Solanum nigrum-derived extracellular vesicles as well as extraction and purification method and application thereof

Abstract

The invention discloses a solanum nigrum-derived extracellular vesicle as well as an extraction and purification method and application thereof, and belongs to the technical field of biomedicine, the method comprises the following steps: mixing cleaned solanum nigrum leaves and fruits with PBS according to a certain ratio, juicing, centrifuging for multiple times, gradually filtering, concentrating twice, and finally purifying to obtain the solanum nigrum-derived extracellular vesicle. The solanum nigrum-derived extracellular vesicles prepared by the method can significantly improve the purity and biological activity of the extracellular vesicles, effectively inhibit the proliferation of tumor cells, especially HCT15 and RKO colon cancer cells in vitro, and ensure the low toxicity and wide application potential in the field of biological medicines.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a Solanum nigrum-derived extracellular vesicle and an extraction and purification method and application thereof. Background Art

[0002] For the treatment of malignant tumors, modern medicine often uses a variety of methods, including surgical resection, radiotherapy, chemotherapy, targeted therapy and immunotherapy. Although these treatment methods can effectively inhibit tumor growth, there are still problems such as the treatment effect varies from individual to individual, drug resistance develops, and side effects are obvious. The research of new anti-tumor drugs is an important part of the field of tumor research, and there is a long way to go to find safe and effective anti-tumor drugs. Therefore, the development of new therapies to achieve precise treatment, improve efficacy and reduce side effects is an important topic in the field of malignant tumor treatment.

[0003] Plant extracellular vesicles (PDEVs) are nanoscale membrane vesicles secreted by plant cells. They are rich in bioactive substances such as proteins, lipids, and RNA. They participate in various physiological and pathological processes by releasing and absorbing these active substances, affecting the occurrence and development of multiple diseases. In addition, plant extracellular vesicles are also rich in a variety of bioactive ingredients such as alkaloids, phenolic compounds, flavonoids, saponins, and other secondary metabolites. These ingredients are closely related to the medicinal value of plants, such as anti-tumor effects. In recent years, PDEVs have shown great application potential in anti-tumor therapy. At present, PDEVs have been successfully isolated and extracted from a variety of plants, and their main components have been identified. Further studies have found that the active ingredients in PDEVs can inhibit the proliferation, migration, and invasion of tumor cells through a variety of mechanisms, such as inducing cell apoptosis, inhibiting angiogenesis, and regulating immune responses. Although the research on PDEVs is still in its infancy, its superior biocompatibility, low toxicity, and potential targeting make it a new hope for the development of anti-tumor drugs.

[0004] Solanum nigrum is an annual herbaceous plant of the genus Solanum in the Solanaceae family. Its fruit tastes sweet and sour, is rich in vitamins and minerals, and can be eaten directly. The whole plant of Solanum nigrum is used as medicine, which has the effects of clearing away heat and detoxifying, promoting blood circulation and reducing swelling. Modern research shows that the whole plant of Solanum nigrum contains a variety of alkaloids such as solanine, solanine, solanine and alkaloid glycosides, and has anti-tumor, antibacterial, lipid-lowering and vasodilation, antihypertensive, antitussive and liver and kidney protection effects, especially in anti-tumor. Although plant extracellular vesicles (PDEVs) have shown potential in anti-tumor therapy, existing technologies still have shortcomings in many aspects. First, the separation and extraction process of PDEVs has not been standardized, resulting in inconsistent extraction efficiency, quality and concentration among different laboratories. In addition, the bioactive components of PDEVs are complex and diverse, and the current research on their biological functions is still very limited, and there is a lack of in-depth research on anti-tumor mechanisms. In addition, key pharmacokinetic parameters such as biocompatibility and metabolic pathways of PDEVs need to be further verified. In terms of application, the large-scale production and purification technology of PDEVs still need to be optimized to meet the needs of future clinical applications.

