A method for preparing fig extracellular exosome-like vesicles and application thereof

Abstract

The invention provides a method for preparing extracellular exosome-like vesicles of figs and application thereof. In the method, a differential-ultracentrifugation method is adopted to process fig juice, the fig juice is centrifuged at 2000×g for 20 min, and supernatant A is collected; supernatant A is centrifuged at 3000×g for 20 min, and supernatant B is collected; supernatant B is centrifuged at 10000×g for 60 min, and supernatant C is collected; supernatant C is filtered to obtain a filtrate; the filtrate is ultracentrifuged at 100000-120000×g for 90-120 min, and a precipitate is collected; the precipitate is resuspended in a PBS solution that is filtered and sterilized to obtain the extracellular exosome-like vesicles; the fig extracellular exosome-like vesicles are a natural product and can be used as a medicinal raw material; the medicinal raw material can be used in the preparation of a drug for treating inflammatory bowelitis and has good biocompatibility.

Description

Technical Field

[0001] The present invention relates to the technical field of exosome biological preparations, and in particular to a method for preparing fig extracellular exosome vesicles and applications thereof. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic, nonspecific intestinal inflammatory disease that affects an increasing number of people worldwide. It mainly includes ulcerative colitis (UC) and Crohn's disease (CD). UC is characterized by diffuse inflammation of the colonic mucosa, and the course of the disease is repeated remission. Current treatments for UC are only somewhat effective, but there is still a certain unacceptable risk of adverse events. More effective methods are needed clinically to treat UC. Therefore, there is an urgent need to develop better UC therapeutic drugs, especially drugs that can act locally in the inflamed colon without systemic exposure and related side effects, which are of great significance for the treatment of UC colitis.

[0003] Extracellular vesicles (EVs) are unit membrane-wrapped structures secreted by cells under physiological or pathological conditions. They are a general term for various types of vesicles, among which the most studied are exosomes with a diameter of 30-150nm secreted by animal cells. Exosomes carry a large amount of proteins, lipids, mRNA, siRNA and other substances in their lipid bimolecular structure, which play an important role in cell communication, cell migration, and the occurrence and development of cancer diseases.

[0004] The saucer-like vesicles similar to exosomes derived from plant cells are named Plant exosome-like vesicles (PDELVs). With the deepening of research, PDELVs have gradually attracted increasing interest in the scientific community. Many studies have found that exosome-like nanovesicles extracted from plants such as grapes, ginger and broccoli can improve intestinal enteritis in mice.

[0005] Fig( Ficus carica ) is a food and medicine ingredient with rich nutritional value. Its fruit contains a variety of nutrients, such as vitamins, proteins, mineral elements, flavonoids and sugars, and contains a variety of anti-cancer active ingredients, which can effectively fight cancer, tumors and oxidation, and have the effects of clearing heat and promoting body fluid, strengthening the spleen and appetizing, clearing heat and detoxifying. At present, there is no research on fig exosomes or extracellular vesicles. Therefore, it is necessary to develop the use of fig exosomes or extracellular vesicles for the treatment of inflammatory bowel disease. Summary of the invention

[0006] In view of this, the present invention provides a method for preparing extracellular exosome-like vesicles of fig and application thereof. In the method, fig juice is treated by differential-ultracentrifugation method to obtain extracellular exosome-like vesicles of fig; the extracellular exosome-like vesicles of fig are a natural product and can be used as a medicinal raw material; the medicinal raw material can be used in the preparation of a drug for treating inflammatory bowel inflammation and has good biocompatibility.

[0007] The technical solution of the present invention is as follows:

[0008] A method for preparing extracellular exosome-like vesicles from figs, which is prepared by differential-ultracentrifugation method, wherein the process of differential-ultracentrifugation is:

[0009] The fig juice was centrifuged at 2000×g for 20 min, and the supernatant A was collected;

[0010] Centrifuge supernatant A at 3000×g for 20 min and collect supernatant B;

[0011] Centrifuge supernatant B at 10,000 × g for 60 min and collect supernatant C;

[0012] Filtering the supernatant C to obtain a filtrate;

[0013] The filtrate is ultracentrifuged at 100,000-120,000×g for 90-120 min, and the precipitate is collected to obtain the extracellular exosome-like vesicles of fig.

