (2). Drying can be carried out by a drying method conventionally selected in the art. Preferably, the drying method is freeze-drying.

[0059] In the present invention, the exosome-like vesicles obtained by drying can be stored at low temperature. For example, they can be stored under the condition of -70°C to -80°C.

[0060] According to a particularly preferred embodiment of the present invention, a method for extracting exosome-like vesicles is provided, including the following steps:

[0061] (1) After pulverizing spinach to obtain spinach juice, mixing the spinach juice and phosphate buffer in a mass ratio of 1:1.5 - 2, and centrifuging and separating for 20 - 30 min at a temperature of 2 - 5°C and a centrifugal force of 2000 - 3000 g to obtain a supernatant, and then centrifuging and separating the supernatant for 50 - 70 min at a temperature of 2 - 5°C and a centrifugal force of 8000 - 10000 g to obtain a supernatant;

[0062] (2) Passing the supernatant obtained in step (1) through a filter membrane with a pore size of 1 - 2.2 μm to obtain a filtrate, subjecting the filtrate to a first ultrafiltration using an ultrafiltration cup with a molecular weight cut-off of 90 - 100 kDa and collecting the ultrafiltrate, and subjecting the ultrafiltrate to a second ultrafiltration using an ultrafiltration cup with a molecular weight cut-off of 2 - 4 kDa and collecting the ultrafiltrate.

[0063] Through the above particularly preferred embodiment, the method for extracting exosome-like vesicles from spinach is simple, and the extracted exosome-like vesicles have self-adaptive surface characteristics, can play a role in alleviating nerve inflammation damage, and have significant market prospects.

[0064] In the third aspect, the present invention provides the application of the exosome-like vesicles described in the first aspect and / or the exosome-like vesicles obtained by the extraction method described in the second aspect in the preparation of products for alleviating nerve inflammation damage.

[0065] According to the present invention, preferably, the product for alleviating nerve inflammation damage is a product that down-regulates the secretion amount of pro-inflammatory factors and / or up-regulates the release amount of anti-inflammatory factors.

[0066] According to the present invention, the pro-inflammatory factors can be TNF-α, IFN-γ, IL-6 or IL-17, etc., and the anti-inflammatory factors can be IL-4 or IL-10, etc. Preferably, the pro-inflammatory factors are selected from at least one of TNF-α, IFN-γ and IL-6, and the anti-inflammatory factor is IL-10.

[0067] Fourthly, the present invention provides a composition for alleviating nerve inflammatory damage, which contains the exosome-like vesicles described in the first aspect above and / or the exosome-like vesicles obtained by the extraction method described in the second aspect above.

[0068] The present invention will be described in detail below with reference to the embodiments, but the scope of the present invention is not limited thereby.

[0069] In the following examples and comparative examples, unless otherwise specified, the raw materials are all commercially available.

[0070] Example 1

[0071] (1) After washing fresh spinach clean, juice it, and dilute it with phosphate buffer solution (PBS solution) with a pH of 7.4 according to the juice concentration. The mass ratio of the juice to the phosphate buffer solution is 1:1.8;

[0072] (2) Centrifuge the above spinach juice at 4°C and 3000g for 30 min, take the supernatant, then centrifuge at 10000g for 60 min, take the supernatant, and discard the precipitate;

[0073] (3) Place a membrane with a pore size of 1 μm on a sterile filter, and filter the supernatant obtained in the previous step, and collect the filtrate.

[0074] (4) Pour the filtrate obtained in the previous step into an ultrafiltration cup equipped with a 100 kDa membrane, and perform magnetic stirring at a speed of 1400 rpm. When the volume of the filtrate reaches 1 / 2 of the total volume of the added liquid, collect the filtrate;

[0075] (5) Take the filtrate collected in the previous step and pour it into an ultrafiltration cup equipped with a 3 kDa membrane, and perform magnetic stirring at a speed of 1400 rpm. When the volume of the filtrate reaches 1 / 3 of the total volume of the added liquid, collect the solution in the ultrafiltration cup;

[0076] (6) Place the collected solution in a freeze dryer for freeze drying to obtain freeze-dried powder of spinach exosome-like vesicles. 2 g of freeze-dried powder of spinach exosome-like vesicles can be extracted from every 100 g of spinach, and the yield is 2%.