[0005] In view of this, the inventor conducted in-depth research on this demand, and thus the present case was produced. Summary of the invention

[0006] In order to overcome the problems in the prior art that the extraction and purification process of extracellular vesicles derived from Solanum nigrum has not yet been standardized, and there is a lack of research on its biological functions and in-depth research on its anti-tumor mechanism, the present invention provides an extracellular vesicle derived from Solanum nigrum and an extraction and purification method and application thereof, which can effectively inhibit the growth and proliferation of tumor cell lines.

[0007] To achieve the above objectives, the experimental plan is as follows:

[0008] A method for extracting and purifying extracellular vesicles derived from Solanum nigrum, comprising the following steps:

[0009] Step (1): Pick fresh Solanum nigrum leaves and fruits, wash them with running water, and then drain and dry them;

[0010] Step (2): mixing the dried Solanum nigrum leaves and fruits in step (1) with PBS, squeezing the juice, and filtering and collecting the juice;

[0011] Step (3): placing the juice of step (2) into a centrifuge and centrifuging at a speed of 1000-1500 g at 3-6° C. for 10-15 min, taking the supernatant, centrifuging at a speed of 2000-3000 g at 3-6° C. for 10-15 min, taking the supernatant, centrifuging at a speed of 8000-10000 g at 3-6° C. for 10-15 min, and taking the supernatant;

[0012] Step (4): passing the supernatant of step (3) through 70 μm, 40 μm, 0.45 μm and 0.22 μm filters in sequence to obtain a filtrate;

[0013] Step (5): The filtrate of step (4) is concentrated twice by using an exosome concentration kit, i.e., firstly adding the mixture according to the ratio in the instruction manual, standing at 4°C overnight, then performing differential centrifugation, discarding the supernatant, and then adding PBS to resuspend the precipitate to obtain a primary concentrate, and then repeating the operation to obtain a secondary concentrate;

[0014] Step (6): The concentrated solution of step (5) is purified by an exosome purification kit, that is, column equilibration and PBS pretreatment are first performed, then the mixture is added according to the ratio in the instruction manual, and finally the effluent is collected to obtain extracellular vesicles derived from Solanum nigrum.

[0015] Preferably, in step (2), the mass volume ratio of the Solanum nigrum leaves and fruits uniformly mixed with PBS is 1 g: (1.5-2) mL.

[0016] Preferably, the fresh filtrate in step (4) can be stored at -80°C for 3 to 6 months if no further operation is performed.

[0017] Preferably, the step (5) can be specifically concentrated according to the instructions of the Enze Kangtai cell supernatant high-fold concentration kit (ES8500), taking 35 mL of the filtrate, 10 mL of solution A in the kit, and 5 mL of solution B in a 50 mL centrifuge tube, mixing and standing at 4°C overnight, centrifuging at a speed of 10000-12000g and a temperature of 3-6°C for 50-60min, discarding the supernatant, and adding 1 mL of PBS to resuspend the precipitate to obtain a concentrated solution.

[0018] Preferably, the step (6) can be specifically purified according to the instructions of the Enze Kangtai Exosome Purification Kit (ES914), column equilibration and PBS pretreatment are performed before purification, and then 1 mL of concentrate is added. After all the samples enter the bottom of the sieve plate and no liquid flows out, the eluent PBS is added, 500 μL is added each time, and after the previous 500 μL eluent has completely entered the exclusion column, the next 500 μL eluent is added, and the effluent is collected in a collection tube. Starting from the first drop, each 500 μL is recorded as 1 fraction, and the first 3 fractions are not collected. The subsequent fractions are collected to obtain Solanum nigrum-derived extracellular vesicles.

[0019] The present invention also provides a Solanum nigrum-derived extracellular vesicle, which is prepared by adopting the above-mentioned method for extracting and purifying the Solanum nigrum-derived extracellular vesicle.

