Black bean exosome and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a black bean exosome and application thereof. The black bean exosome is extracted according to the following steps: soaking black beans in water, performing juicing treatment by taking PBS as a solvent, centrifuging the obtained juice, and collecting supernate; mixing and incubating the supernate and polyethylene glycol, centrifuging, and collecting the precipitate, thereby obtaining the product. Tests prove that the extracted exosome has anti-oxidation and anti-tyrosinase activity, and can be used for preparing functional cosmetics or anti-oxidation drugs.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biotechnology, and particularly relates to a black bean exosome and application thereof. BACKGROUND

[0002] Extracellular vesicles (EVs) are a general term for various types of vesicles secreted by cells, have a membrane structure, and can act as information carriers to participate in cell-to-cell communication. The International Society for Extracellular Vesicles (ISEV) classifies the extracellular vesicles secreted by cells into three types, of which the largest with a diameter of 1000-5000 nm is called apoptotic body, which is a membrane vesicle released from cells during late-stage programmed cell death. The other two types of cell vesicles are microvesicles with a diameter of 100-1000 nm directly budding from the plasma membrane and exosome-like nanoparticles (ELNs) (30-150 nm in diameter) released from cells after fusion of multivesicles with the plasma membrane. Exosomes are nanovesicles with a phospholipid bilayer structure, containing various biological components such as proteins, lipids and nucleic acids.

[0003] In 1946, CHARGAFF et al. discovered small particles in platelets, which opened the door to the study of exosomes. In 1987, Johnstone et al. named this type of vesicular structure as exosome. With the continuous study of animal cell exosomes, similar nanovesicles to exosomes were also found in plant cells, and plant-derived exosome-like nanoparticles (PELNs) are usually referred to as plant-derived exosome-like nanoparticles. PELNs have similar structure and biological activity to mammalian-derived exosomes, have the advantages of wide source, low immunogenicity and easy acquisition, and about 80 types of PELNs have been reported, of which about 58% are derived from edible plants and 28% are derived from medicinal plants. Studies have shown that PELNs have pharmacological effects such as anti-inflammatory, anti-tumor and immune regulation, and can be used for the treatment of tumors, inflammatory diseases and bacterial infections, antioxidant, antiviral, and as a transport carrier for drug targeted delivery. Plant exosomes can also be applied in the field of cosmetics, and their research has broad prospects.

[0004] Black beans were first recorded in Shennong's Herbal Classic, and all dynasties believed that only the black seed coat could be used as medicine. In the 2020 edition of Chinese Pharmacopoeia, black beans are the legume Glycine max (L.) Merr. ( Glycine maxThe dried mature seeds of Glycine max (L.) merr) are 6-12 mm long and 5-9 mm in diameter, and are black or gray-black in color. The seeds are sweet and flat in nature, and are associated with the spleen and kidney channels. They have the functions of nourishing essence and brightening eyesight, nourishing blood and dispelling wind, promoting water, and detoxifying. They are used to treat dizziness and blurred vision, yin deficiency and thirst, arthralgia and spasm, physical weakness and excessive sweating, numbness of the hands and feet, edema and oliguria, and kidney deficiency and lumbago. In addition, research has found that black beans contain rich anthocyanins, which have antioxidant and anti-inflammatory effects, and can effectively scavenge free radicals in the body. They are widely used in the fields of medicine, food, cosmetics, etc. However, the exosomes contained in black beans still need to be developed and researched. SUMMARY

[0005] The present application provides a kind of black soybean exosome and its application.

[0006] The specific technical scheme provided by the present application is as follows: In the first aspect of the present application, a black soybean exosome is provided, which is extracted according to the following steps: After the black soybeans are soaked in water, juice is extracted with PBS buffer as the solvent. The obtained juice is sequentially subjected to centrifugal treatment at 1500xg for 15-25 min, 3000xg for 25-35 min, and 10000xg for 55-65 min. The final supernatant is collected. After the supernatant is mixed with polyethylene glycol and incubated, it is centrifuged at 8000xg-10000xg for 30 min. The precipitate is collected, which is the black soybean exosome.

[0007] The exosome precipitate obtained needs to be resuspended with PBS for subsequent storage, detection or experimental use. In addition, the osmotic pressure of PBS is close to the in vivo environment, which can avoid the rupture or deformation of exosomes due to changes in osmotic pressure. Polyethylene glycol (PEG) can change the solubility and dispersibility of exosomes, promote the aggregation of exosomes, and increase the particle size of aggregated exosomes. The exosomes can be separated from the solution by low-speed centrifugation, and preliminary separation from other small molecular impurities can be achieved.