[0014] The precipitate was resuspended in a sterilized PBS solution to obtain a resuspension of extracellular exosome-like vesicles from figs, which was then preserved and identified. The total protein content of fig PDELVs was determined using the BCA method, and the protein concentration was used as the concentration of PDELVs for subsequent applications.

[0015] Preferably, the fig juice is one or a mixture of fig fruit juice, fig leaf juice, fig stem and leaf juice, and fig serum.

[0016] Preferably, the preparation process of fig fruit juice, fig leaf juice and fig stem and leaf juice is:

[0017] (1) Cleaning: The fig material was initially cleaned with water and then rinsed with PBS solution;

[0018] (2) Juice making: Cut the washed figs into small pieces of 15±5 mm, add PBS solution, mix thoroughly, and crush in a juicer. Filter through a mesh to remove the fig residue and collect the mixture.

[0019] Preferably, when the fig material is fig fruit and needs to be stored for a long time, fresh fig fruit is not easy to preserve due to its high sugar content. Even if it is refrigerated at 4°C, it is easy to rot and deteriorate. Therefore, fresh fig fruit is stored by low-temperature freezing; this storage method not only prolongs the storage time of fresh fig fruit, but also allows more extracellular exosome-like vesicles in cells to be released after freeze-thaw stimulation, thereby effectively increasing the yield of extracellular exosome-like vesicles from fresh fig fruit.

[0020] Preferably, in step (1), the washing process is carried out at room temperature, and the fresh fig fruits are commercially available green-skin and / or purple-skin fresh figs; the figs are rinsed with PBS solution three times.

[0021] Preferably, the fresh fig fruits are washed and then frozen at -20°C or -80°C for 3-90 days.

[0022] Preferably, in step (2), the weight-to-volume (g / mL) ratio of the fig material to PBS is 1:3.

[0023] Preferably, the differential-ultracentrifugation process is carried out at 4±0.5°C.

[0024] Preferably, during the differential-ultracentrifugation process, the supernatant C is filtered through 0.45µm and 0.22µm filter membranes in sequence to obtain a filtrate.

[0025] Application of fig extracellular exosome-like vesicles as medicinal raw materials.

[0026] Preferably, the specific application is to prepare the medicinal raw materials into medicines.

[0027] Preferably, the fig extracellular exosome-like vesicles are used in the preparation of a drug for treating inflammatory bowel inflammation.

[0028] Preferably, the dosage form of the drug is any one or more of injection, capsule, tablet, oral liquid, granule and traditional Chinese medicine.

[0029] Compared with the prior art, the beneficial effects of the present invention are:

[0030] In the present invention, a low-temperature freezing method is used to pre-treat fig fruit, thereby increasing the yield of extracellular exosome-like vesicles in fig fruit and solving the problem of low yield of extracellular exosome-like vesicles in fig fruit.

[0031] Fig juice is obtained by washing and juicing fig materials, and fig juice is prepared by differential-ultracentrifugation method to obtain fig extracellular exosome-like vesicles; the fig extracellular exosome-like vesicles have good biocompatibility and antioxidant properties; the medicine prepared by using the fig extracellular exosome-like vesicles can effectively relieve ulcerative colitis and is expected to be prepared into a natural medicine product that can treat inflammatory bowel inflammation;

[0032] The size of the fig extracellular exosome-like vesicles obtained by the present invention is 50-150nm; the small-particle fig extracellular exosome-like vesicles can be better absorbed and utilized when used as a drug raw material;

[0033] The fig extracellular exosome-like vesicles of the present invention are used as a medicinal raw material and then prepared into a drug. When forming a specific product, the drug can be prepared into any dosage form of the above by conventional means to meet different taking or intake requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] 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, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0035] Figure 1 This is the TEM structural characterization of the extracellular exosome-like vesicles of fig fruit prepared in Example 1, wherein the scale bar in the figure is 200 nm.

[0036] Figure 2 This is the distribution of extracellular exosome-like vesicle proteins in fig fruit prepared in Example 1. In the figure, M is a marker, and 1, 2 and 3 are all samples of Example 1.

[0037] Figure 3 This is the tracing of the extracellular exosome-like vesicles of fig fruit prepared in Example 1 in macrophages RAW264.7.

[0038] Figure 4 The figure shows the effect of the extracellular exosome-like vesicles of fig fruit prepared in Example 1 on the proliferation activity of RAW264.7 cells at different concentrations.