[0020] The present invention also provides the use of the Solanum nigrum-derived extracellular vesicles in the preparation of drugs for preventing and treating tumors.

[0021] Preferably, the Solanum nigrum-derived extracellular vesicles can inhibit the growth and proliferation of one or more tumor cell lines in vitro, especially inhibit the growth and proliferation of colon cancer cell lines.

[0022] Preferably, the Solanum nigrum-derived extracellular vesicles can inhibit the proliferation of colon cancer cell lines HCT15 and RKO cells.

[0023] Preferably, the Solanum nigrum-derived extracellular vesicles contain at least one carrier acceptable in pharmaceuticals, food science or nutraceuticals.

[0024] Preferably, the dosage form of the anti-tumor drug is any one of injection, oral solution, capsule, tablet, spray or ointment.

[0025] The beneficial effects produced by the technical solution of the present invention are as follows:

[0026] The present invention provides a method for extracting and purifying extracellular vesicles derived from Solanum nigrum, which can significantly improve the purity and biological activity of the extracellular vesicles. The Solanum nigrum derived extracellular vesicles prepared by the method can effectively inhibit the proliferation of tumor cells, especially HCT15 and RKO colon cancer cells in vitro, while ensuring its low toxicity and wide application potential in the field of biomedicine.

[0027] In addition, unlike the single effects of compound active ingredients contained in the drug itself, such as alkaloids, volatile oils or saponins, these extracellular vesicles derived from Solanum nigrum contain rich plant metabolites of bioactive substances such as proteins, lipids, RNA, etc., which can be used as a delivery system for active ingredients. In application, they can be combined to form a variety of acceptable pharmaceutical carriers to make different forms of anti-tumor drug preparations, such as injections, oral solutions, capsules, etc., which not only improve the medicinal effects of plants, but also enhance the breadth and convenience of their clinical applications. Therefore, the present invention has important potential application value in the development of new plant-derived anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 This is a transmission electron microscopy image of the Solanum nigrum extracellular vesicles prepared in Example 1;

[0030] Figure 2 This is a particle size diagram of the Solanum nigrum extracellular vesicles prepared in Example 1;

[0031] Figure 3 This is a graph showing the BCA protein concentration detection of Solanum nigrum extracellular vesicles prepared in Example 1;

[0032] Figure 4 This is a microscopic picture of Solanum nigrum extracellular vesicles prepared in Example 1 intervening colon cancer cells for 48 hours;

[0033] Figure 5 This is the CCK8 result after Solanum nigrum extracellular vesicles prepared in Example 1 intervened in colon cancer cells for 48 hours. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Therefore, the following detailed description of the embodiments provided by the present invention is not intended to limit the scope of the claimed invention, but merely represents the selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] This embodiment fully analyzes the existing problems of extracellular vesicles derived from Solanum nigrum in the prior art, such as the extraction and purification process has not been standardized, and there is a lack of research on its biological functions and in-depth research on its anti-tumor mechanism. The present invention provides an extracellular vesicle derived from Solanum nigrum and an extraction and purification method and application thereof, which can effectively inhibit the growth and proliferation of tumor cell lines.

[0036] The present invention is further described in detail below in conjunction with specific embodiments:

[0037] Example 1

[0038] This embodiment 1 provides a method for extracting and purifying extracellular vesicles derived from Solanum nigrum, comprising the following steps:

[0039] Step (1): Pick fresh Solanum nigrum leaves and fruits, wash them with running water, drain the water and slightly dry them;

[0040] Step (2): 1 kg of Solanum nigrum leaves and fruits dried in step (1) were mixed evenly with 1500 mL of PBS, added to a juicer in five batches for juicing for 5 minutes, and the juice was filtered and collected;

[0041] Step (3): Place the juice obtained in step (2) in a centrifuge and centrifuge at a speed of 1000 g at 4°C for 10 min, take the supernatant, centrifuge at a speed of 3000 g at 4°C for 10 min, take the supernatant, centrifuge at a speed of 10000 g at 4°C for 10 min, and take the supernatant;

