Composition containing plant-derived exosome as well as preparation method and application of composition

Abstract

The invention belongs to the technical field of biology, and particularly relates to a composition containing plant-derived exosomes as well as a preparation method and application of the composition. The invention provides a composition containing plant-derived exosomes. The composition is prepared from the following components in parts by mass: 1-60 parts of a plant-derived exosome preparation, 1-60 parts of chitosan oligosaccharide and 1-60 parts of probiotics. According to the composition provided by the invention, the plant-derived exosome, the probiotics and the chitosan oligosaccharide are compounded according to a specific proportion, so that the abundance of effective flora can be increased for a long time, the composition of intestinal flora can be improved, the structural balance of the intestinal flora can be regulated and maintained, the intestinal inflammation can be remarkably inhibited, the intestinal leakage can be improved, and the oxidative stress can be resisted; the repair of damaged intestinal mucosa and the elimination of inflammation are facilitated. The composition is safe and stable in raw materials, free of toxic and side effects and capable of being taken as daily diet.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a composition containing plant-derived exosomes, a preparation method thereof, and applications thereof. Background Art

[0002] Inflammatory bowel disease is a group of chronic non-specific intestinal inflammatory diseases with unclear etiologies, including ulcerative colitis (UC) and Crohn's disease (CD). These two diseases are different in clinical manifestations, pathological features, and treatments, but both involve chronic inflammatory reactions in the intestine. Intestinal flora can affect human functions such as metabolism, immunity, and nutrition, interact with the human body for a long time, co-evolve, and are closely related to human intestinal health. Once the intestinal flora is dysregulated, it will lead to intestinal dysfunction, trigger various intestinal diseases, such as diarrhea, constipation, decreased human immunity, and in severe cases, it will cause inflammatory bowel disease, enterotoxemia, and even the risk of developing intestinal cancer. Therefore, regulating the balance of intestinal flora is particularly important for improving intestinal inflammation and the treatment of inflammatory bowel disease.

[0003] Currently, existing regulators for regulating the balance of intestinal flora contain probiotics, but usually have poor effects. Moreover, directly orally taking probiotics will also cause problems such as ineffective colonization in the intestine, little residence and occupation, easy loss, and easy recurrence of harmful bacteria after discontinuation. Coupled with the extremely complex human intestinal environment, there may be great differences in the therapeutic effects of different probiotics in different populations. For example, the probiotic preparation in Chinese Patent CN105454966A has a very limited improvement effect on intestinal inflammatory diseases. How to ensure that orally taken probiotics play an effective role in regulating intestinal flora and improving intestinal inflammatory diseases is particularly important. Summary of the Invention

[0004] The purpose of the present invention is to provide a composition containing plant-derived exosomes, a preparation method thereof, and applications thereof. The composition containing plant-derived exosomes has safe and stable raw materials, no toxic and side effects, can be taken as daily diet, can increase the abundance of beneficial flora, improve the composition of intestinal flora, regulate and maintain the balance of intestinal flora structure, repair damaged intestinal mucosa, inhibit intestinal inflammation, improve intestinal leakage, and resist oxidative stress.

[0005] The present invention provides a composition containing plant-derived exosomes, which comprises the following components in parts by mass: 1 to 60 parts of a plant-derived exosome preparation, 1 to 60 parts of chitosan oligosaccharide, and 1 to 60 parts of probiotics.

[0006] Preferably, it comprises the following components in parts by mass: 5 to 20 parts of a plant-derived exosome preparation, 30 to 60 parts of chitosan oligosaccharide, and 20 to 60 parts of probiotics.

[0007] Preferably, the plant-derived exosome preparation includes a freeze-dried powder preparation of plant-derived exosomes.

[0008] Preferably, the plant includes one or more of ginger, turmeric, and yam.

[0009] Preferably, the degree of polymerization of the chitosan oligosaccharide is 2 to 20.

[0010] Preferably, the probiotics include one or more of Bifidobacterium, Lactobacillus, Lacticaseibacillus, Limosilactobacillus, Lactiplantibacillus, Ligilactobacillus, Latilactobacillus, Streptococcus, Lactococcus, Propionibacterium, Acidipropionibacterium, Leuconostoc, Pediococcus, Weizmannia, Mammaliicoccus, Staphylococcus, and Kluyveromyces.