[0039] Figure 5 This is a graph showing the effect of low-temperature storage temperature on the yield of extracellular exosome-like vesicles of fig fruit prepared in Example 2.

[0040] Figure 6 This is the TEM structural characterization of the extracellular exosome-like vesicles of fig milk plasma prepared in Example 3; wherein the scale bar in the figure is 200 nm.

[0041] Figure 7 This is the particle size distribution of the extracellular exosome-like vesicles prepared in Example 3.

[0042] Figure 8 This is the distribution of exosome-like vesicle proteins outside the cells of fig milk plasma prepared in Example 3. In the figure, M is a marker, and 1, 2 and 3 are samples of Example 3.

[0043] Fig. 9 This is a graph showing the weight changes of mice in Example 4.

[0044] Fig.10 The figure shows the change in colon length of mice in Example 4. DETAILED DESCRIPTION

[0045] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0046] Example 1 Preparation, characterization and intracellular tracing of extracellular exosome-like vesicles from fig fruit

[0047] 1.1 Preparation of extracellular exosome-like vesicles from fig fruit

[0048] (1) Washing: Wash 100 g of ripe fresh purple fig fruit with purified water at room temperature, and then rinse it with PBS solution three times;

[0049] (2) Juice preparation: Cut the washed figs into small pieces of 15±1 mm, add 300 mL of sterile PBS buffer at a weight-to-volume (g / mL) ratio of 1:3, mix thoroughly and crush in a juicer for 7 min, filter through a gauze to remove the fig fruit residue, and collect the fig juice;

[0050] (3) Differential-ultracentrifugation:

[0051] At 4°C, the fig juice was centrifuged at 2000×g for 20 min, and the supernatant A was collected;

[0052] Centrifuge supernatant A at 3000×g for 20 min and collect supernatant B;

[0053] Centrifuge supernatant B at 10,000 × g for 60 min and collect supernatant C;

[0054] The supernatant C was filtered through 0.45µm and 0.22µm filter membranes in sequence to obtain a filtrate;

[0055] The filtrate was ultracentrifuged at 120,000 × g for 110 min, and the precipitate was collected, which was the extracellular exosome-like vesicles of fig fruit;

[0056] The precipitate was resuspended in a sterilized PBS solution to obtain a resuspension of extracellular exosome-like vesicles in fig fruit, which was then stored and identified in a refrigerator at -80°C. The total protein content of fig PDELVs was determined to be 5.4 mg / mL using the BCA method, and the protein concentration was used as the concentration of PDELVs for subsequent applications.

[0057] 1.2 Characterization of extracellular exosome-like vesicles from fig fruit: TEM, size distribution, and protein distribution

[0058] (1) Transmission electron microscopy (TEM) structural characterization

[0059] Take 10 μL of the fresh resuspension of extracellular exosome-like vesicles of fig fruit prepared by the above method, drop it on a clean culture dish, place the membrane surface of the copper mesh on the resuspension, suspend for 10 minutes, and slowly absorb with filter paper; transfer the copper mesh to a drop of 2.5% glutaraldehyde, suspend for 5 minutes, and absorb with filter paper; transfer the copper mesh to a drop of ultrapure water, 2 minutes each time, 8 times, and absorb with filter paper each time; then transfer the copper mesh to a drop of 2% uranyl acetate, 5 minutes, and absorb with filter paper; continue to transfer the copper mesh to a drop of 2% carboxymethyl cellulose, incubate on ice for 10 minutes, and absorb with filter paper; after natural drying, observe with a Hitachi transmission electron microscope HT7800, see Figure 1 .

[0060] The general state under the transmission electron microscope is saucer-shaped or concave round cake-shaped, such as Figure 1 As shown, the obtained fig fruit extracellular exosome-like vesicles have a clear structure observed by transmission electron microscopy, mostly in the shape of concave round cakes, with a relatively clean background, and a size distribution in the range of 50-150nm.