[0042] Step (4): passing the supernatant of step (3) through 70 μm, 40 μm, 0.45 μm and 0.22 μm filters in sequence to obtain a filtrate;

[0043] Step (5): The filtrate of step (4) was concentrated twice according to the instructions of Enze Kangtai Cell Supernatant High-fold Concentration Kit (ES8500), 35 mL of the filtrate, 10 mL of solution A in the kit, and 5 mL of solution B were placed in a 50 mL centrifuge tube, mixed and allowed to stand at 4°C overnight, centrifuged at a speed of 10000g at 4°C for 60 min, the supernatant was discarded, 1 mL of PBS was added to resuspend the precipitate, and the primary concentrate was obtained. The above operation was repeated to obtain a secondary concentrate;

[0044] Step (6): Purify the concentrate of step (5) according to the instructions of Enze Kangtai Exosome Purification Kit (ES914). Before the start of purification, perform column equilibration and PBS pretreatment, then add 1 mL of concentrate, and after the concentrate has completely entered the bottom of the sieve plate and no liquid has flowed out, start adding eluent PBS, add 500 μL each time, and after the previous 500 μL eluent has completely entered the exclusion column, add the next 500 μL eluent, collect the effluent in the collection tube, and start counting from the first drop. Each 500 μL is recorded as 1 fraction. The first 3 fractions are not collected, and the subsequent 4 to 8 fractions are collected to obtain extracellular vesicles derived from Solanum nigrum.

[0045] Example 2

[0046] This embodiment 2 provides a method for extracting and purifying extracellular vesicles derived from Solanum nigrum, comprising the following steps:

[0047] Step (1): Pick fresh Solanum nigrum leaves and fruits, wash them with running water, drain the water and slightly dry them;

[0048] Step (2): 1 kg of Solanum nigrum leaves and fruits dried in step (1) were mixed evenly with 2000 mL of PBS, added to a juicer in six batches for juicing for 5 minutes, and the juice was filtered and collected;

[0049] Step (3): Place the juice obtained in step (2) into a centrifuge and centrifuge at a speed of 1300 g at 5° C. for 13 min, take the supernatant, centrifuge at a speed of 2500 g at 5° C. for 13 min, take the supernatant, centrifuge at a speed of 9000 g at 5° C. for 13 min, and take the supernatant;

[0050] Step (4): passing the supernatant of step (3) through 70 μm, 40 μm, 0.45 μm and 0.22 μm filters in sequence to obtain a filtrate;

[0051] Step (5): The filtrate of step (4) was concentrated twice according to the instructions of Enze Kangtai Cell Supernatant High-fold Concentration Kit (ES8500), 35 mL of the filtrate, 10 mL of solution A in the kit, and 5 mL of solution B were placed in a 50 mL centrifuge tube, mixed and allowed to stand overnight at 4°C, centrifuged at a speed of 11000g and 5°C for 55 min, the supernatant was discarded, 1 mL of PBS was added to resuspend the precipitate, and the primary concentrate was obtained. The above operation was repeated to obtain a secondary concentrate;

[0052] Step (6): Purify the concentrate of step (5) according to the instructions of Enze Kangtai Exosome Purification Kit (ES914). Before the start of purification, perform column equilibration and PBS pretreatment, then add 1 mL of concentrate, and after the concentrate has completely entered the bottom of the sieve plate and no liquid has flowed out, start adding eluent PBS, add 500 μL each time, and after the previous 500 μL eluent has completely entered the exclusion column, add the next 500 μL eluent, collect the effluent in the collection tube, and start counting from the first drop. Each 500 μL is recorded as 1 fraction. The first 3 fractions are not collected, and the subsequent 4 to 8 fractions are collected to obtain extracellular vesicles derived from Solanum nigrum.