[0077] The flow chart for extracting spinach exosome-like vesicles in Example 1 is as Figure 1 shown.

[0078] Example 2

[0079] (1) After washing fresh spinach clean, juice it, and dilute it with phosphate buffer solution with a pH of 7.4 according to the juice concentration. The mass ratio of the juice to the phosphate buffer solution is 1:1.5;

[0080] (2) The above spinach juice is centrifuged at 2000 g for 20 min at 2 °C, the supernatant is taken, and then centrifuged at 8000 g for 70 min, the supernatant is taken, and the precipitate is discarded;

[0081] (3) Place a membrane with a pore size of 1 μm on a sterile filter, filter the supernatant obtained in the previous step, and collect the filtrate.

[0082] (4) Pour the filtrate obtained in the previous step into an ultrafiltration cup equipped with a 90 kDa membrane, and perform magnetic stirring at a speed of 1400 rpm. When the volume of the filtrate reaches 1 / 2 of the total volume of the added liquid, collect the filtrate;

[0083] (5) Take the filtrate collected in the previous step and pour it into an ultrafiltration cup equipped with a 4 kDa membrane, and perform magnetic stirring at a speed of 1400 rpm. When the volume of the filtrate reaches 1 / 3 of the total volume of the added liquid, collect the solution in the ultrafiltration cup;

[0084] (6) The collected solution is placed in a freeze dryer for freeze-drying to obtain freeze-dried powder of spinach exosome-like vesicles. 1.71 g of freeze-dried powder of spinach exosome-like vesicles can be extracted from every 100 g of spinach, and the yield is 1.71%.

[0085] Example 3

[0086] (1) After washing fresh spinach and juicing it, dilute it with phosphate buffer (PBS solution) with a pH value of 7.4 according to the juice concentration, and the mass ratio of the juice to the phosphate buffer is 1:2;

[0087] (2) The above spinach juice is centrifuged at 2500 g for 25 min at 5 °C, the supernatant is taken, and then centrifuged at 9000 g for 50 min, the supernatant is taken, and the precipitate is discarded;

[0088] (3) Place a membrane with a pore size of 1 μm on a sterile filter, filter the supernatant obtained in the previous step, and collect the filtrate.

[0089] (4) Pour the filtrate obtained in the previous step into an ultrafiltration cup equipped with a 95 kDa membrane, and perform magnetic stirring at a speed of 1400 rpm. When the volume of the filtrate reaches 1 / 2 of the total volume of the added liquid, collect the filtrate;

[0090] (5) Take the filtrate collected in the previous step and pour it into an ultrafiltration cup equipped with a 2 kDa membrane, and perform magnetic stirring at a speed of 1400 rpm. When the volume of the filtrate reaches 1 / 3 of the total volume of the added liquid, collect the solution in the ultrafiltration cup;

[0091] (6) The collected solution is placed in a freeze dryer for freeze-drying to obtain freeze-dried powder of spinach exosome-like vesicles. 1.58 g of freeze-dried powder of spinach exosome-like vesicles can be extracted from every 100 g of spinach, and the yield is 1.58%.

[0092] Example 4

[0093] (1) After washing fresh spinach clean, juice it and dilute with phosphate buffer solution with a pH of 7.4 according to the juice concentration.

[0094] (2) Centrifuge the above spinach juice at 4 °C and 3000 g for 30 min, take the supernatant, then centrifuge at 10000 g for 60 min, take the supernatant and discard the precipitate.

[0095] (3) Place a membrane with a pore size of 1 μm on a sterile filter, filter the supernatant obtained in the previous step, and collect the filtrate.

[0096] (4) Centrifuge the filtrate obtained in the previous step at 4 °C and 150000 g for 120 min, discard the supernatant to obtain a precipitate.

[0097] (5) Resuspend the precipitate in 2 mL of PBS solution with a pH of 7.4 to obtain a suspension, transfer the suspension to a sucrose gradient solution with mass fractions of 8 / 30 / 45 / 60%, centrifuge at 150000 g for 120 min, and collect the solutions in the bands between the 8 / 30% layer, 30 / 45% layer, and 45 / 60% layer into EP tubes respectively.

[0098] (6) According to the density of exosomes being 1.13 - 1.19 g / mL, it is known that the band at the 30 / 45% interface is enriched with exosome-like extracellular vesicles.