[0061] (2) SDS-PAGE characterization of protein distribution of extracellular exosome-like vesicles in fig fruit

[0062] The prepared fig fruit PDELVs sample was mixed with 4× Loading buffer in a volume ratio of 3:1, and heated in a 95°C water bath for 5 min before use; 10% SDS-PAGE separation gel + 5% SDS concentration gel were prepared, and 30 μg of protein was loaded into each well according to the protein concentration determined by the BCA method; 10 μL of Rainbow Maker was added to the first loading well on the left as a positive control, and the results are shown in Figure 2 ; Figure 2The results showed that the proteins contained in the extracellular exosome-like vesicles of fig fruit had a small molecular weight and were roughly distributed in the range of 5KD~25KD with a small number of bands.

[0063] 1.3 Intracellular tracing of extracellular exosome-like vesicles from fig fruit

[0064] Because fluorescent dyes are involved, intracellular tracing requires the entire process to be done in the dark.

[0065] The specific method is: prepare the dye according to the instructions: 1μL PKH26 dye + 9μL Diulent C;

[0066] The above-mentioned fig fruit extracellular exosome-like vesicles were added to the dye prepared above, and the control group used 10 μL of PBS buffer to replace the dye. After fully mixing, they were incubated at room temperature in the dark for 10 minutes; 1 mL of PBS solution was added to each group of solutions, and centrifuged at 4°C and 100,000 g for 2 hours to remove the dye not attached to the vesicles;

[0067] RAW264.7 macrophages were cultured at 1×10 6 / mL density was spread on the confocal dish. After the cells adhered to the wall, the stained extracellular exosome-like vesicles of the fruit were added to the cells and incubated in the cell culture incubator for 0.5h, 1h and 2h respectively. After the staining was completed, the stain solution was discarded and the cells were washed three times with PBS buffer. Then 4wt% paraformaldehyde was added to fix at room temperature for 30min, washed three times with PBS buffer, and examined under a fluorescence microscope. The results are shown in Figure 3 ; Combine Figure 3 It can be seen that the extracellular exosome-like vesicles of fig fruit can enter macrophages in 0.5h, and the amount of intracellular fluorescence increases with time; this is because the extracellular exosome-like vesicles of the fruit have a micro-nanoscale size and a typical lipid bilayer structure, which makes it easy to enter the cell.

[0068] 1.4 Effects of Fig Fruit Extracellular Exosome-Like Vesicles on Macrophage Activity

[0069] RAW264.7 cells were collected and cultured at 1 × 10 5 / mL density for plating operation, after the cells adhere to the wall, the original culture medium is discarded, and the extracellular exosome vesicles of fig fruit are added for 24h; the concentration of the extracellular exosome vesicles of fig fruit in the experimental group is set to 5, 10, and 20μg / mL, and the drug in the positive control group is hydrogen peroxide (100μM); after culturing for 24h, the medium containing the drug is discarded, and the medium containing 10% CCK-8 reagent is added, incubated at 37℃ for 2h, and the microplate reader is used to detect at 450nm to calculate the cell survival rate. The results are as follows Figure 4 shown; combined Figure 4It can be seen that when the concentration of extracellular exosome-like vesicles in fig fruit is 5-20μg / mL, the survival rate of RAW264.7 cells continues to increase with the increase of concentration, and at the concentration of 10μg / mL and 20μg / mL, the cell survival rate is significantly increased, and the difference is statistically significant (P < 0.001).

[0070] The experimental data were statistically analyzed and plotted using GraphPad Prism 8.0.2 software. The P value was calculated using the T-test function, with P < 0.05 indicating a statistically significant difference (*P < 0.05, **P < 0.01, ***P < 0.001).

[0071] Example 2 Low temperature freezing increases the yield of extracellular exosome-like vesicles in fig fruit

[0072] Experiment 1: The difference from Example 1 is:

[0073] In step (2), 100 g of fresh fig fruit is washed and stored at 4° C. for 3 days, taken out, thawed, cut into small pieces of 15±1 mm, added with 300 mL of sterile PBS buffer, mixed thoroughly, crushed in a juicer, filtered through a gauze to remove fig residue, and collected fig juice;

[0074] The remaining steps are the same as in Example 1;

[0075] Experiment 2: The difference from Example 1 is:

[0076] In step (2), 100 g of fresh fig fruit is washed and frozen at -20°C for 3 days, taken out, thawed and cut into small pieces of 15±1 mm, added with 300 mL of sterile PBS buffer, mixed thoroughly and crushed in a juicer, filtered through a gauze to remove fig residues, and collected fig juice;

[0077] The remaining steps are the same as in Example 1;

[0078] Experiment 3: The difference from Example 1 is:

[0079] In step (2), 100 g of fresh fig fruit is washed and frozen at -80°C for 3 days, taken out, thawed and cut into small pieces of 15±1 mm, added with 300 mL of sterile PBS buffer, mixed thoroughly and crushed in a juicer, filtered through a gauze to remove fig residue, and collected fig juice;

[0080] The remaining steps are the same as in Example 1;

[0081] The protein concentration of extracellular exosome-like vesicles was quantified by the BCA method, and the total protein content was used as the basis for calculating the yield according to the following formula:

[0082] Extracellular exosome-like vesicle yield (mg / kg) = protein amount (mg) / fig weight (g) × 1000;

[0083] The results are as follows Figure 5 As shown, combined Figure 5 It can be seen that the extracellular exosome vesicle yield of fig fruit stored at 4°C (Experiment 1) was 35.40±3.42 mg / kg, while after low-temperature freezing at -20°C (Experiment 2) and -80°C (Experiment 3), the extracellular exosome vesicle yield increased to 53.88±0.43 mg / kg and 61.65±2.24 mg / kg, with a significant increase of 52% (P<0.05) and 74% (P<0.01). The above data were statistically analyzed using SPSS software, and the quantitative data were expressed as mean±standard deviation (x±s). The t-test was used, and the inter-group comparison was performed using one-way analysis of variance. P<0.05 indicated a significant difference.

[0084] Example 3 Preparation, identification and characterization of exosome-like vesicles outside the cells of fig milk plasma

[0085] 3.1 Preparation of exosome-like vesicles from fig milk plasma

[0086] (1) Take 5 mL of fig serum, add 25 mL of PBS solution at a ratio of 1:5 (mL / mL), mix well and centrifuge directly to obtain fig juice;

[0087] (2) Differential-ultracentrifugation:

[0088] At 4°C, the fig juice was centrifuged at 2000×g for 20 min, and the supernatant A was collected;

[0089] Centrifuge supernatant A at 3000×g for 20 min and collect supernatant B;

[0090] Centrifuge supernatant B at 10,000 × g for 60 min and collect supernatant C;

[0091] The supernatant C was filtered through 0.45µm and 0.22µm filter membranes in sequence to obtain a filtrate;

[0092] The filtrate was ultracentrifuged at 120,000 × g for 110 min, and the precipitate was collected, which was the extracellular exosome-like vesicles of fig milk plasma;

[0093] The precipitate was resuspended in a sterilized PBS solution to obtain a resuspension of extracellular exosome-like vesicles from fig serum, which was then stored and identified in a refrigerator at -80°C. The total protein content of fig serum PDELVs was determined to be 5.2 mg / mL using the BCA method, and the protein concentration was used as the concentration of fig serum PDELVs for subsequent applications.

[0094] 3.2 Characterization of the particle size, morphology and protein distribution of exosome-like vesicles from fig milk plasma

[0095] The sample preparation and observation method of transmission electron microscopy (TEM) was consistent with that of the extracellular exosome-like vesicles of fig fruit. Figure 6 shown; combined Figure 6 It can be seen that the exosome-like vesicles outside the fig milk plasma cells under the transmission electron microscope are saucer-shaped or concave round cake-shaped, with a relatively clean background, and the size distribution is in the range of 50-150nm.

[0096] The results of the nano-flow cytometry test were shown in Figure 7 , Figure 7 The analysis showed that the particles in the sample were unimodal in distribution, and the average particle size distribution range of the exosome-like vesicles outside the fig milk plasma cells was 64.4±42.9nm, which was close to the size detected by TEM; the concentration of the exosome-like vesicles outside the fig milk plasma cells was 7.2×10 8 Pieces / mL;

[0097] The prepared fig serum PDELVs sample was mixed with 4× Loading buffer in a volume ratio of 3:1, and heated in a 95°C water bath for 5 min before use; 10% SDS-PAGE separation gel + 5% SDS concentration gel were prepared; according to the protein concentration determined by the BCA method, 30 μg of protein was loaded into each well, and 10 μL of Rainbow Maker was added to the first loading well on the left as a positive control. The results are shown in Figure 8 ; Figure 8 The results showed that the proteins contained in the extracellular exosome-like vesicles of fig serum were unevenly distributed, with large differences between samples from different batches, but their protein content was significantly higher than that of fig fruit PDELVs; the protein molecular weight of fig serum PDELVs No. 2 and No. 3 was relatively small, roughly distributed in the range of 5~25kDa, and the bands were brighter, while sample No. 1 also had more protein distribution between 48 and 245kDa.