[0011] Preferably, the Bifidobacterium includes one or more of Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, and Bifidobacterium longum;

[0012] The lactobacilli include one or more of Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus bulgaricus, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, and Lactobacillus kefiranofaciens;

[0013] The lacticaseibacilli include one or more of Lacticaseibacillus casei, Lacticaseibacillus paracasei, and Lacticaseibacillus rhamnosus;

[0014] The limosilactobacilli include Limosilactobacillus fermentum and / or Limosilactobacillus reuteri;

[0015] The lactiplantibacilli include Lactiplantibacillus plantarum;

[0016] The ligilactobacilli include Ligilactobacillus salivarius;

[0017] The latilactobacilli include Latilactobacillus curvatus and / or Latilactobacillus sakei;

[0018] The streptococci include Streptococcus salivarius;

[0019] The lactococci include Lactococcus lactis and / or Lactococcus cremoris;

[0020] The propionibacteria include Propionibacterium freudenreichii;

[0021] The propionibacteria include Acidipropionibacterium acidipropionici;

[0022] The leuconostocs include Leuconostoc mesenteroides;

[0023] The pediococci include Pediococcus acidilactici and / or Pediococcus pentosaceus;

[0024] The weizmannias include Weizmannia coagulans;

[0025] The mammaliicocci include Mammaliicoccus vitulinus;

[0026] The staphylococci include Staphylococcus xylosus and / or Staphylococcus carnosus;

[0027] The kluyveromyces include Kluyveromyces marxianus.

[0028] Preferably, based on the mass parts of the plant-derived exosome preparation, the composition further includes 5 - 15 parts of other components;

[0029] The other components include zinc gluconate and / or taurine.

[0030] The present invention also provides a preparation method of the composition according to the above technical solution, including the following steps:

[0031] Mix the components to obtain the composition.

[0032] The present invention also provides the application of the composition according to the above technical solution or the composition obtained by the preparation method according to the above technical solution in one or more of the following;

[0033] (1) Preparing health foods, functional foods or drugs for regulating the balance of intestinal flora;

[0034] (2) Preparing drugs for treating intestinal inflammation.

[0035] Beneficial effects:

[0036] The present invention provides a composition containing plant-derived exosomes, comprising the following components in parts by mass: 1 to 60 parts of a plant-derived exosome preparation, 1 to 60 parts of chitosan oligosaccharide, and 1 to 60 parts of probiotics. The present invention uses a compound of a plant-derived exosome preparation, chitosan oligosaccharide, and probiotics in a specific mass ratio, and there is a synergistic effect among the components, which can expand the application range and improve the combined application effect. As a stable loading carrier, plant-derived exosomes are beneficial to the release and absorption of active ingredients. Chitosan oligosaccharide can improve the utilization of probiotic metabolites by the body, and is also beneficial to the rapid absorption of active ingredients into the intestine to exert pharmacological effects. Probiotics can regulate the disordered flora structure of the human intestine, inhibit the colonization of pathogenic bacteria in the intestine and the reduction of microbial diversity and the decrease of species abundance of the intestinal flora caused by inflammatory bowel disease. At the same time, it helps the host establish a healthy intestinal mucosal protection layer, enhance the intestinal barrier function, improve the intestinal flora environment, and after the intestinal microecological environment is improved, it is also beneficial to the better absorption and utilization of functional substances such as amino acids, vitamins, and short-chain fatty acids in probiotic metabolites, so as to better exert the health care effect of regulating the physiological functions of the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0038] Figure 1 It is a result diagram of ginger-derived exosomes taken by a transmission electron microscope (TEM);

[0039] Figure 2 It is a flow chart for the construction of a cell line enteritis model and the detection of drug efficacy;

[0040] Figure 3 It is the expression of inflammatory genes in cell lines of different treatment groups; among them, * indicates P < 0.05; *** indicates P < 0.001;

[0041] Figure 4 It is the immunofluorescence test results of cell lines of different treatment groups;

[0042] Figure 5 It is the detection results of the contents of MTT, DAO, and D-LA in cells of cell lines of different treatment groups; among them, * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0043] The present invention provides a composition containing plant-derived exosomes, comprising the following components in parts by mass: 1 to 60 parts of a plant-derived exosome preparation, 1 to 60 parts of chitosan oligosaccharide, and 1 to 60 parts of probiotics.

[0044] By mass parts, the composition containing plant-derived exosomes of the present invention comprises 1 to 60 parts of a plant-derived exosome preparation. As an embodiment, the composition containing plant-derived exosomes comprises 5 to 20 parts of a plant-derived exosome preparation; as another embodiment, the composition containing plant-derived exosomes comprises 8 to 15 parts of a plant-derived exosome preparation; as another embodiment, the composition containing plant-derived exosomes comprises 10 parts of a plant-derived exosome preparation.

[0045] In the present invention, the plant-derived exosomes can serve as an effective therapeutic carrier for genes and drugs, maintaining an intact particle form during gastrointestinal transport, thereby enabling the effective and stable absorption of the encapsulated contents. Therefore, it has an excellent effect in improving intestinal inflammatory symptoms. Compared with exosomes from other sources (such as exosomes from milk), the use of plant-derived exosomes has the advantage of lower immunogenicity.

[0046] As an embodiment, the number of particles of the plant-derived exosomes of the present invention is 10 9 ~10 12 particles / mg; as another embodiment, the number of particles of the plant-derived exosomes of the present invention is 10 11 particles / mg.