[0053] Example 3

[0054] This embodiment 3 provides a method for extracting and purifying extracellular vesicles derived from Solanum nigrum, comprising the following steps:

[0055] Step (1): Pick fresh Solanum nigrum leaves and fruits, wash them with running water, drain the water and slightly dry them;

[0056] Step (2): 1 kg of Solanum nigrum leaves and fruits dried in step (1) were mixed evenly with 1700 mL of PBS, added to a juicer in six batches for juicing for 5 minutes, and the juice was filtered and collected;

[0057] Step (3): Place the juice obtained in step (2) in a centrifuge and centrifuge at a speed of 1500 g at 6° C. for 15 min, take the supernatant, centrifuge at a speed of 2000 g at 6° C. for 15 min, take the supernatant, centrifuge at a speed of 8000 g at 6° C. for 15 min, and take the supernatant;

[0058] Step (4): passing the supernatant of step (3) through 70 μm, 40 μm, 0.45 μm and 0.22 μm filters in sequence to obtain a filtrate;

[0059] Step (5): The filtrate of step (4) was concentrated twice according to the instructions of Enze Kangtai Cell Supernatant High-fold Concentration Kit (ES8500), 35 mL of the filtrate, 10 mL of solution A and 5 mL of solution B in the kit were placed in a 50 mL centrifuge tube, mixed and allowed to stand overnight at 4°C, centrifuged at a speed of 12000g and 6°C for 50 min, the supernatant was discarded, 1 mL of PBS was added to resuspend the precipitate, and the primary concentrate was obtained. The above operation was repeated to obtain a secondary concentrate;

[0060] Step (6): Purify the concentrate of step (5) according to the instructions of Enze Kangtai Exosome Purification Kit (ES914). Before the start of purification, perform column equilibration and PBS pretreatment, then add 1 mL of concentrate, and after the concentrate has completely entered the bottom of the sieve plate and no liquid has flowed out, start adding eluent PBS, add 500 μL each time, and after the previous 500 μL eluent has completely entered the exclusion column, add the next 500 μL eluent, collect the effluent in the collection tube, and start counting from the first drop. Each 500 μL is recorded as 1 fraction. The first 3 fractions are not collected, and the subsequent 4 to 8 fractions are collected to obtain extracellular vesicles derived from Solanum nigrum.

[0061] Experiment 1: Study on the preventive and therapeutic effects of Solanum nigrum extracellular vesicles extracted and purified in preferred embodiment 1 on colon cancer in vitro:

[0062] 1. Cultivation of colon cancer HCT15 and RKO tumor cells

[0063] (1) Cell recovery: Prepare the items needed for the experiment and sterilize them in a clean bench under ultraviolet for 30 minutes. Wipe the clean bench with 75% alcohol and preheat the water bath to 37°C. Take out the frozen cells and quickly place them in a water bath and shake them. After they are completely thawed, wipe off the liquid on the surface of the cryopreserved tube, spray them with 75% alcohol for disinfection and place them in a clean bench. Use a 1mL pipette to pipette the cell suspension into a centrifuge tube containing 2mL of complete culture medium and mix them by beating. The speed is 1000rpm / min, centrifuge for 5min, discard the supernatant, pipette 1mL of complete culture medium and beating the cells to a single cell suspension, inoculate them into a 10cm cell culture dish containing 8mL of complete culture medium, shake them evenly, observe under a microscope for no abnormalities, and place them in a 37°C, 5% CO2 incubator for culture. Complete culture medium configuration: RPMI 1640+10%FBS+1%P / S.

[0064] (2) Cell culture medium replacement: Observe the cell status under a microscope to determine whether the culture medium needs to be replaced. Sterilize the experimental supplies in a clean bench for 30 minutes. Spray the culture dish with alcohol and place it in the clean bench. Discard the old culture medium, add an appropriate amount of PBS to wash twice, add 8 mL of complete culture medium, observe under a microscope for any abnormalities, and place it in the incubator to continue culturing.