[0008] As a preferred embodiment of the present application, the mass ratio of black soybeans to water is 0.5-1:1.

[0009] As a preferred embodiment of the present application, the dosage ratio of PBS buffer to black soybeans is 1 mL:0.5-1 g.

[0010] As a preferred embodiment of the present application, the centrifugal conditions for collecting the supernatant are as follows: 1500xg for 20 min, 3000xg for 30 min, and 10000xg for 60 min at 4°C.

[0011] As a preferred embodiment of the present application, the mass concentration of the polyethylene glycol solution is 12% to 24%, and the polyethylene glycol solution is mixed with the supernatant in equal volume.

[0012] As a preferred embodiment of the present application, the incubation is overnight incubation at 4℃.

[0013] As a preferred embodiment of the present application, the centrifugal condition for collecting the exosomes is 10000xg centrifugation for 30 min.

[0014] As a preferred embodiment of the present application, the supernatant is filtered by using a filter membrane with a pore size of 0.45 μm, and the obtained filtrate is mixed with the polyethylene glycol.

[0015] In the second aspect of the present application, the use of the black bean exosomes in the preparation of antioxidant products is provided.

[0016] In the third aspect of the present application, the use of the black bean exosomes in the preparation of whitening or anti-pigmentation products is provided.

[0017] Compared with the prior art, the present application has the following beneficial effects: In the present application, plant exosomes are extracted by using polymer precipitation method combined with differential centrifugation method. The morphology and size of the extracted exosomes are characterized by transmission electron microscopy and nanoparticle size and potential analyzer, and the antioxidant and anti-tyrosinase activities are evaluated by combining DPPH free radical scavenging experiment and tyrosinase inhibition experiment. The results show that: by adjusting the extraction process, the plant source exosomes are successfully separated from black beans, and the morphology, particle size and biological activity are systematically characterized. The present application establishes a green and efficient extraction process of plant source exosomes, and confirms that they have certain biological activity, which provides an experimental basis for the application of exosomes in the field of functional cosmetics and medicine. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Figure 1 is a schematic diagram of black bean exosome extraction in Example 1; A, original solution; B, 1500xg centrifugation; C, 3000xg; D, supernatant 10000xg; E, incubation liquid 10000xg; Figure 2 Figure 2 is a schematic diagram of black bean exosome extraction in Example 2; A, original solution; B, 1500xg; C, 3000xg; D, supernatant 10000xg; E, incubation liquid 10000xg; Figure 3 Figure 3 is a schematic diagram of black bean exosome extraction in Example 3; A, original solution; B, 1500xg; C, 3000xg; D, supernatant 10000xg; E, incubation liquid 10000xg; Figure 4is a transmission electron microscope image of black bean exosomes in Example 1; the arrow in the figure refers to the exosome; Figure 5 is a particle size distribution graph of black bean exosomes in Example 1; Figure 6 is a potential graph of black bean exosomes in Example 1; Figure 7 is a transmission electron microscope image of black bean exosomes in Example 2; the arrow in the figure refers to the exosome; Figure 8 is a particle size distribution graph of black bean exosomes in Example 2; Figure 9 is a potential graph of black bean exosomes in Example 2; Figure 10 is a transmission electron microscope image of black bean exosomes in Example 3; the arrow in the figure refers to the exosome; Figure 11 is a particle size distribution graph of black bean exosomes in Example 3; Figure 12 is a potential graph of black bean exosomes in Example 3; Figure 13 is a DPPH free radical scavenging curve of black bean exosomes; Figure 14 is a tyrosinase inhibition curve of black bean exosomes. DETAILED DESCRIPTION

[0019] In the following description, specific details of the application are further set forth for the purpose of fully understanding the application. The description used in the specification is only for the purpose of explaining the advantages and features of the application, and is not intended to limit the application.

[0020] Unless otherwise defined, all professional and scientific terms used in the application are the same as understood by those skilled in the art. Unless otherwise specified, the drugs or reagents used in the application are used according to the product instructions or using conventional methods in the art. The technical solutions of the application are further described according to the drawings and specific embodiments in the specification.

[0021] The materials and reagents involved in the embodiments of the application are as follows: Black beans (purchased from Shaanxi) are divided into large grains (100-grain weight 18 g or more), medium grains (100-grain weight 12-17.9 g), and small grains (100-grain weight 11.9 g or less) according to the grain weight in the Chinese Agricultural Encyclopedia, which classifies Chinese soybean varieties according to the grain weight. The sample was weighed to be 12.34 g, which is a medium grain.