[0099] (7) Add an equal volume of PBS solution with a pH of 7.4 to the solution of the 30 / 45% interface band collected, mix well and then perform ultrafiltration centrifugation at 1500 g, 4 °C for 20 min. The molecular weight cut-off of the filter membrane is 100 kDa to remove sucrose, and collect the final filtrate.

[0100] (8) Place the collected filtrate in a freeze dryer for freeze drying to obtain freeze-dried powder of spinach exosome-like vesicles. 1.24 g of freeze-dried powder of spinach exosome-like vesicles can be extracted from 100 g of spinach, and the yield is 1.24%.

[0101] Comparative Example

[0102] Extract exosome-like vesicles according to the method of Example 1, except that spinach is replaced with green amaranth.

[0103] 1.12 g of freeze-dried powder of green amaranth exosome-like vesicles can be extracted from 100 g of green amaranth, and the yield is 1.12%.

[0104] Test Example 1

[0105] Observe the morphology of the spinach exosome-like vesicles extracted in Example 1 by transmission electron microscopy. The transmission electron microscopy results are as Figure 2 shown.

[0106] The testing process includes: taking 0.1 g of the spinach exosome-like vesicles obtained by extracting Example 1 and resuspending them in 0.2 mL of phosphate buffer with a pH value of 7.4. Taking 10 μL of the sample and dropping it onto a 300-mesh copper grid with a supporting film, standing for 15 min, then dropping 2% phosphotungstic acid by volume for negative staining for 1 min, sucking the excess liquid from the edge of the droplet with a filter paper strip, standing for drying, and imaging with an HT-7800 transmission electron microscope.

[0107] As shown by Figure 2 It can be seen that in the electron microscope field of view, there are clearly visible saucer-like membrane vesicle structures with a bilayer membrane and a particle size less than 200 nm.

[0108] Test Example 2

[0109] The particle size of the spinach exosome-like vesicles obtained by extracting Example 1 was tested. The testing process includes: resuspending 0.1 g of spinach exosome-like vesicles in 1 mL of phosphate buffer with a pH value of 7.4, filtering with a 0.22 μm filter membrane after diluting 20 times, sucking 1 mL of the diluted solution with a syringe and slowly injecting it into the sample cell, and using a nanoparticle tracking analyzer to detect the particle size and concentration. Each sample was measured 3 times and the average value was taken. The results are as Figure 3 shown.

[0110] From Figure 3 the results, it can be seen that the average particle size of the spinach exosome-like vesicles is 146.8 nm, which belongs to the size range of exosomes (50 - 200 nm).

[0111] Test Example 3

[0112] The contents of proteins, lipids, and nucleic acids in the spinach exosome-like vesicles obtained by extracting Example 1 were tested respectively, and the results are shown in Table 1.

[0113] The process for determining the protein content in spinach exosome-like vesicles includes: weighing 100 mg of freeze-dried spinach exosome-like vesicles and dissolving them in 2 mL of phosphate buffer with a pH of 7.4, adding protein lysis buffer, centrifuging to obtain the protein supernatant, and centrifuging at 4000 r / min for 20 min; preparing BSA standard products with concentrations of 2 mg / mL, 1.5 mg / mL, 1 mg / mL, 0.75 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 0.025 mg / mL, and 0 mg / mL respectively. Take 5 μL of each of the above BSA standard product solutions into a 96-well plate, and then add 200 μL of Coomassie Brilliant Blue reagent (G250), and measure the absorbance at 595 nm on an enzyme-linked immunosorbent assay (ELISA) reader. Plot a BSA standard curve with the concentration of the BSA standard product as the abscissa and the corresponding absorbance at 595 nm as the ordinate; make three parallels of the sample supernatant, add 5 μL of the sample and 200 μL of G250 reagent to each well, mix well, and measure the absorbance at 595 nm on an ELISA reader; substitute the measured value of the sample into the BSA standard curve to obtain the protein concentration.

[0114] The process for determining the protein content in spinach exosome-like vesicles includes: according to the Soxhlet extraction method, wash and dry the test tube, weigh and record it, weigh 1 g of freeze-dried spinach exosome-like vesicles on filter paper, package it and tie it with absorbent cotton string, put it into a Soxhlet extractor, continuously extract with petroleum ether for 2 h, evaporate to dryness, then place it in an oven and dry for another 30 min, cool and weigh, and subtract to obtain the total lipid content.