[0047] As an embodiment, the particle size of the plant-derived exosomes of the present invention is 80 to 120 nm; as another embodiment, the particle size of the plant-derived exosomes of the present invention is 90 to 110 nm; as an embodiment, the particle size of the plant-derived exosomes of the present invention is 100 nm.

[0048] As an embodiment, the plant-derived exosome preparation of the present invention comprises a plant-derived exosome freeze-dried powder preparation. The present invention places no special restrictions on the source of the plant-derived exosome preparation, which can be purchased conventionally or prepared by itself. For example, the plant-derived exosome freeze-dried powder preparation can be obtained by freeze-drying the plant-derived exosomes.

[0049] As an implementation method, the preparation method of the freeze-dried powder preparation of plant-derived exosomes includes the following steps: homogenize the mixture of plants and water to obtain a homogenate; perform low-speed centrifugation on the homogenate to obtain a first supernatant; perform medium-speed centrifugation on the first supernatant to obtain a second supernatant; perform ultra-high-speed centrifugation on the second supernatant, collect the precipitate, resuspend and wash it to obtain the plant-derived exosomes; perform freeze-drying treatment on the plant-derived exosomes to obtain the freeze-dried powder preparation of plant-derived exosomes. As an implementation method, the mass-volume ratio of the plants to water is 1 g: 5 mL. As an implementation method, the rotation speed of the low-speed centrifugation is 5000×g. As an implementation method, the time of the low-speed centrifugation is 10 min. As an implementation method, the rotation speed of the medium-speed centrifugation is 10000×g. As an implementation method, the time of the medium-speed centrifugation is 20 min. As an implementation method, the rotation speed of the ultra-high-speed centrifugation is 100000×g. As an implementation method, the freeze-drying treatment includes freeze-treatment at -50°C to -40°C for 1 to 2 h, then sucking out the moisture by vacuum, and maintaining at -80°C for 24 to 48 h.

[0050] As an implementation method, the present invention further includes purifying the plant-derived exosomes after the freeze-drying treatment. As an implementation method, the purification of the present invention uses a 0.22 μm filter membrane.

[0051] As an implementation method, the plants of the present invention include one or more of ginger, turmeric and yam; as another implementation method, the plants of the present invention include ginger.

[0052] Based on the mass fraction of the plant-derived exosome preparation, the composition containing plant-derived exosomes of the present invention includes 1 to 60 parts of chitosan oligosaccharide. As an implementation method, the composition containing plant-derived exosomes includes 30 to 60 parts of chitosan oligosaccharide; as an implementation method, the composition containing plant-derived exosomes includes 40 to 50 parts of chitosan oligosaccharide; as an implementation method, the composition containing plant-derived exosomes includes 45 parts of chitosan oligosaccharide. The present invention has no strict requirements on the source of the chitosan oligosaccharide, and conventional commercially available chitosan oligosaccharide products can be used.

[0053] In the present invention, chitosan oligosaccharide is utilized in the intestine, can play the role of prebiotic, be utilized and metabolized by beneficial intestinal bacteria, proliferate beneficial human flora, and can directly inhibit pathogenic bacteria such as Escherichia coli and Staphylococcus aureus in the intestine, thereby assisting in maintaining the microecological balance of the entire intestine, fundamentally solving various diseases caused by intestinal flora imbalance. In addition, chitosan oligosaccharide can also stimulate defense genes in the body and enhance the immune function of the body. Moreover, the amino group structure in the unique molecular structure of chitosan oligosaccharide has complexing property, ion exchange property and sustained release property, and can combine with components such as probiotics and exosomes, not only can make the above components continuously play their effects in the body, and the action time is long.

[0054] As an embodiment, the degree of polymerization of the chitosan oligosaccharide in the present invention is 2 to 20; as another embodiment, the degree of polymerization of the chitosan oligosaccharide in the present invention is 3 to 15; as another embodiment, the degree of polymerization of the chitosan oligosaccharide in the present invention is 5 to 10; as another embodiment, the degree of polymerization of the chitosan oligosaccharide in the present invention is 8.

[0055] Based on the mass fraction of the plant-derived exosome preparation, the composition containing plant-derived exosomes in the present invention comprises 1 to 60 parts of probiotics. As an embodiment, the composition containing plant-derived exosomes in the present invention comprises 10 to 55 parts of probiotics; as another embodiment, the composition containing plant-derived exosomes in the present invention comprises 20 to 50 parts of probiotics; as another embodiment, the composition containing plant-derived exosomes in the present invention comprises 30 to 45 parts of probiotics.