[0098] Example 4: Therapeutic effect of extracellular exosome-like vesicles from fig fruit on ulcerative colitis model in mice

[0099] 4.1 Model Construction

[0100] Female C57BL / 6 mice, 6-8 weeks old, weighing 18-20 g, were randomly divided into a control group, a DSS model group, and an experimental group (DSS+fig fruit PDELVs group), with 8 mice in each group.

[0101] The mice in the DSS model group and the experimental group drank 2.5% dextran sulfate sodium (DSS) solution freely for 6 consecutive days, and then switched to single distilled water for free drinking for 3 days;

[0102] The mice in the control group were given the same volume of PBS as DSS for 6 consecutive days and then were given distilled water to drink freely for 3 days.

[0103] The model was established. Control group: mice were allowed to drink water freely every day.

[0104] DSS model group: mice had free access to water every day;

[0105] Experimental group: The fig fruit extracellular exosome vesicles PDELVs obtained in Example 1 were gavaged from the same day, once a day for 7 consecutive days, and the dosage (calculated as PDELVs protein) was 250 μg / mouse / time;

[0106] The mice were observed for blood in stool and weight changes every day, and were weighed and killed on the 13th day.

[0107] The weight change, disease activity index (Table 1) and colon length of mice are all important indicators of colitis; the weight change, disease activity index and colon length of mice were counted during these 13 days.

[0108] Table 1 DAI score table

[0109]

[0110] 4.2 Results and Statistics

[0111] The weight changes of mice within 13 days are shown in Fig. 9 ;like Fig. 9 As shown in the figure, starting from the 5th day of drug addition, the body weights of mice in the three groups began to fluctuate continuously: the body weights of mice in the control group remained basically unchanged; the body weights of mice in the DSS model group gradually decreased with daily feeding of 2.5% DSS, and then reached the lowest value around the 12th day, and their body weights gradually recovered and tended to be stable, but were still lower than those of mice in the control group.

[0112] In the experiment of DSS-induced enteritis in the experimental group, the mortality rate of the group of mice treated with the extracellular exosome vesicles of fig fruit in Example 1 for enteritis within 14 days was significantly lower than that of the mice in the DSS model group, indicating that fig PDELVs can significantly prevent the occurrence of ulcerative enteritis in mice and has a certain therapeutic effect on IBD inflammatory bowel inflammation in mice.

[0113] The disease activity index is shown in Table 2, as follows:

[0114] Table 2 Disease Activity Index (DAI) score

[0115]

[0116] Combined with Table 2, it can be seen that the disease activity index (DAI) score of colitis in the DSS model group was significantly higher than that in the control group, indicating that the DSS-induced colitis model in mice was successfully established; the DAI score of the mice in the experimental group was significantly lower than that in the model group, indicating that fig fruit PDELVs can alleviate the symptoms of DSS-induced colitis in mice.

[0117] Colon length Fig.10 ;like Fig.10 As shown in the figure, there was a significant difference in the colon length of mice among the three groups. The results showed that fig extracellular exosome vesicles have a good therapeutic effect in preventing inflammatory bowel inflammation, and can restore the large intestine damage induced by DSS to a certain extent, effectively preventing the occurrence and development of inflammation; at the same time, it also played a vital role in DAI; in addition, fig extracellular exosome vesicles prevented the shortening of the colon, indicating that fig extracellular exosome vesicles have a good anti-inflammatory effect.

[0118] Example 5 Preparation of extracellular exosome-like vesicles from fig fruit

[0119] (1) Washing: Wash 50 g of ripe fresh purple fig fruit with purified water at room temperature, and then rinse it with PBS solution three times;

[0120] (2) Juice preparation: Cut the washed figs into small pieces of 15±1 mm, add 150 mL of sterile PBS buffer at a ratio of 1:3 (g / mL), mix thoroughly and crush in a juicer for 5 min, filter through a gauze to remove the fig fruit residue, and collect the fig juice;

[0121] (3) Differential-ultracentrifugation:

[0122] At 4°C, the fig juice was centrifuged at 2000×g for 20 min, and the supernatant A was collected;