[0115] The process for determining the nucleic acid content in spinach exosome-like vesicles includes: after grinding the freeze-dried spinach exosome-like vesicles, weigh 50 mg of the powder into a 1.5 mL EP tube, add 1 mL of Trizol at low temperature, and shake well; let it stand for 30 min, vortex once every 5 min; centrifuge at 12000 g and 4 °C for 10 min, and take the supernatant; add 200 μL of chloroform, invert violently, shake well and let it stand for 15 min; centrifuge at 12000 g and 4 °C for 15 min, and take the supernatant; add 500 μL of isopropanol, shake gently, and let it stand for 10 min; centrifuge at 12000 g and 4 °C for 10 min. After centrifugation, flocculent precipitates can be seen on the side wall and bottom of the tube, which is RNA; discard the supernatant, add 1 mL of 75% ethanol by volume to wash the precipitate; centrifuge at 8000 g and 4 °C for 10 min, let it stand for 10 min, wait for the 75% ethanol to volatilize, and dry the RNA; add 50 μL of ultrapure water to dissolve the RNA, and measure the RNA concentration with a micro-spectrophotometer.

[0116] Test Example 4

[0117] Test the adaptive surface properties of spinach exosome-like vesicles, and the results are as Figure 4As shown in the figure, where (A) shows the results of the hydrated particle size of spinach exosome-like vesicles under different pH conditions, (B) shows the results of the ζ-potential changes of spinach exosome-like vesicles under different pH conditions, (C) shows the results of the complexation constants of spinach exosome-like vesicles under different incubation times, and (D) shows the results of the surface hydrophilicity of spinach exosome-like vesicles evaluated by the mucin affinity experiment under different pH conditions. Note: Compared with the blank group, *** P < 0.001, **** P < 0.0001.

[0118] (1) The hydrated particle size and ζ-potential changes of spinach exosome-like vesicles (SL-ELNs) extracted in Example 1 under different pH conditions were tested. The test process included: dissolving 0.1 g of SL-ELNs in PBS solutions with pH 5.5, pH 6.0, pH 6.8, and pH 7.4 respectively, and incubating them with shaking at 37 °C for 1, 2, 4, 6, and 8 h; collecting the solutions at each time point for centrifugation, discarding the supernatant, and resuspending the precipitate in PBS with pH 7.4, and then measuring the hydrated particle size and ζ-potential of the vesicles in the solutions respectively.

[0119] From Figure 4 (A), it can be seen that in the neutral environment, the hydrated particle size of SL-ELNs changes little and tends to be stable; in the weakly acidic condition, the hydrated particle size of SL-ELNs shows a decreasing trend with the increase of the action time, which may be related to the detachment of the hydrated layer on the vesicle surface. At the same time, from Figure 4 (B), it can be seen that in the neutral environment, the ζ-potential of SL-ELNs hardly changes; in the weak acid condition, the negative value of the ζ-potential on the surface of SL-ELNs gradually increases with the prolongation of time, and the stronger the acidity, the more obvious the change, indicating that SL-ELNs can overcome the influence of the acidic environment and make the vesicles more stable.

[0120] (2) The surface hydrophobicity of spinach exosome-like vesicles (SL-ELNs) extracted in Example 1 under different incubation times was tested. The Rose Bengal (RB) adsorption method was used to determine the surface hydrophobicity of SL-ELNs at physiological pH. The test process included:

[0121] a. Prepare RB solutions with different concentrations (10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μg / mL), measure the absorption of RB at 543 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and draw a standard curve;

[0122] b. Take 1 mg / mL SL-ELNs and resuspend them in PBS solution with pH 5.5. After incubating with shaking at 37 °C for 1, 2, 4, 6, and 8 h, collect the sample solution, perform centrifugation (12000 g, 20 min), discard the supernatant, and resuspend the precipitate in PBS solution with pH 7.4;

[0123] c. Incubate the above solutions with RB solutions at different concentrations (10 - 100 μg / mL) at 37 °C for 3 h. Centrifuge the mixed solutions (12,000 g, 30 min), take the supernatant, and measure the absorbance of the supernatant at 543 nm using a microplate reader;

[0124] d. Substitute the absorbance values measured for the supernatant samples into the standard curve to obtain the concentration corresponding to free RB;

[0125] e. According to the calculation formula r / a = K*N - K*r, where r is the amount of RB adsorbed per milligram of vesicle particles (μg / mg), a is the equilibrium concentration of RB (μg / mL), and N is the maximum binding amount of RB (mg / mg), calculate the binding constant K (mL / μg).