[0056] As an embodiment, the total number of effective viable bacteria in the probiotics in the present invention ≥ 10 10 CFU / g; as another embodiment, the total number of effective viable bacteria in the probiotics in the present invention is 10 10 CFU / g. The present invention has no special limitation on the source of the strains of the probiotics, and known strains in the art can be used, such as powders of various commercially available bacterial strains or active probiotic powders prepared by liquid culturing, centrifugal collection, drying, etc. of various commercially available bacterial strains.

[0057] In the present invention, the probiotics can regulate the disordered flora structure of the human intestine, inhibit the colonization of pathogenic bacteria in the intestine and the reduction of the microbial diversity and the decrease of the species abundance of the intestinal flora caused by inflammatory bowel disease, and at the same time help the host establish a healthy intestinal mucosal protective layer, enhance the intestinal barrier function, improve the intestinal flora environment, and further regulate the immune system and metabolic function of the host, and can also delay the progress of enteritis diseases and reduce the inflammation level.

[0058] As an embodiment, the probiotics of the present invention include one or more of Bifidobacterium, Lactobacillus, Lactobacillus casei, Lactobacillus mucosae, Lactiplantibacillus plantarum, Limosilactobacillus fermentum, Lactobacillus paraplantarum, Streptococcus, Lactococcus, Propionibacterium, Propionibacterium acidipropionici, Leuconostoc, Pediococcus, Weissella, Aerococcus, Staphylococcus, and Kluyveromyces.

[0059] As an embodiment, the Bifidobacterium of the present invention includes one or more of Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, and Bifidobacterium longum. As an embodiment, the Lactobacillus of the present invention includes one or more of Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus delbrueckii, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, and Lactobacillus kefiranofaciens. As an embodiment, the Lactobacillus casei of the present invention includes one or more of Lactobacillus casei casei, Lactobacillus casei paracasei, and Lactobacillus casei rhamnosus. As an embodiment, the Lactobacillus mucosae of the present invention includes Lactobacillus mucosae fermentum and / or Lactobacillus reuteri. As an embodiment, the Lactiplantibacillus plantarum of the present invention includes Lactiplantibacillus plantarum plantarum. As an embodiment, the Limosilactobacillus fermentum of the present invention includes Limosilactobacillus fermentum salivarius. As an embodiment, the Lactobacillus paraplantarum of the present invention includes Lactobacillus paraplantarum curvatus and / or Lactobacillus paraplantarum sakei. As an embodiment, the Streptococcus of the present invention includes Streptococcus salivarius. As an embodiment, the Lactococcus of the present invention includes Lactococcus lactis and / or Lactococcus cremoris. As an embodiment, the Propionibacterium of the present invention includes Propionibacterium freudenreichii. As an embodiment, the Propionibacterium acidipropionici of the present invention includes Propionibacterium acidipropionici acidipropionici. As an embodiment, the Leuconostoc of the present invention includes Leuconostoc mesenteroides. As an embodiment, the Pediococcus of the present invention includes Pediococcus acidilactici and / or Pediococcus pentosaceus. As an embodiment, the Weissella of the present invention includes Weissella cibaria. As an embodiment, the Aerococcus of the present invention includes Aerococcus viridans var. homari. As an embodiment, the Staphylococcus of the present invention includes Staphylococcus xylosus and / or Staphylococcus carnosus. As an embodiment, the Kluyveromyces of the present invention includes Kluyveromyces marxianus.

[0060] As an implementation manner, the Bifidobacterium animalis of the present invention includes Bifidobacterium animalis subsp. animalis and / or Bifidobacterium animalis subsp. lactis. As an implementation manner, the Bifidobacterium longum includes Bifidobacterium longum subsp. longum and / or Bifidobacterium longum subsp. infantis. As an implementation manner, the Lactobacillus delbrueckii includes Lactobacillus delbrueckii subsp. bulgaricus and / or Lactobacillus delbrueckii subsp. lactis. As an implementation manner, the Lactobacillus kefiranofaciens includes Lactobacillus kefiranofaciens subsp. kefiranofaciens. As an implementation manner, the Streptococcus salivarius includes Streptococcus salivarius subsp. thermophilus. As an implementation manner, the Lactococcus lactis includes Lactococcus lactis subsp. lactis and / or Lactococcus lactis subsp. diacetylactis. As an implementation manner, the Propionibacterium freudenreichii includes Propionibacterium freudenreichii subsp. shermanii.

[0061] As an implementation manner, based on the mass parts of the plant-derived exosome preparation, the composition containing plant-derived exosomes of the present invention further includes 5-15 parts of other components; the other components include zinc gluconate and / or taurine; as another implementation manner, based on the mass parts of the plant-derived exosome preparation, the composition containing plant-derived exosomes of the present invention further includes 10 parts of other components. The present invention does not have special limitations on the sources of the zinc gluconate and / or taurine, and they can be purchased conventionally. The present invention utilizes zinc gluconate and / or taurine to have an auxiliary synergistic effect.