[0022] PBS phosphate buffer (dry powder, batch number BL601A, Hefei Baishabiological Technology Co., Ltd.), polyethylene glycol 6000 (analytical pure, Tianjin BinHai Chemical Factory), phosphotungstic acid (AR, Shanghai Honggu Biological MedicalTechnology Co., Ltd.), 1,1-diphenyl-2-trinitrobenzene hydrazine (abbreviation DPPH, batch number D0909, Chengdu Aikeda Chemical Reagent Co., Ltd.), ascorbic acid (analytical pure, Tianjin Tianli Chemical Reagent Co., Ltd.), tyrosinase (9002-10-2, Baoji Chenlight Biological Technology Co., Ltd.), analytical methanol (AR purity ≥ 99.5%, Tianjin Damao Chemical Reagent Factory), L-tyrosine (60-18-4, Saen Chemical Technology (Shanghai) Co., Ltd.), arbutin (Guangzhou Xinyan Cosmetics Co., Ltd.).

[0023] Example 1 A black bean exosome was extracted according to the following steps: Take 200g black beans and soak overnight with water 1:1. Add 200ml PBS and juice in a juicer, filter to get juice. At 4℃, centrifuge 1500xg for 20min, 3000xg for 30min, and 10000xg for 60min once to get supernatant. Take PEG6000 dissolved in ultrapure water to prepare a 16% solution. Mix the filtered PEG6000 solution with the previous supernatant in equal volume, incubate at 4℃ overnight, then centrifuge at 10000xg for 30min to get the precipitate. Resuspend the precipitate with appropriate amount of pre-cooled PBS to get sample one, store in-80℃ for standby, see Figure 1 .

[0024] Example 2 A black bean exosome was extracted according to the following steps: Take 50g black beans and soak overnight with water 0.5:1. Add 100ml PBS and juice in a juicer, filter to get juice. At 4℃, centrifuge 1500xg for 15min, 3000xg for 25min, and 10000xg for 55min once to get supernatant. Take PEG6000 dissolved in ultrapure water to prepare a 24% solution. Mix the filtered PEG6000 solution with the previous supernatant in equal volume, incubate at 4℃ overnight, then centrifuge at 10000xg for 30min to get the precipitate. Resuspend the precipitate with appropriate amount of pre-cooled PBS to get sample two, store in-80℃ for standby, see Figure 2 .

[0025] Example 3 A black bean exosome was extracted according to the following steps: Take 100 g black beans and soak overnight with water 1:1. Add 100 ml PBS and juice in a juicer, filter to get juice. At 4℃, centrifuge 1500 x g for 25 min, 3000 x g for 35 min, and 10000 x g for 65 min to get supernatant, filter with 0.45 μm filter membrane. Take PEG6000 to dissolve in ultrapure water to prepare a 12% solution, mix the PEG6000 solution with the supernatant in equal volume, incubate at 4℃ overnight, centrifuge at 10000 x g for 30 min to get the precipitate, resuspend the precipitate with appropriate amount of pre-cooled PBS, filter with 0.45 μm filter membrane, get sample three, store in -80℃, see Figure 3 .

[0026] Experimental Example 1 Exosome characterization 1. Particle size potential analysis Take the exosome precipitate obtained by centrifugation and dilute with appropriate amount of PBS, then ultrasonicate the diluted sample to fully dissolve and disperse. Filter through a 0.45 μm filter membrane. Place the sample cell containing the sample in the measurement area of the instrument, allow the sample to equilibrate in the measurement environment for a period of time to allow the particles to reach a stable dispersion state, then use the Malvern nanoparticle size and zeta potential analyzer to perform particle size and potential analysis.

[0027] 2. Transmission electron microscope observation of exosome morphology Phosphotungstic acid staining solution preparation: take appropriate amount of phosphotungstic acid solid and dissolve in distilled water to prepare a 2% solution by mass fraction for standby.

[0028] After centrifugation to extract the exosome precipitate, resuspend the exosomes with pre-cooled PBS, filter through a 0.45 μm filter membrane for standby; place clean filter paper in a petri dish, use forceps to take out the copper mesh and place it on the filter paper (at this time the copper mesh is not distinguished as front and back, both sides can be upwards); take 10 μL of exosome suspension and drop it onto the copper mesh, react for 3 min, wash with distilled water for 2 times, 30 s each time (at this time the copper mesh starts to distinguish front and back, the sample drop side is the front); let stand for 10 min to dry (after drying, if there is still a small amount of solution, the drying time can be appropriately extended; unless there is a lot of solution, try not to use a small filter paper strip to absorb the excess solution); drop 5 μL of phosphotungstic acid negative staining reagent on the filter paper, place the front copper mesh downwards on the negative staining reagent, negative stain for 3 min; take out the copper mesh and place it in the petri dish containing the filter paper, dry in a 65℃ oven or air dry; perform measurement.