[0065] (3) Cell passaging: When the contact density of the monolayer adherent cells is about 80% under microscope observation, the cell culture is carried out. The items required for the experiment are sterilized by conventional ultraviolet light, the culture dish is sprayed with 75% alcohol and placed in the clean bench, the old culture medium is discarded, and an appropriate amount of PBS is added to rinse twice. 1 mL of 0.25% trypsin is added to the 10 cm cell culture dish, and the dish is placed in the incubator for digestion for 1 min. When the cells become round and float under the microscope, 2 mL of complete culture medium is added to terminate the digestion. The cell suspension is pipetted into a centrifuge tube at a speed of 1000 rpm / min. Centrifuge for 5 min, discard the supernatant, and resuspend the cell suspension with 1 mL of complete culture medium to a single cell suspension. The cells are transferred to a new culture dish containing 8 mL of complete culture medium at a ratio of 1:3 of cell suspension: fresh complete culture medium, and placed in the incubator for continued culture.

[0066] (4) Cell cryopreservation: The operation steps are the same as (3) cell passage. After centrifugation, add serum-free cell cryopreservation solution, resuspend the cells, seal with sealing glue, and mark the cell name, generation number, date and operator. Store in a -80℃ refrigerator for 48 hours, and then transfer the cells to liquid nitrogen for storage.

[0067] (5) Cell counting: The operation before counting is the same as that of cell passaging in (3). After centrifugation, resuspend the cells in 1 mL of basal culture medium and pipette to a single cell suspension. Dilute the cell suspension 10-fold in a sterile EP tube and pipette to mix. Pipette 10 μL of the diluted single cell suspension and add the cell suspension along the edge of the cover glass of the counting pool. If bubbles appear in the cover glass and the counting plate, re-add the sample. Under the 10× eyepiece, 4 large squares can be seen in the counting pool. Counting follows the principle of counting the top but not the bottom, and counting the left but not the right. Count the total number of cells in the 4 large squares and calculate the cell density according to the following formula: number of cells / mL = total number of cells in the 4 large squares / 4×dilution factor×104.

[0068] 2. Dilution and filtration of extracellular vesicles derived from Solanum nigrum

[0069] The gradient dilution concentrations were determined to be 15, 10, 7.5, 5, and 3.75 μg / mL. According to the BCA protein quantification results, the original concentration of Solanum nigrum-derived extracellular vesicles was 1.44 μg / μL, which was diluted to 15 μg / mL using complete medium and then filtered using a 0.22 nm filter to continue to prepare other concentrations.

[0070] 3. CCK-8 experiment

[0071] (1) Preliminary experiment: Solanum nigrum-derived extracellular vesicles were used to treat HCT15 and RKO cells for 48 h. The density of the plates (96-well plates) was 1000 cells / well.

[0072] (2) Experimental groups:

[0073] Experimental groups: HCT15 and RKO cells + different dilution concentrations of Solanum nigrum-derived extracellular vesicles;

[0074] Blank group: 10% CCK-8 prepared in basal medium;

[0075] The experimental group was set up with 6 replicate wells, and the blank control group was set up with 3 replicate wells.

[0076] (3) Cell plating: When the cell growth density is about 80%, digest and collect the cells by conventional centrifugation. After centrifugation, discard the supernatant and add 1 mL of basal culture medium to make a single cell suspension. Count the cells with a counting plate and prepare a cell stock solution with a concentration of 1×104 / mL. Mix the cell stock solution before plating. Use a 100 μL pipette to aspirate 100 μL / well of the cell suspension and inoculate it into a 96-well plate. Inoculate an equal volume of PBS in the peripheral wells around the 96-well plate to avoid evaporation during CCK-8 incubation and affect the test results.