[0062] The present invention also provides a preparation method of the composition described in the above technical solution, including the following steps: mixing the components to obtain the composition. The present invention does not have strict requirements on the mixing method, and it is only necessary to mix the plant-derived exosome preparation, chitosan oligosaccharide and probiotics evenly.

[0063] The composition provided by the present invention can increase the abundance of beneficial flora, improve the intestinal flora composition, regulate and maintain the balance of the intestinal flora structure, and has a good improvement effect on related diseases such as intestinal physiological function disorders and pathological damages caused by abnormal intestinal flora. Moreover, the composition of the present invention also has the effects of significantly inhibiting intestinal inflammation and antioxidant stress, which helps the repair of damaged mucosa and inhibits the development of gastrointestinal inflammation. In addition, the composition of the present invention has a good effect of moistening the intestines and relieving constipation, can improve the intestinal homeostasis environment, avoid or improve symptoms such as diarrhea and constipation, soothe the signs of gastrointestinal sensitivity, and has an obvious and delicate care effect on the gastrointestinal tract. Furthermore, the raw materials of the composition of the present invention are safe and stable, non-toxic and side-effect-free, and can be taken daily as a health food or functional food for enhancing human immunity and strengthening the stomach for a long time.

[0064] In view of the effects of the composition provided by the present invention, the application of the composition described in the above technical solution or the composition obtained by the preparation method described in the above technical solution in one or more of the following also belongs to the protection scope of the present invention: (1) preparing health foods, functional foods or drugs for regulating the balance of intestinal flora; (2) preparing drugs for treating intestinal inflammation.

[0065] The health food or functional food prepared by using the composition of the present invention can regulate the intestinal flora, increase the abundance of beneficial bacteria in the intestine, reduce the proportion of harmful bacteria, and help the beneficial bacteria attach and colonize on the intestinal epithelial cells. The drug prepared by using the composition of the present invention can enhance the intestinal regulatory immunity and intestinal barrier function, promote the secretion of anti-inflammatory cytokines, and reduce the level of pro-inflammatory cytokines in the colon tissue, thereby alleviating the intestinal oxidative stress and inflammatory response, showing a strong anti-inflammatory effect.

[0066] As an embodiment, the drug for treating intestinal inflammation of the present invention has a significant effect in treating gastrointestinal discomfort symptoms such as diarrhea, loose stools, constipation, abdominal pain and other intestinal problems. As an embodiment, the drugs of the present invention can be prepared by different conventional preparation methods according to different dosage forms. It should be noted that when preparing the composition into a drug, pharmaceutically acceptable auxiliary components need to be added, and the added auxiliary components are selected according to the dosage form required, which is known in the art. It has certain physiological activities, but the addition of this component will not change the dominant position of the above composition, but only play an auxiliary effect. These auxiliary effects are only the utilization of the known activities of this component and are common auxiliary treatment methods in the medical field.

[0067] To further illustrate the present invention, a composition containing plant-derived exosomes, its preparation method and application provided by the present invention will be described in detail below with reference to the drawings and examples, but they should not be construed as limiting the protection scope of the present invention.

[0068] Example 1

[0069] Isolation and purification of ginger-derived exosomes

[0070] 1. Sample preparation

[0071] (1) Washing and cutting: Ginger (purchased from Ultra-Extraction Biotechnology (Xianning) Co., Ltd., batch number: 20241031-1) was thoroughly washed and cut into small pieces with a sterile scalpel. (2) Homogenization: The cut ginger was put into PBS and mixed at a ratio of 1:5 (weight / volume), and processed with a homogenizer to prepare a homogenate.

[0072] 2. Preliminary impurity removal

[0073] (1) Low-speed centrifugation: The homogenate was centrifuged at 5000×g for 10 min to remove impurities such as coarse particles and cell debris. (2) Medium-speed centrifugation: The supernatant was taken and centrifuged at 10000×g for 20 min to further remove large particle impurities.

[0074] 3. Exosome extraction

[0075] (1) Ultracentrifugation: The supernatant was placed in an ultracentrifuge and centrifuged at 100000×g for 90 min. Note: Fixed-angle rotors or well-balanced ultracentrifuge tubes are required for ultracentrifugation. (2) Washing: The pellet was removed, resuspended in PBS, and centrifuged again at 100000×g for 90 min. (3) Collection of the pellet: Exosomes will be retained in the form of a pellet.

[0076] 4. Purification

[0077] 4.1. Filtration through a membrane: The exosome suspension was filtered through a 0.22-μm filter membrane to remove large particle contamination.

[0078] 4.2. Density gradient centrifugation: Sucrose gradient or iodixanol gradient was used to further purify exosomes. The specific steps are as follows:

[0079] (1) Preparation of sucrose gradient solution: Three layers of sucrose solution were prepared to form a gradient of 0.5 M sucrose, 1.0 M sucrose, and 1.5 M sucrose from the bottom to the top.