[0126] From Figure 4 it can be seen from (C) that as the incubation time increases, although the binding constant K of RB to SL-ELNs shows an increasing trend, there is no significant difference, indicating that the surface hydrophobicity of SL-ELNs is relatively stable in a weakly acidic environment.

[0127] (3) Mucin affinity experiment to evaluate the surface hydrophilicity of spinach exosome-like vesicles (SL-ELNs) obtained in Example 1. The test process includes:

[0128] a. Dissolve porcine mucin at a mass-to-volume ratio of 5% in PBS solution with pH = 12, and adjust the pH value to 5.5, 6.0, 6.8, and 7.4 using dilute hydrochloric acid solution for standby;

[0129] b. Add PKH67-labeled SL-ELNs to the above mucin solutions with different pH values, and incubate in a shaker at 37 °C for 1 h. Then, centrifuge the mixed solutions at 12,000 g for 60 min, and take the supernatant;

[0130] c. After treating the supernatant with 1% Triton X-100, measure the concentration of PKH67 in the supernatant using a microplate reader, and record the result as A 1 ;

[0131] d. Do a set of control groups according to the same steps. Add PKH67-labeled SL-ELNs to PBS solutions with pH values of 5.5, 6.0, 6.8, and 7.4, incubate in a shaker at 37 °C for 1 h, centrifuge at 12,000 g for 60 min, and take the supernatant; measure the concentration of PKH67 in the supernatant using a microplate reader, and record the result as A 2 ;

[0132] e. The degree of mucin aggregation is represented by A 1 / A2 The ratio is determined to reflect the surface hydrophilicity of SL-ELNs.

[0133] From Figure 4 As can be seen from (D), at pH 6.8 and 7.4, SL-ELNs showed good resistance to mucin, with a lower degree of mucin aggregation. While at pH 5.5 and pH 6.0, SL-ELNs showed stronger hydrophobicity, resulting in a higher degree of mucin aggregation. This indicates that SL-ELNs can regulate their own hydrophilicity under different pH conditions.

[0134] Test Example 5

[0135] The uptake of spinach exosome-like vesicles by murine microglial cells (BV2) was tested.

[0136] The test procedure included: Spinach exosome-like vesicles (SL-ELNs) (200 ng / mL, 1 mL) obtained by extraction in Example 1 were incubated with PKH67 dye (10 μL) at 4 °C for 10 min, and the excess dye was removed for later use; BV-2 cell suspension with a density of 2×10 5 cells / mL was inoculated into a φ20 mm glass-bottom dish, 1 mL of complete medium was added, and it was cultured overnight in an incubator; 1 mL of the PKH67-labeled SL-ELNs solution was added to the cell culture dish and co-incubated with the cells. Samples were taken at 4 h and 8 h respectively. The cells were washed with DMEM, the cell nuclei were stained with DAPI for 20 min, the staining solution was discarded, the cells were washed with DMEM, and the cells were imaged using a laser confocal imager. The results are shown in Figure 5 .

[0137] From Figure 5 it can be seen that no green fluorescence appeared in the blank control group, while when PKH67-labeled SL-ELNs were co-incubated with the cells for 4 h, green fluorescence appeared in the cytoplasm; as the time increased to 8 h, the green fluorescence in the cells was significantly enhanced, indicating that SL-ELNs were successfully taken up by BV2 cells, and the uptake of SL-ELNs by BV-2 cells was time-dependent.