[0077] (4) Intervention with extracellular vesicles derived from Solanum nigrum: After the cells adhered to the wall for 12 hours and the cells were evenly spread under a microscope, a gradient of extracellular vesicles derived from Solanum nigrum were prepared in complete culture medium at concentrations of 15, 10, 7.5, 5, 3.75, and 0 μg / mL. The old culture medium was discarded, and 100 μL / well of extracellular vesicles derived from Solanum nigrum were added to the cells for 48 hours.

[0078] (5) CCK-8 incubation: After the intervention, the old complete culture medium containing Solanum nigrum-derived extracellular vesicles was discarded, the cells were washed twice with basal culture medium at room temperature, and 100 μL / well of CCK-8 solution prepared in basal culture medium was added and incubated at 37°C in the dark for 1 h.

[0079] (6) Detection of OD450 value by microplate reader: Preheat the microplate reader at 37°C, calibrate the experimental groups, and incubate with CCK-8 for 1 hour before testing. Terminate the CCK-8 incubation when the OD450 value of the control group is around 1.0.

[0080] (7) Calculation of cell survival rate: The average OD450 value of each experimental group was obtained, and the cell survival rate was calculated according to the following formula: Cell survival rate (%) = [(experimental well - blank well) / (control well - blank well)] × 100%. The results of at least three independent repeated experiments were imported into Graphpadprism 9.0 software to draw statistical graphs.

[0081] 4. Cell morphology observation

[0082] (1) Cell seeding: HCT15 and RKO cells were seeded at 1×106 cells / 10 cm dish.

[0083] (2) Solanum nigrum-derived extracellular vesicles intervention: Different concentrations of Solanum nigrum-derived extracellular vesicles were prepared. After HCT15 and RKO cells adhered to the cell wall for 12 hours, 15 and 10 μg / mL of Solanum nigrum-derived extracellular vesicles were added at 8 mL / 10 cm dish for 48 hours.

[0084] (3) Bright field photography: Bright field images of cells in the intervention groups with different concentrations of Solanum nigrum-derived extracellular vesicles were taken under a 40× optical microscope and saved.

[0085] 5. Results Analysis

[0086] (1) Figures 1 to 3 As shown, the extracellular vesicles derived from Solanum nigrum have regular and intact morphology, low breakage rate, and a quasi-round vesicle structure under transmission electron microscopy. The average particle size distribution is 81.3nm, the protein concentration is 1.435412μg / μL, and the particle size distribution is uniform and concentrated, which is consistent with the characteristics of plant-derived extracellular vesicles with negative potential shown by many research studies and high stability.

[0087] (2) Figure 4 As shown, three different concentrations of Solanum nigrum-derived extracellular vesicles had inhibitory effects on two colon cancer cell lines, HCT15 and RKO. Solanum nigrum-derived extracellular vesicles at concentrations of 10 μg / mL and 15 μg / mL could significantly inhibit the growth of both HCT15 and RKO cell lines.

[0088] (3) Figure 5 As shown, different concentrations of extracellular vesicles derived from Solanum nigrum have an effect on the survival rate of two colon cancer cell lines, HCT15 and RKO. The picture shows that the survival rates of HCT15 cells at concentrations of 0μg / mL, 3.75μg / mL, 5μg / mL, 10μg / mL and 15μg / mL of extracellular vesicles derived from Solanum nigrum are 100%, 99.2%, 98.1%, 62.7% and 43.1% respectively; the survival rates of RKO cells are 100%, 98.3%, 62.8%, 39.6% and 8.3% respectively. It can be concluded that the higher the concentration of extracellular vesicles derived from Solanum nigrum, the stronger the inhibitory ability against colon cancer cell lines.