[0080] (2) Sample loading: The exosome sample was evenly added to the upper layer of the density gradient, avoiding mixing.

[0081] (3) Centrifugation: Using an ultracentrifuge, centrifuge at a speed of 100000×g for 2 - 3 h.

[0082] (4) Collection of exosomes: After centrifugation, observe the turbidity of the liquid layer and carefully remove the turbid liquid layer using a pipette.

[0083] (5) Washing: The collected exosomes were resuspended in PBS buffer and then centrifuged at 100000×g for 90 minutes to remove unpurified impurities in the solvent.

[0084] 5. Identification of ginger-derived exosomes

[0085] (1) The basic morphology of the ginger-derived exosomes obtained by the above separation and purification was observed using a transmission electron microscope (TEM). The specific method includes: taking 20 μL of the prepared exosomes, thoroughly mixing them, and then dropping them onto a copper grid for loading with a diameter of 2 mm. After standing at room temperature for 5 min, the residual liquid at the edge of the copper grid was gently blotted with filter paper. Then, the copper grid was inverted onto a droplet of 30 g / L phosphotungstic acid (pH 6.8), and negatively stained at room temperature for 5 min. Finally, the copper grid was dried under an incandescent lamp and placed under the transmission electron microscope for observation and photography. The results are as Figure 1 shown. According to Figure 1 , it can be seen that the average particle size of the ginger-derived exosomes prepared in this example is 100 nm, and they have a typical cup-shaped structure of exosomes, achieving the large-scale extraction of exosomes.

[0086] (2) A nanoparticle tracking analyzer (NTA) was used to detect the particle size distribution and concentration of the exosomes. The results showed that the average diameter of the exosomes was about 100 nm.

[0087] 6. Storage

[0088] The suspension of the extracted ginger-derived exosomes was aliquoted into sterile freeze-drying bottles or sterile EP tubes, with an aliquot volume of about 1 - 2 mL per bottle, and stored at -80 °C to avoid repeated freezing and thawing.

[0089] Example 2

[0090] Preparation of a composition containing ginger-derived exosomes

[0091] 1. Freezing: The ginger-derived exosomes from Example 1 were frozen at -80 °C for 24 h using an -80 °C refrigerator to ensure that the samples were completely frozen.

[0092] 2. Freeze-drying process: The frozen ginger-derived exosomes from Example 1 were carefully transferred to the sample rack of the freeze-dryer, ensuring that each bottle or tube did not collide with each other to facilitate air circulation; the freeze-dryer was started for preliminary freezing treatment and drying treatment to obtain a freeze-dried powder preparation of ginger-derived exosomes;

[0093] Among them, the temperature of the preliminary freezing treatment was -50 °C to -40 °C, and the time was 1 - 2 h; during the drying treatment, the water in the sample was sucked out by vacuum, and the sample temperature was maintained at -80 °C for 24 - 48 h to avoid damage to the structure of the exosomes.

[0094] 2. Freeze-dried powder preparation of ginger-derived exosomes, chitosan oligosaccharide (purchased from Ultra-Extraction Biotechnology (Xianning) Co., Ltd., batch number: 2023815-1, degree of polymerization 5-10), and probiotics (composed of Lactobacillus acidophilus LA85 and Bifidobacterium infantis B145, purchased from Ultra-Extraction Biotechnology (Xianning) Co., Ltd., batch number: 20230623-25) were mixed at a mass ratio of 10:45:45 to obtain a composition containing ginger-derived exosomes.

[0095] Test Example 1

[0096] According to Figure 2 the following process, the cell line enteritis model was constructed and the drug efficacy was detected. The specific steps are as follows:

[0097] 1. HT-29 cell line culture and subculture

[0098] (1) Subculture was performed when the density of the HT-29 cell line in the cell culture plate reached over 80%.

[0099] (2) Digestion: Discard the old medium, add 1-2 mL of 0.25% trypsin to the culture plate, place it in a 37°C incubator for digestion for 2-4 minutes, observe the cell digestion under a microscope. If most of the cells become round and detached, quickly take it back to the operation table, gently tap the culture flask a few times, and then add 1-2 mL of complete medium to terminate digestion. Gently pipette the cells, aspirate them into a centrifuge tube after complete detachment, and centrifuge (1000 rpm, 5 minutes).

[0100] (3) Seeding: Discard the supernatant, resuspend with 1-3 mL of complete medium. Transfer the resuspended cell suspension to two new cell culture plates.

[0101] 2. Establishment of the cell line inflammation model and drug administration plan

[0102] (1) The HT-29 cell line was subcultured into the well plates for experiments. When the HT-29 cell line was cultured to a density of about 70%, the experiment could begin.