[0138] Test Example 6

[0139] The effect of the spinach exosome-like vesicles obtained by extraction in Example 1 on the proliferation of microglial cells was tested. The test procedure included:

[0140] (1) BV2 cell suspension with a density of 8×10 4 cells / mL was inoculated in a 96-well culture plate (8000 cells / 100 μL), with 6 replicates in each group. After the cells adhered for 18 h, it was reserved for later use;

[0141] (2) The following groups were set:

[0142] Experimental group: BV2 cells were added with SL-ELNs at different concentrations (1, 5, 10, 20, 50, 80, 100, 200 ng / mL); negative control group: BV2 cells were added with culture medium; blank control group: culture medium;

[0143] (3) After culturing for 24 h, 20 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5 mg / mL) was added to each well, incubated at 37 °C for 4 h, the culture medium in the well was carefully aspirated, 100 μL of dimethyl sulfoxide was added to each well to dissolve the purple crystals, incubated at 37 °C for 15 min, and the optical density (OD) value of each well at 490 nm was measured with an enzyme-linked immunosorbent assay reader; the blank control group was recorded as OD 空白 , the negative control group was recorded as OD 对照 , the experimental group was recorded as OD 样品 ;

[0144] (4) The cell viability under the action of SL-ELNs at different concentrations was calculated according to the following formula:

[0145] Cell viability = (OD 样品 - OD 空白 ) / (OD 对照 - OD 空白 ) * 100%.

[0146] It can be seen from Figure 6 that when the dose of spinach exosome-like vesicles is 1 - 200 ng / mL, the viability of BV2 cells is about 100%, with no significant difference, indicating that spinach exosome-like vesicles have no effect on the growth and proliferation of BV2 cells, that is, the biocompatibility of spinach exosome-like vesicles is good.

[0147] Test Example 7

[0148] The effect of the spinach exosome-like vesicles obtained by extraction in Example 1 on the secretion level of cytokines after LPS-induced inflammation in BV2 cells was tested. The test process included:

[0149] (1) The BV2 cell suspension with a density of 1×10 4 cells / mL was inoculated in a 24-well culture plate (2000 cells / 100 μL), with 3 replicates in each group. After the cells adhered for 18 h, they were reserved for use;

[0150] (2) The following groups were set:

[0151] Experimental group: BV2 cells were cultured in a culture medium containing different concentrations of SL-ELNs (25, 50, 100 ng / mL) for 4 h, then lipopolysaccharide (LPS, 1 μg / mL) was added and the cells were cultured for another 20 h; LPS group: BV2 cells were cultured in a culture medium for 4 h, then lipopolysaccharide (LPS, 1 μg / mL) was added and the cells were cultured for another 20 h;

[0152] (3) Take 100 μL of the cell culture supernatant, and according to the ELISA kit instructions, measure the secretion levels of cytokines TNF-α, IFN-γ, IL-6 and IL-10 in the supernatant. The results are as Figure 7 shown. Among them, (A) is the bar chart of the TNF-α secretion level in lipopolysaccharide-induced nerve cells before and after treatment with spinach exosome-like vesicles, (B) is the bar chart of the IFN-γ secretion level in lipopolysaccharide-induced nerve cells before and after treatment with spinach exosome-like vesicles, (C) is the bar chart of the IL-6 secretion level in lipopolysaccharide-induced nerve cells before and after treatment with spinach exosome-like vesicles, and (D) is the bar chart of the IL-10 secretion level in lipopolysaccharide-induced nerve cells before and after treatment with spinach exosome-like vesicles. Note: Compared with the blank group, #### P < 0.0001; compared with the LPS group, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

[0153] Through the LPS signal transduction pathway, the pro-inflammatory factors TNF-α and IFN-γ are overexpressed in cells. It can be seen that the secretion levels of TNF-α, IFN-γ and IL-6 in the LPS group cells are significantly higher than those in the blank group. Compared with the LPS group, the secretion levels of TNF-α, IFN-γ and IL-6 in the SL-ELNs treatment group cells are significantly decreased. The results show that SL-ELNs can inhibit the excessive secretion of pro-inflammatory factors in BV-2 cells and exhibit anti-inflammatory activity. IL-10 is an anti-inflammatory cytokine produced by cells in response to inflammation. The secretion level of IL-10 is significantly reduced in the LPS-induced BV-2 cell inflammation model, and SL-ELNs at 25 - 100 ng / mL can up-regulate the secretion of IL-10. The results show that spinach exosome-like vesicles can protect nerve cells and relieve nerve inflammation damage by up-regulating the release of anti-inflammatory factors and down-regulating the secretion of pro-inflammatory factors. Figure 7

[0154] Based on the above analysis, spinach exosome-like vesicles can protect nerve cells by up-regulating the release of anti-inflammatory factors and down-regulating the secretion of pro-inflammatory factors, and have the effect of relieving nerve inflammation damage.