[0089] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for extracting and purifying extracellular vesicles derived from Solanum nigrum, characterized in that: The following steps are involved: Step (1): Pick fresh Solanum nigrum leaves and fruits, wash them with running water, and then drain and dry them; Step (2): Evenly mix the dried Solanum nigrum leaves and fruits in step (1) with PBS, squeeze the juice, and filter and collect the juice; Step (3): placing the juice of step (2) into a centrifuge and centrifuging at a speed of 1000-1500 g at 3-6° C. for 10-15 min, taking the supernatant, centrifuging at a speed of 2000-3000 g at 3-6° C. for 10-15 min, taking the supernatant, centrifuging at a speed of 8000-10000 g at 3-6° C. for 10-15 min, and taking the supernatant; Step (4): passing the supernatant of step (3) through 70 μm, 40 μm, 0.45 μm and 0.22 μm filters in sequence to obtain a filtrate; Step (5): The filtrate of step (4) is concentrated twice by using an exosome concentration kit, i.e., firstly adding the mixture according to the ratio in the instruction manual, standing at 4°C overnight, then performing differential centrifugation, discarding the supernatant, and then adding PBS to resuspend the precipitate to obtain a primary concentrate, and then repeating the operation to obtain a secondary concentrate; Step (6): The concentrated solution of step (5) is purified by an exosome purification kit, that is, column equilibration and PBS pretreatment are first performed, then the mixture is added according to the ratio in the instruction manual, and finally the effluent is collected to obtain extracellular vesicles derived from Solanum nigrum.

2. The method for extracting and purifying extracellular vesicles derived from Solanum nigrum according to claim 1, characterized in that: In the step (2), the mass volume ratio of the uniform mixture of Solanum nigrum leaves and fruits and PBS is 1 g: (1.5-2) mL; if the fresh filtrate in the step (4) is not subjected to the next step, it can be stored at a temperature of -80°C for 3-6 months.

3. The method for extracting and purifying extracellular vesicles derived from Solanum nigrum according to claim 1, characterized in that: The differential centrifugation in step (5) is specifically performed at a speed of 10000-12000 g and a temperature of 3-6° C. for 50-60 min, the supernatant is discarded, and 1 mL of PBS is added to resuspend the precipitate to obtain a concentrated solution.

4. The method for extracting and purifying extracellular vesicles derived from Solanum nigrum according to claim 1, characterized in that: The collecting of the effluent in the step (6) is specifically to add 1 mL of concentrated solution, and after all the samples enter the bottom of the sieve plate without liquid flowing out, start adding the eluent PBS, adding 500 μL each time, and after all the previous 500 μL of eluent enters the exclusion column, add the next 500 μL of eluent, collect the effluent in the collection tube, and count from the first drop, each 500 μL is recorded as 1 fraction, the first 3 fractions are not collected, and the subsequent fractions are collected to obtain Solanum nigrum extracellular vesicles.

5. An extracellular vesicle derived from Solanum nigrum, characterized in that: Prepared by the extraction and purification method according to any one of claims 1 to 4.

6. Use of the Solanum nigrum-derived extracellular vesicles according to claim 5 in the preparation of drugs for preventing and treating tumors.

7. The use of the Solanum nigrum-derived extracellular vesicles in the preparation of anti-tumor drugs according to claim 6, characterized in that: The Solanum nigrum-derived extracellular vesicles can inhibit one or more growth and proliferation of tumor cell lines in vitro.

8. The use of the Solanum nigrum-derived extracellular vesicles in the preparation of anti-tumor drugs according to claim 7, characterized in that: The Solanum nigrum-derived extracellular vesicles can inhibit the proliferation of colon cancer cell lines HCT15 and RKO cells.

9. The use of the Solanum nigrum-derived extracellular vesicles in the preparation of anti-tumor drugs according to claim 8, characterized in that: The Solanum nigrum-derived extracellular vesicles contain at least one carrier acceptable in pharmacy, food science or health care products.

10. The use of the Solanum nigrum-derived extracellular vesicles in the preparation of anti-tumor drugs according to claim 6, characterized in that: The dosage form of the anti-tumor drug is any one of injection, oral solution, capsule, tablet, spray or ointment.

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