[0103] (2) The HT-29 cell line was randomly divided into a control group (CTRL), an inflammation treatment group (IMF), a positive drug treatment group (IMF + 5-ASA), a 24-hour experimental group (IMF + 300 24h), and a 48-hour experimental group (IMF + 300 48h); among them, the HT-29 cell lines in the inflammation treatment group, the positive drug treatment group, the 24-hour experimental group, and the 48-hour experimental group were pre-induced with complete medium containing 20 ng / mL IFN-γ for 9 hours, and then induced with complete medium containing 1 μg / mL LPS for 15 hours, while the control group was cultured normally; after the induction culture ended, the positive drug treatment group treated the HT-29 cell line with 500 μg / mL 5-aminosalicylic acid (5-ASA) for 24 hours; the 24-hour experimental group treated the HT-29 cell line with the composition containing ginger-derived exosomes obtained in Example 2 at 300 μg / mL for 24 hours; the 48-hour experimental group treated the HT-29 cell line with the composition containing ginger-derived exosomes obtained in Example 2 at 300 μg / mL for 48 hours.

[0104] 3. Result verification

[0105] After the treatment in Step 2, the cells were collected to obtain the test samples, and the following detections were carried out:

[0106] (1) RT-qPCR verification

[0107] The three genes of IL-16, IL-1B, and TNF-α are all cell inflammation genes. After extracting the RNA of the test samples using an RNA kit and reverse-transcribing it into cDNA, RT-qPCR was performed to detect the intestinal inflammation marker genes, and the results are as Figure 3 shown.

[0108] According to Figure 3 it can be seen that compared with the control group, the expression of the three inflammatory genes increased significantly after the inflammation treatment group induced the inflammatory response, and the expression of the COX-2 gene representing intestinal inflammation also increased significantly, proving that after the induction with INF-γ and LPS, the cells had an obvious inflammatory response. After inducing the inflammatory response, after adding 5-ASA to the cell culture for 24 hours, a certain down-regulation of the expression of genes such as IL-1B, TNF-α, and COX-2 was observed, proving that the drug had a certain effect in the inflammatory cell model. After adding the composition containing ginger-derived exosomes obtained in Example 2 to the culture system, a certain degree of reduction in the cell inflammatory genes was also observed, proving that the composition containing ginger-derived exosomes obtained in Example 2 may have a similar effect to the positive anti-inflammatory drug 5-ASA in terms of cell inflammation.

[0109] (2) Immunofluorescence verification.

[0110] The samples to be tested were plated on the cell slides, and the cell slides were fixed-blocked-stained-sealed and immunofluorescence stained. Images were collected using a fluorescence microscope. The results were as follows: Figure 4 shown.

[0111] COX-2 (cyclooxygenase-2) plays an important role in cell apoptosis and tumorigenesis. In ulcerative colitis, COX-2 expression increases. Figure 4 It can be seen that after the inflammation model was established (inflammation treatment group), COX-2 fluorescence ( Figure 4 The red fluorescence in the upper middle row) increased compared with the control group, and the fluorescence expression decreased after using 5-ASA and the composition containing ginger-derived exosomes obtained in Example 2, proving that the expression of intestinal inflammatory response proteins decreased after using the drugs.

[0112] CDX-2 is an intestinal-specific transcription factor and a key regulatory protein for intestinal epithelial formation and differentiation. ZO-1 is an important structural protein for maintaining the integrity of the intestinal barrier. It is related to intestinal leakage and plays an important role in cell connection and signal transduction. When ZO-1 expression is lost, it proves that the intestinal barrier is damaged. Figure 4 It can be seen that in the control group, CDX-2 ( Figure 4 Red fluorescence in the middle and lower row), ZO-1( Figure 4 Green fluorescence in the middle and lower rows) are all clearly expressed. When the cells are in an inflammatory state (IMF group), the expression of cell CDX-2 and ZO-1 is significantly reduced. After using the drug 5-ASA, the expression of CDX-2 in the cells increased compared with the inflammation group. When the composition containing ginger-derived exosomes obtained in Example 2 was added, the expression of ZO-1 increased significantly. It is proved that the use of the composition containing ginger-derived exosomes obtained in Example 2 can rescue the inflammatory state of cells to a certain extent and restore cell activity and characteristics to a certain extent.

[0113] (3) MTT, DAO and D-LA content detection

[0114] a. MTT (thiazolyl blue) test.

[0115] The samples to be tested were plated in a flat-bottom 96-well plate, with 5000 cells per well. After the experiment, MTT dissolution reagent was added according to the instructions of the MTT kit. After the dissolution was completed, the absorbance value was detected using an ELISA instrument and the data was processed. The results are shown in the figure. Figure 5 shown.

[0116] b.DAO (diamine oxidase) test

[0117] The samples to be tested were processed, interrupted, and developed according to the instructions of the DAO kit. The samples were added to a blank 96-well plate and the absorbance was tested using an ELISA reader. The data were processed and the results were as follows. Figure 5as shown

[0118] c. Detection of D-LA (D-lactic acid).