[0155] Test Example 8 ​

[0156] According to the method of Test Example 7, the effect of exosome-like vesicles on the secretion level of cytokines after LPS-induced inflammation in BV2 cells was tested. The difference was that the spinach exosome-like vesicles obtained in Example 1 were replaced with the green amaranth exosome-like vesicles obtained in the comparative example, and the secretion amounts of IL-6 and IL-10 in the cytokines in the supernatant were measured. The results are shown in Figure 8 , in which, (A) is the bar chart of the secretion level of IL-6 in lipopolysaccharide-induced nerve cells before and after treatment with green amaranth exosome-like vesicles, and (B) is the bar chart of the secretion level of IL-10 in lipopolysaccharide-induced nerve cells before and after treatment with green amaranth exosome-like vesicles.

[0157] According to Figure 8 the results, compared with the LPS group, although the secretion amount of IL-6 in the cells of the green amaranth exosome-like vesicle treatment group decreased, it was not significant; the change in the secretion amount of IL-10 in the cells of the green amaranth exosome-like vesicle treatment group was also not significant. The results show that the anti-inflammatory effect of the green amaranth exosome-like vesicles obtained by the same extraction method is less than that of the spinach exosome-like vesicles.

[0158] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. An exosome-like vesicle, characterized in that The exosome-like vesicles are extracted and separated from spinach.

2. The exosome-like vesicle according to claim 1, characterized in that The particle size of the exosome-like vesicles is 50-200 nm; Preferably, the exosome-like vesicles have a tray-shaped structure.

3. The exosome-like vesicle according to claim 1 or 2, characterized in that The exosome-like vesicles contain proteins, lipids, and nucleic acids; Preferably, based on 100 mg of the exosome-like vesicles, the protein content is 3-5 mg, the lipid content is 0.5-1 mg, and the nucleic acid content is 15-25 μg.

4. A method for extracting exosome-like vesicles, characterized in that: The following steps are involved: The spinach was extracted and separated.

5. The extraction method according to claim 4, characterized in that The method for extracting exosome-like vesicles comprises the following steps: (1) crushing the spinach to obtain spinach juice, and subjecting the spinach juice to solid-liquid separation to obtain a supernatant; (2) Passing the supernatant through a filter membrane to obtain a filtrate, and subjecting the filtrate to ultrafiltration.

6. The extraction method according to claim 5, characterized in that In step (1), the solid-liquid separation includes a first centrifugal separation and a second centrifugal separation; the conditions of the first centrifugal separation include at least: a temperature of 2-5°C, a centrifugal force of 2000-3000g, and a time of 20-30min; the conditions of the second centrifugal separation include at least: a temperature of 2-5°C, a centrifugal force of 8000-10000g, and a time of 50-70min; Preferably, before the centrifugal separation, the spinach juice and phosphate buffer are mixed; Preferably, the mass ratio of the spinach juice to the phosphate buffer is 1:1.5-2.

7. The extraction method according to claim 5, characterized in that In step (2), the filter membrane has a filter diameter of 1-2.2 μm; Preferably, the ultrafiltration process comprises: subjecting the filtrate to a first ultrafiltration through an ultrafiltration cup having a filter membrane with a molecular weight cutoff of 90-100 kDa and collecting the ultrafiltrate, and subjecting the ultrafiltrate to a second ultrafiltration through an ultrafiltration cup having a filter membrane with a molecular weight cutoff of 2-4 kDa.

8. Use of the exosome-like vesicles described in any one of claims 1 to 3 and / or the exosome-like vesicles obtained by the extraction method described in any one of claims 4 to 7 in the preparation of a product for alleviating neuroinflammatory damage.

9. The use according to claim 8, characterized in that: The product for alleviating neuroinflammatory damage is a product that downregulates the secretion of pro-inflammatory factors and / or upregulates the release of anti-inflammatory factors; Preferably, the pro-inflammatory factor is selected from at least one of TNF-α, IFN-γ and IL-6, and the anti-inflammatory factor is IL-10.

10. A composition for alleviating neuroinflammatory damage, characterized in that: The composition contains the exosome-like vesicles according to any one of claims 1 to 3 and / or the exosome-like vesicles obtained by the extraction method according to any one of claims 4 to 7.

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