[0029] 0. Results (1) Sample one characterization results The morphology of black bean exosomes was observed by transmission electron microscope, as shown in Figure 4 The black bean exosomes showed a round cup-shaped double-membrane structure.

[0030] The Malvern Zetasizer nano particle size and potential analyzer was used to analyze the particle size distribution and potential of black bean exosomes. The particle size distribution is shown in Figure 5 The results showed that the particle size range was 50-300 nm, and the average particle size was 126.7 nm. The average potential was -8.1 mV, as shown in Figure 6 .

[0031] (2) Characterization results of sample two The morphology of black bean exosomes was observed by transmission electron microscopy, as shown in Figure 7 The black bean exosomes showed a circular cup-shaped double-membrane structure.

[0032] The Malvern Zetasizer nano particle size and potential analyzer was used to analyze the particle size distribution and potential of black bean exosomes. The particle size distribution is shown in Figure 8 The results showed that the particle size range was 100-300 nm, and the average particle size was 238.7 nm. The average potential was -6.5 mV, as shown in Figure 9 .

[0033] (3) Characterization results of sample three The morphology of black bean exosomes was observed by transmission electron microscopy, as shown in Figure 10 The black bean exosomes showed a circular or oval cup-shaped double-membrane structure.

[0034] The Malvern Zetasizer nano particle size and potential analyzer was used to analyze the particle size distribution and potential of black bean exosomes. The particle size distribution is shown in Figure 11 The results showed that the particle size range was 30-300 nm, and the average particle size was 86.01 nm. The average potential was -11.1 mV, as shown in Figure 12 .

[0035] In summary, sample three had the smallest average particle size, the widest distribution range, and the most efficient vesicle separation or the least aggregation. In addition, the potential of sample three indicated that the surface charge stability of exosomes was higher, which may be due to the fact that filtration reduced the interference of impurities. Therefore, the black bean exosomes prepared by the extraction scheme provided in Example 3 were used for subsequent experimental research.

[0036] Experimental Example 2 In vitro activity determination of black bean-derived exosomes 1. Antioxidant activity determination Sample solution preparation: Take an appropriate amount of the above-mentioned extracted resuspension, centrifuge to obtain a precipitate, resuspend the precipitate with anhydrous methanol in a 10 ml capacity bottle, add anhydrous methanol to constant volume, ultrasonic the sample to make it uniformly dispersed, and obtain a sample solution (the volume ratio of precipitate to solution is 1:10). Further dilution with anhydrous methanol obtains a series of sample solutions with concentrations (25%, 50%, 75%, 100%) for use.

[0037] Preparation of control solution: accurately weigh 5 mg of ascorbic acid into a 50 ml capacity bottle, add anhydrous methanol to constant volume to obtain a control solution. Further dilution with anhydrous methanol obtains a series of control solutions with concentrations (25%, 50%, 75%, 100%) for use.

[0038] Preparation of DPPH solution: accurately weigh 5 mg of DPPH solid under light protection, dissolve in a 50 mL capacity bottle with anhydrous methanol to obtain a DPPH solution with a concentration of 0.1 g / L, store in the dark, and reserve for use.

[0039] Take 20 μL of the sample to be tested and mix with 180 μL of 0.1 g / L DPPH solution (A1); take 20 μL of anhydrous methanol and mix with 180 μL of 0.1 g / L DPPH solution (A2); take 180 μL of anhydrous methanol and mix with 20 μL of the sample to be tested (A3). After 30 min of reaction in the dark, measure the absorbance values of A1, A2, and A3 at 517 nm, repeat the measurement 3 times for each sample and take the average value. The calculation formula of clearance rate is as follows:

[0040] In the formula, A1 is the absorbance of the sample solution, A2 is the absorbance value of the sample without adding the sample solution and replacing it with anhydrous methanol, and A3 is the absorbance value of the background of the sample to be tested. According to the formula, the DPPH free radical clearance rate of the sample is calculated.

[0041] 2. In vitro tyrosinase inhibitory activity study Weigh 2 mg of tyrosinase into a 10 mL capacity bottle and add PBS buffer to constant volume to obtain a 0.2 g / L tyrosinase solution. Weigh 14 mg of L-tyrosine into a 10 mL capacity bottle and add PBS buffer to constant volume to obtain a 1.4 g / L tyrosine substrate solution.