[0123] Centrifuge supernatant A at 3000×g for 20 min and collect supernatant B;

[0124] Centrifuge supernatant B at 10,000 × g for 60 min and collect supernatant C;

[0125] The supernatant C was filtered through 0.45µm and 0.22µm filter membranes in sequence to obtain a filtrate;

[0126] The filtrate was ultracentrifuged at 100,000 × g for 90 min, and the precipitate was collected, which was the extracellular exosome-like vesicles of fig fruit;

[0127] The precipitate was resuspended in a sterilized PBS solution to obtain a resuspension of extracellular exosome-like vesicles from fig milk plasma, and the resuspension was stored and identified; the resuspension was stored in a refrigerator at -80°C.

[0128] Example 6 Preparation of extracellular exosome-like vesicles from fig fruit

[0129] (1) Washing: 200 g of ripe fresh purple fig fruit was washed with purified water at room temperature, and then rinsed with PBS solution three times;

[0130] (2) Juice preparation: Cut the washed figs into small pieces of 15±1 mm, add 600 mL of sterile PBS buffer at a ratio of 1:3 (g / mL), mix thoroughly and crush in a juicer for 10 min, filter through a gauze to remove the fig fruit residue, and collect the fig juice;

[0131] (3) Differential-ultracentrifugation:

[0132] At 4°C, the fig juice was centrifuged at 2000×g for 20 min, and the supernatant A was collected;

[0133] Centrifuge supernatant A at 3000×g for 20 min and collect supernatant B;

[0134] Centrifuge supernatant B at 10,000 × g for 60 min and collect supernatant C;

[0135] The supernatant C was filtered through 0.45µm and 0.22µm filter membranes in sequence to obtain a filtrate;

[0136] The filtrate was ultracentrifuged at 120,000 × g for 120 min, and the precipitate was collected, which was the extracellular exosome-like vesicles of fig fruit;

[0137] The precipitate was resuspended in a sterilized PBS solution to obtain a resuspension of extracellular exosome-like vesicles from fig milk plasma, and the resuspension was stored and identified; the resuspension was stored in a refrigerator at -80°C.

[0138] Although the present invention has been described in detail by reference to the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions shall be within the scope of the present invention. Any person of ordinary skill in the art may easily think of changes or substitutions within the technical scope disclosed by the present invention, and these shall be within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope of protection of the claims.

Claims

1. An application of fig extracellular exosome-like vesicles in the preparation of a drug for treating ulcerative colitis, characterized in that: Fig extracellular exosome-like vesicles were prepared from fig fruit juice.

2. The use according to claim 1, characterized in that The dosage form of the drug is any one or more of injection, capsule, tablet, oral liquid, and granule.

3. The use according to claim 1, characterized in that Fig extracellular exosome-like vesicles were prepared by differential-ultracentrifugation method, wherein the differential-ultracentrifugation process is as follows: The fig fruit juice was centrifuged at 2000×g for 20 min, and the supernatant A was collected; Centrifuge supernatant A at 3000×g for 20 min and collect supernatant B; Centrifuge supernatant B at 10,000 × g for 60 min and collect supernatant C; Filtering the supernatant C to obtain a filtrate; The filtrate is ultracentrifuged at 100,000-120,000×g for 90-120 min, and the precipitate is collected to obtain the extracellular exosome-like vesicles of fig.

4. The use according to claim 3, characterized in that The preparation process of fig fruit juice is: (1) Washing: Wash the fig fruit with water and then rinse it with PBS solution; (2) Juice making: Cut the washed figs into small pieces of 15±5 mm, add PBS solution, mix thoroughly, and crush in a juicer. Filter through a mesh to remove the fig residue and collect the juice.

5. The use according to claim 4, characterized in that Fresh fig fruits are stored by low-temperature freezing.

6. The use according to claim 4, characterized in that The washing process is carried out at room temperature. The fresh fig fruits are commercially available green-skin and / or purple-skin fresh figs. The figs are rinsed with PBS solution for 3 times.

7. The use according to claim 3, characterized in that The differential-ultracentrifugation process was carried out at 4±0.5°C.

8. The use according to claim 3, characterized in that During the differential-ultracentrifugation process, the supernatant C was filtered through 0.45µm and 0.22µm filter membranes in sequence to obtain a filtrate.

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