[0119] The collected cell samples were processed, disrupted, and colored respectively according to the D-LA kit instructions. The samples were added to a blank 96-well plate and the absorbance value was measured using an enzyme-linked immunosorbent assay (ELISA) reader, and the data was processed. The results are as Figure 5 shown

[0120] According to Figure 5 It can be seen that when the intestinal inflammation model was completed (inflammatory treatment group), the MTT reading decreased significantly. After using 5-ASA and the composition containing exosomes derived from ginger obtained in Example 2, the MTT reading rebounded to a level similar to that of the control group. When the intestinal inflammation model was completed (inflammatory treatment group), the DAO content increased. After using 5-ASA and the composition containing exosomes derived from ginger obtained in Example 2, the DAO content decreased. When the intestinal inflammation model was completed (inflammatory treatment group), the D-LA content increased. When using a positive anti-inflammatory drug and the test drug, the D-LA content decreased to a level similar to that of the control group. The detection results of MTT, DAO, and D-LA contents indicate that the use of 5-ASA and the composition containing exosomes derived from ginger obtained in Example 2 has a certain anti-inflammatory effect on intestinal cell inflammation.

[0121] According to the above content, it can be seen that the composition provided by the present invention can increase the abundance of beneficial bacteria, improve the intestinal flora composition, regulate and maintain the balance of the intestinal flora structure, repair damaged intestinal mucosa, inhibit intestinal inflammation, improve intestinal leakage, and resist oxidative stress.

[0122] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A composition containing plant-derived exosomes, characterized in that: The invention comprises the following components in parts by weight: 1 to 60 parts of plant-derived exosome preparation, 1 to 60 parts of chitosan oligosaccharide and 1 to 60 parts of probiotics.

2. The composition according to claim 1, characterized in that The invention comprises the following components in parts by weight: 5 to 20 parts of plant-derived exosome preparation, 30 to 60 parts of chitosan oligosaccharide and 20 to 60 parts of probiotics.

3. The composition according to claim 1 or 2, characterized in that The plant-derived exosome preparation includes a plant-derived exosome lyophilized powder preparation.

4. The composition according to claim 3, characterized in that The plants include one or more of ginger, turmeric and yam.

5. The composition according to claim 1 or 2, characterized in that The polymerization degree of the chitosan oligosaccharide is 2-20.

6. The composition according to claim 1 or 2, characterized in that The probiotics include one or more of Bifidobacterium, Lactobacillus, Lactaseibacillus, Limosilactobacillus, Lactiplantibacillus, Ligilactobacillus, Latilactobacillus, Streptococcus, Lactococcus, Propionibacterium, Acidipropionibacterium, Leuconostoc, Pediococcus, Weizmannia, Mammaliicoccus, Staphylococcus and Kluyveromyces.

7. The composition according to claim 6, characterized in that The bifidobacterium includes one or more of Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve and Bifidobacterium longum; The lactobacillus comprises one or more of Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus bulgaricus, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii and Lactobacillus kefiranofaciens; The lactobacillus includes one or more of Lacticaseibacillus casei, Lacticaseibacillus paracasei and Lacticaseibacillus rhamnosus; The mucus lactobacillus includes Limosilactobacillus fermentum and / or Limosilactobacillus reuteri; The lactobacillus includes Lactobacillus plantarum; The genus Lactobacillus includes Ligilactobacillus salivarius; The lactobacillus includes Latilactobacillus curvatus and / or Latilactobacillus sakei; The streptococci include Streptococcus salivarius; The lactococci include Lactococcus lactis and / or Lactococcus cremoris; The propionibacterium includes Propionibacterium freudenreichii; The propionic acid bacteria include Acidipropionibacterium acidipropionici; The Leuconostoc includes Leuconostoc mesenteroides; The Pediococcus includes Pediococcus acidilactici and / or Pediococcus pentosaceus; The Weizmannia includes Weizmannia coagulans; The animal cocci include Mammaliicoccus vitulinus; The staphylococci include Staphylococcus xylosus and / or Staphylococcus carnosus; The Kluyveromyces yeast includes Kluyveromyces marxianus.

8. The composition according to claim 1 or 2, characterized in that Based on the mass percentage of the plant-derived exosome preparation, the composition further includes 5 to 15 parts of other components; The other components include zinc gluconate and / or taurine.

9. The method for preparing the composition according to any one of claims 1 to 8, characterized in that: The following steps are involved: The components are mixed to obtain a composition.

10. Use of the composition according to any one of claims 1 to 8 or the composition obtained by the preparation method according to claim 9 in one or more of the following; (1) Preparation of health foods, functional foods or medicines for regulating the balance of intestinal flora; (2) Preparation of drugs for treating intestinal inflammation.

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