[0042] Take an appropriate amount of the extracted resuspension, place it in a 10 mL capacity bottle, add PBS to constant volume, ultrasonic the sample to make it uniformly dispersed, and obtain a sample solution (the volume ratio of precipitate to solution is 1:10). Further dilution with PBS obtains a series of sample solutions with concentrations (25%, 50%, 75%, 100%) for use.

[0043] Accurately weigh 100 mg of arbutin and place it in a 50 ml volumetric flask. Add PBS to make up the control solution. Further dilute with PBS to obtain a series of concentrations (25%, 50%, 75%, 100%) of the control solution for use.

[0044] In a 96-well plate, add the following: 50 μL of sample solution at different dilution concentrations, 80 μL of PBS buffer, and 50 μL of tyrosinase solution. Set three parallel values for each. Incubate at 37°C for 10 min, then add 20 μL of substrate to each well, incubate for 30 min, and finally measure the absorbance at 475 nm using an enzyme marker. Repeat the measurement three times for each sample and record the absorbance. The formula for calculating the tyrosinase inhibition rate is as follows:

[0045] In the formula, A is the blank control group (PBS 130 μL + enzyme 50 μL + substrate 20 μL); B is the blank background group (PBS 180 μL + substrate 20 μL); C is the sample group at different concentrations (PBS 80 μL + enzyme 50 μL + sample solution 50 μL + substrate 20 μL); and D is the experimental background group (PBS 130 μL + sample solution 50 μL + substrate 20 μL). Calculate the tyrosinase inhibition rate of the sample according to the formula.

[0046] 3. Data processing The measured values are processed and plotted using Origin Pro software.

[0047] 4. Results and analysis The results are shown in Figure 13 and Figure 14 As can be seen from Figures 13-14 , the clearance rate and inhibition rate gradually increase with increasing concentration, indicating that the black bean-derived exosomes have certain effects on inhibiting tyrosinase and scavenging DPPH free radicals.

[0048] In summary, the black bean exosomes extracted by the present application have a circular or oval cup-shaped double-membrane structure, which meets the typical morphological characteristics of exosomes. Nanoparticle size analysis shows that the optimized extraction process significantly reduces exosome aggregation and improves separation efficiency.

[0049] The clearance rate of DPPH free radicals by black bean exosomes increases with increasing concentration, confirming its antioxidant capacity. The inhibition rate of tyrosinase by exosomes is also concentration-dependent, indicating its potential application value in whitening or anti-pigmentation. Black bean-derived exosomes have broad application potential in functional cosmetics such as antioxidant and whitening products, and in the medical field such as anti-inflammatory and targeted drug delivery carriers due to their low immunogenicity, high biological activity, and green extraction process.

[0050] The above detailed description of the embodiments of the present application is only preferred embodiments of the present application, and should not be considered as limiting the scope of the present application. Any equivalent changes and improvements made according to the scope of the present application should still belong to the patent coverage of the present application.

Claims

1. A black bean exosome, characterized in that, It is extracted according to the following steps: After the black beans are soaked with water, juice is extracted with PBS buffer as the solvent, and the obtained juice is sequentially subjected to centrifugal treatment in the following manner: 1500xg centrifugation for 15-25min, 3000xg centrifugation for 25-35min, and 10000xg centrifugation for 55-65min, and the final supernatant is collected; The supernatant is mixed with polyethylene glycol and incubated, and then centrifuged at 8000xg-10000xg for 30min, and the precipitate is collected, thereby obtaining the black bean exosomes.

2. The black bean exosome of claim 1, wherein, The mass ratio of the black beans to water is 0.5-1:

1.

3. The black bean exosome of claim 1, wherein, The dosage ratio of PBS buffer to black beans is 1mL:0.5g-1g.

4. The black bean exosome of claim 1, wherein, The centrifugal conditions for collecting the supernatant are as follows: sequentially subjected to 1500xg centrifugation for 20min, 3000xg centrifugation for 30min, and 10000xg centrifugation for 60min at 4°C.

5. The black bean exosome of claim 1, wherein, The mass concentration of the polyethylene glycol solution is 12%-24%, and the polyethylene glycol solution is mixed with the supernatant in equal volume.

6. The black bean exosome of claim 1, wherein, The incubation is overnight incubation at 3-5°C.

7. The black bean exosome of claim 1, wherein, The centrifugal conditions for collecting the exosomes are 10000xg centrifugation for 30min.

8. The black bean exosome of claim 1, wherein, The supernatant is filtered with a 0.45μm filter membrane, and the obtained filtrate is mixed with polyethylene glycol.

9. Use of the black bean exosomes of claim 1 in the preparation of an antioxidant product.

10. Use of the black bean exosomes of claim 1 in the preparation of a whitening or anti-pigmentation product.

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